Riboflavin production

The present invention provides a recombinant bacterium for the over-production of riboflavin. The recombinant bacterium has has been transformed by three or four vectors, two of which each comprise either a DNA sequence coding for the riboflavin synthesizing enzymatic activities of Bacillus subtilis or a DNA sequence which is substantially homologous and one or more transcription elements and a third and/or fourth vector comprising either a DNA sequence coding for the ribA gene product of Bacillus subtilis or a DNA sequence which is substantially homologous and optionally a transcription element whereby one or a plurality of copies of each of these vectors has/have been integrated at three or four different sites within the bacterium's chromosome.

BACKGROUND OF THE INVENTION
 Riboflavin (vitamin B.sub.2) is synthesized by all plants and many
 microorganisms but is not produced by higher animals. Because it is a
 precursor to coenzymes such as flavin adenine dinucleotide and flavin
 mononucleotide, that are required in the enzymatic oxidation of
 carbohydrates, riboflavin is essential to basic metabolism. In higher
 animals, insufficient riboflavin can cause loss of hair, inflammation of
 the skin, vision deterioration, and growth failure.
 Riboflavin can be commercially produced either by a complete chemical
 synthesis, starting with ribose, or by fermentation with the fungi
 Eremothecium ashbyii or Ashbya gossypii (The Merck Index, Windholz et al.,
 eds., Merck & Co., p. 1183, 1983). Mutants of Bacillus subtilis, selected
 by exposure to the purine analogs azaguanine and azaxanthine, have been
 reported to produce riboflavin in recoverable amounts (U.S. Pat. No.
 3,900,368, Enei et al., 1975). In general, exposure to purine or
 riboflavin analogs selects for deregulated mutants that exhibit increased
 riboflavin biosynthesis, because the mutations allow the microorganism to
 "compete out" the analog by increased production (Matsui et al., Agric.
 Biol. Chem. 46:2003, 1982). A purine-requiring mutant of Saccharomyces
 cerevisiae that produces riboflavin has also been reported (U.S. Pat. No.
 4,794,081, Kawai et al., 1988). Rabinovich et al. (Genetika 14:1696
 (1978)) report that the riboflavin operon (rib operon) of B. subtilis is
 contained within a 7 megadalton (Md) EcoRI fragment (later referred to as
 a 6.3 Md fragment in Chikindas et al., Mol. Genet. Mik. Virusol. no. 2:20
 (1987)). It is reported that amplification of the rib operon may have been
 achieved in E. coli by cloning the operon into a plasmid that conferred
 resistance to ampicillin and exposing bacteria containing that plasmid to
 increasing amounts of the antibiotic. The only evidence for rib
 amplification is a coincident increase in the presence of a
 green-fluorescing substance in the medium; the authors present a number of
 alternative possibilities besides an actual amplification of the operon to
 explain the phenomenon observed.
 French Patent Application No. 2,546,907, by Stepanov et al. (published Dec.
 7, 1984), discloses a method for producing riboflavin that utilizes a
 mutant strain of B. subtilis which has been exposed to azaguanine and
 roseoflavin and that is transformed with a plasmid containing a copy of
 the rib operon.
 Morozov et al. (Mol. Genet. Mik. Virusol. no. 7:42 (1984)) describe the
 mapping of the B. subtilis rib operon by assaying the ability of cloned B.
 subtilis rib fragments to complement E. coli riboflavin auxotrophs or to
 marker-rescue B. subtilis riboflavin auxotrophs. Based on the known
 functions of the E. coli rib genes, the following model was proposed for
 the B. subtilis operon: ribG (encoding a deaminase)--ribO (the control
 element)--ribB (a synthetase)--ribF--ribA (a GTP-cyclohydrolase)--ribT/D
 (a reductase and an isomerase, respectively)--ribH (a synthetase).
 Morozov et al. (Mol. Genet. Mik. Virusol. no. 11:11 (1984)) describe the
 use of plasmids containing the B. subtilis rib operon with either
 wild-type (ribO.sup.+) or constitutive (ribO 335) operator regions to
 assay their ability to complement B. subtilis riboflavin auxotrophs. From
 the results, a revised model of the rib operon was proposed, with ribO now
 located upstream of all of the structural genes, including ribG, and with
 the existence of an additional operator hypothesized, possibly located
 just upstream of ribA.
 Morozov et al. (Mol. Genet. Mik. Virusol. no. 12:14 (1985)) report that the
 B. subtilis rib operon contains a total of three different promoters (in
 addition to a fourth "promoter" that is only active in E. coli). The
 primary promoter of the operon was reported to be located within the ribO
 region, with the two secondary promoters reported between the ribB and
 ribF genes and within the region of the ribTD and ribH genes,
 respectively.
 Chikindas et al. (Mol. Genet. Mik. Virusol. no. 2:20 (1987)) propose a
 restriction enzyme map for a 6.3 Md DNA fragment that contains the rib
 operon of B. subtilis. Sites are indicated for the enzymes EcoRI, PstI,
 SalI, EcoRV, PvuII and HindIII.
 Chikindas et al. (Mol. Genet. Mik. Virusol. no. 4:22 (1987) report that all
 of the structural genes of the B. subtilis rib operon are located on a 2.8
 Md BglII-HindIII fragment and that the BglII site is located between the
 primary promoter of the operon and the ribosomal-binding site of its first
 structural gene. As described infra, Applicants show that this BglII site
 is actually located within the most-5' open reading frame of the rib
 operon, so that the 2.8 Md fragment described does not contain all of the
 rib structural genes. Thus, in contrast to the report of Chikindas et al.,
 the 1.3 Md BglII fragment does not contain the ribosomal-binding site of
 the first structural gene; insertions at this site lead to a
 riboflavin-negative phenotype. Consequently, any attempt to use this BglII
 site to engineer the rib operon in order to increase expression, for
 example by replacing the 5' regulatory region with a stronger promoter,
 would actually destroy the integrity of the first structural gene and thus
 the operon as well.
 Chikindas et al. (Dokl. Akad. Nauk 5 SSSR 298:997 (1988)) disclose another
 model of the B. subtilis rib operon, containing the primary promoter,
 p.sub.1, and two minor promoters, p.sub.2 and p.sub.3 :
 ribO(p.sub.1)-ribG-ribB-p.sub.2 -ribF-ribA-ribT-ribD-p.sub.3 -ribH. As
 before, it is incorrectly reported that the 1.3 Md BglII fragment contains
 the entire first structural gene of the operon and that this proximal
 BglII site maps within the primary regulatory region.
 SUMMARY OF THE INVENTION
 It is therefore an object of the present invention to provide a recombinant
 bacterium comprising a bacterium which has been transformed by three or
 four vectors, two of which each comprise either a DNA sequence coding for
 the riboflavin synthesizing enzymatic activities of Bacillus subtilis or a
 DNA sequence which is substantially homologous and one or more
 transcription elements and a third and/or fourth vector comprising either
 a DNA sequence coding for the ribA gene product of Bacillus subtilis or a
 DNA sequence which is substantially homologous and optionally further
 comprising transcription element whereby one or a plurality of copies of
 each of these vectors has/have been integrated at three or four different
 sites within its chromosome. More preferably it is an object of the
 present invention to provide a recombinant bacterium as described above
 whereby the two vectors which comprise either the DNA sequence coding for
 the riboflavin synthesizing enzymatic activities of Bacillus subtilis or a
 DNA sequence which is substantially homologous further comprise two
 transcription elements for each vector, preferably promoters and the third
 and/or fourth vector comprise either the DNA sequence coding for the ribA
 gene product of Bacillus subtilis or a DNA sequence which is substantially
 homologous and a transcription element, preferably a promotor.
 Furthermore it is an object of the present invention to provide a
 recombinant bacterium as described above whereby the two vectors which
 comprise either the DNA sequence coding for the riboflavin synthesizing
 enzymatic activities of Bacillus subtilis or a DNA sequence which is
 substantially homologous have been integrated at two different sites of
 the chromosome in a plurality of copies and the third and/or fourth
 vector, has/have been integrated at the third and/or fourth site as a
 single copy.
 Furthermore it is an object of the present invention to provide a
 recombinant bacterium characterized therein that the additional DNA
 sequence coding for the ribA gene product of Bacillus substilis or a DNA
 sequence which is substantially homologous is not integrated at a third
 and/or fourth additional site in the chromosome but integrated at the same
 site as one of the two vectors with DNA sequences coding for the
 riboflavin synthesizing enzymatic activities and amplified together with
 this vector in this site. Such constructs can be made by one skilled in
 the art based upon generally available knowledge and detailed teachings as
 given, e.g., in EP 405 370 (EP 370), the corresponding U.S. application
 Ser. No. 370,378, abandoned, (US. 378) filed Jun. 22, 1989 and its
 continuation in part U.S. Ser. No. 07/581,048, abandoned, (US. 048) filed
 Sep. 11, 1990. These applications, EP 405,370, U.S. Pat. No. 370,378 and
 U.S. Pat. No. 581,048 are hereby incorporated by reference, the pertinent
 portions of which are reproduced herein.
 Furthermore it is understood by one skilled in the art that for
 transformation the DNA sequences used need not necessarily be in the form
 of vectors but could also be used without additional vector DNA.
 Furthermore it is an object of the present invention to provide a
 recombinant bacterium as described above which is E. coli or Bacillus,
 preferably Bacillus subtilis, Cyanobacter or Corynebacteria.
 Furthermore it is an object of the present invention to provide a process
 for the production of riboflavin characterized therein that a recombinant
 bacterium as described above is grown under suitable growth conditions and
 the riboflavin secreted into the medium is isolated by methods known in
 the art. It is also an object of the present invention to provide a
 process for the preparation of a food or feed composition characterized
 therein that such a process has been effected and the riboflavin obtained
 thereby is converted into a food or feed composition by methods known in
 the art.

DETAILED DESCRIPTION OF THE INVENTION
 DNA sequences which are useful for the purpose of the present invention
 comprise DNA sequences which code for the riboflavin synthesizing
 enzymatic activities of Bacillus subtilis described above and which are
 selected from the following DNA sequences:
 (a) a DNA sequence which hybridizes under standard conditions with
 sequences defined above;
 (b) a DNA sequence which, because of the degeneracy of the genetic code,
 does not hybridize with sequence (a), but which codes for polypeptides
 having exactly the same amino acid sequences as the polypeptides encoded
 by these DNA sequences; and
 (c) a DNA sequence which is a fragment of the DNA sequences specified in
 (a) or (b) and which codes for a polypeptide having the riboflavin
 synthesizing enzymatic activities of Bacillus subtilis.
 "Standard conditions" for hybridization in this context the conditions
 which are generally used by a man skilled in the art to detect specific
 hybridization signals and which are described, e.g., by Sambrook et al.,
 "Molecular Cloning" second edition, Cold Spring Harbor Laboratory Press
 1989, New York, or preferably so called stringent hybridization and
 non-stringent washing conditions, or more preferably so called stringent
 hybridization and stringent washing conditions of which one skilled in the
 art is familiar with and which are described, e.g., in Sambrook et al.
 (s.a.).
 DNA sequences which can be used for the purpose of the present invention
 are disclosed, e.g., in the specification of EP 370, US 378 and US 048 of
 which, the pertinent portions are hereinbelow reproduced The riboflavin
 biosynthetic genes from various bacteria can be cloned for use in the
 present invention. Yeast or bacterial cells from species including but not
 limited to the genus Bacillus, E. coli and many other gram-positive and
 gram-negative bacteria can potentially serve as the nucleic acid source
 for the molecular cloning of the rib operon. The DNA containing the fib
 operon may be obtained, by standard procedures known in the art, for
 example, from a DNA library prepared by cloning chromosomal DNA or
 fragments thereof, purified from the desired bacterial cell, into a
 suitable vector for propagation of the gene. (See, for example, Maniatis
 et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
 Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1982, DNA
 Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II).
 In the molecular cloning of the gene from chromosomal DNA, fragments are
 generated, some of which will encode the desired rib operon. The DNA may
 be cleaved at specific sites using various restriction enzymes.
 Alternatively, one may use DNAse in the presence of manganese to fragment
 the DNA, or the DNA can be physically sheared, as for example, by
 sonication. The linear DNA fragments can then be separated according to
 size by standard techniques, including but not limited to agarose and
 polyacrylamide gel electrophoresis and density gradient centrifugation.
 Once the DNA fragments are generated, DNA libraries are prepared using an
 appropriate cloning and/or expression vector. A large number of
 vector-host systems known in the art may be used. Possible vectors
 include, but are not limited to, plasmids or modified viruses, but the
 vector system must be compatible with the host cell used. For E. coli such
 vectors include, but are not limited to, bacteriophages such as .lambda.
 derivatives, high-copy plasmids such as pBR322 or pUC plasmids, or
 low-copy plasmids derived from Pseudomonas plasmid RK2. For Bacillus such
 vectors include, but are not limited to, bacteriophages such as .rho.11
 (Dean et al., J. Virol. 20: 339, 1976; Kawamura et al., Gene 5:87, 1979)
 or .phi.105 derivatives (Iijima et al., Gene 2:115, 1980; Errington, J.
 Gen. Microbiology 130:2615, 1984; Dhaese et al., Gene 32: 181, 1984;
 Errington, J. in Bacillus Molecular Biology and Biotechnology
 Applications, A. T. Ganesan and J. A. Hoch, eds. (Academic Press, New
 York,), p. 217, 1986), high-copy plasmids such as pUB110 (Ehrlich, Proc.
 Natl. Acad. Sci. (USA) 74: 1680, 1977) or pBD64, or low-copy plasmids such
 as pE194 derivatives (Gryczan, T. J. in The Molecular Biology of the
 Bacilli, D. A. Dubnau, ed. (Academic Press, New York), pp. 307-329, 1982;
 Horinouchi and Weisblum, J. Bacteriol. 150: 804, 1982). Recombinant
 molecules can be introduced into host cells via transformation,
 transfection, protoplasting, infection, electroporation, etc.
 Once the DNA libraries are generated, identification of the specific clones
 harboring recombinant DNA containing the a operon may be accomplished in a
 number of ways (as described, for example, in Maniatis et. al., supra).
 For example, if an amount of the operon or a fragment thereof is available
 from another bacterial source (e.g., from E. coli) and is sufficiently
 homologous to the riboflavin biosynthetic genes of Bacillus to hybridize
 thereto, that DNA can be purified and labeled, and the generated bank of
 DNA fragments may be screened by nucleic acid hybridization to the labeled
 probes (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. and
 Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Alternatively,
 sequences comprising open reading frames of the endogenous rib operon, or
 subsequences thereof comprising about 10, preferably 15 or more
 nucleotides, may be used as hybridization probes. Such probes can be made
 synthetically, based on a portion of the nucleic acid or amino acid
 sequence (examples of which are provided below) of a gene product known to
 be encoded by the operon ("reverse genetics"). If a purified rib
 operon-specific probe is unavailable, cloned gene libraries of restriction
 fragments (from partial Sau3A-digests, for example) can be made in
 bacteria, especially B. subtilis or E. coli, and the rib operon-containing
 recombinant clones can be identified by either marker-rescue or
 complementation of known rib mutations.
 In a preferred embodiment, the rib operon of B. subtilis can be isolated
 for use from an E. coli plasmid library of B. subtilis DNA. In particular,
 and as described below, the B. subtilis rib operon can be isolated by
 virtue of its homology to a radiolabelled, synthesized nucleotide probe
 that is derived from an internal region of a gene product known to be
 encoded by the operon of B. subtilis. Although a portion of the amino acid
 sequence for .beta.-riboflavin synthase (Ludwig et al., J. Biol. Chem.
 262:1016, 1987) can be the basis for such a probe, with the third
 nucleotide of each codon estimated from frequency of codon usage, a
 similar probe based on another region of this protein or another protein
 from the rib operon can be utilized and would fall within the scope of the
 present invention. The present invention further enables screening by use
 of synthetic probes which are derived from the nucleic acid sequence shown
 in FIG. 3.
 Analogous methods to those detailed here can be used to isolate the rib
 operon of other bacteria, especially other Bacilli or E. coli. In a
 specific embodiment, such clones can be selected by assay for ability to
 hybridize to the labeled B. subtilis rib operon or a hybridizable portion
 thereof. It is well known in the art that starting from an appropriate
 mRNA preparation, cDNA can be prepared; such cDNA can also be used in
 accordance with the present invention to prepare vectors for the
 transformation of appropriate bacteria for riboflavin overproduction.
 Once the host cells with recombinant DNA molecules that include the
 isolated rib operon or a portion thereof are identified, the DNA may be
 obtained in large quantities. This then permits the rib operon to be
 manipulated and its nucleotide sequence to be determined using various
 cloning and sequencing techniques familiar to those knowledgeable in the
 art.
 For example, insertional mutagenesis can be used to locate and characterize
 the rib operon and genes thereof within a cloned piece of DNA. In a
 specific embodiment, rib-biosynthetic containing regions can be identified
 by inserting small cat (chloramphenicol acetyltransferase)-containing
 restriction fragments into several different restriction enzyme sites of
 the cloned DNA, and testing each derivative for insertional inactivation
 of riboflavin biosynthesis in an appropriate host (see below).
 The cloned DNA corresponding to the rib operon can be analyzed by methods
 including but not limited to Southern hybridization (Southern, E. M.,
 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g., Freeman
 et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction
 endonuclease mapping (Maniatis et al., 1982, Molecular Cloning, A
 Laborator Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
 N.Y.), and DNA sequence analysis. Restriction endonuclease mapping can be
 used to roughly determine the genetic structure of rib operon. Restriction
 maps derived by restriction endonuclease cleavage can be confirmed by DNA
 sequence analysis.
 DNA sequence analysis can be performed by any techniques known in the art,
 including but not limited to the method of Maxam and Gilbert (1980, Meth.
 Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al., 1977,
 Proc. Natl. Acad. Sci. U.S.A. 74:5463), or use of an automated DNA
 sequenator (e.g., Applied Biosystems, Foster City, Calif.). As an example,
 the DNA sequence of the rib operon of B. subtilis is presented in FIG. 3.
 Once the nucleotide sequence of the rib operon has been determined,
 putative open reading frames (ORFs) can then be identified along with the
 deduced amino acid sequence of their encoded product. Actual
 identification of the encoded product can be carried out, e.g., by
 performing S-30 coupled in vitro transcription/translation reactions, with
 various ORFs used as templates. Various mutational derivatives of the ORFs
 can also be tested for activity in functional assays of the S-30 reaction
 products, in order to test the function of the encoded products.
 In a specific embodiment of the invention relating to the B. subtilis rib
 operon, and detailed in the examples below, the above-described methods
 were used to determine that B. subtilis riboflavin biosynthesis is
 controlled by a single operon of approximately 4.2 kb containing five
 biosynthetic genes: the .beta. subunit of riboflavin synthase and ORFs
 designated 2, 3, 4, and 5 (see FIG. 4). ORFs 2, 3, 4, and 5 were
 subsequently shown to encode proteins with molecular weights of about 15
 kd, 47 kd, 26 kd, and 44 kd, respectively. As described below, ORF 5 was
 shown to encode a putative rib-specific deaminase that catalyzes the
 reduction of a deaminated pyrimidine to a ribitylamino-linkage in an early
 step in riboflavin biosynthesis. Our data also indicated that ORF 4
 encodes the ax subunit of riboflavin synthase and ORF 3 encodes a GTP
 cyclohydrolase, while ORF 2 possibly encodes a rib-specific reductase. ORF
 1 and ORF 6 were found to be outside the primary transcription unit of the
 rib operon. The primary site for initiation of transcription of the rib
 operon was determined to be probably the apparent .sigma..sup.A promoter
 located 290 bp upstream from the first gene in the operon, ORF 5 (FIG. 4,
 P.sub.1). The coding regions, promoters and transcription termination
 sites of the B. subtilis rib operon are shown in Table VI below.
 The present invention encompasses the nucleotide and amino acid sequences
 of the genes of the rib operon, as well as subsequences thereof encoding
 functionally active peptides, and sequences which are substantially the
 same as such sequences. A functionally active peptide, as used herein,
 shall mean a protein or peptide which is capable of catalysing a reaction
 leading to riboflavin biosynthesis. A functionally active nucleic acid
 sequence shall mean a sequence capable of regulating riboflavin
 biosynthesis. A sequence substantially the same as another sequence shall
 mean a sequence capable of hybridizing to the complementary sequence
 thereof. In addition, a nucleic acid sequence not naturally controlling
 the expression of a second nucleic acid sequence shall mean a sequence
 which does not control the expression of the second sequence in the
 bacterium from which the second sequence is isolated.
 Once the genetic structure of the rib operon is known, it is possible to
 manipulate the structure for optimal use in the present invention. For
 example, the rib operon can be engineered to maximize riboflavin
 production.
 Depending on the host-vector system utilized, any one of a number of
 suitable transcription and translation elements may be used. Promoters
 produced by recombinant DNA or synthetic techniques may also be used to
 provide for transcription of the inserted sequences. When propagating in
 bacteria the regulatory sequences of the rib operon itself may be used. In
 an embodiment in which the entire rib operon, or greater than one gene
 thereof, is desired to be expressed as a polycistronic message, a
 prokaryotic host is required. In an embodiment in which a eukaryotic host
 is to be used, appropriate regulatory sequences (e.g., a promoter) must be
 placed in the recombinant DNA upstream of each gene/ORF that is desired to
 be expressed.
 Specific initiation signals are also required for efficient translation of
 inserted protein coding sequences. These signals include the initiation
 codon (ATG, GTG or TFG) and adjacent sequences, such as the ribosome
 binding site (RBS). It should be noted that the RBS of a given coding
 sequence can be manipulated to effect a more efficient expression of that
 coding sequence at the translational level. In cases where an entire open
 reading frame of the rib operon, including its own initiation codon and
 adjacent regulatory sequences, is inserted into the appropriate expression
 vectors, no additional translational control signals may be needed.
 However, in cases where only a portion of the coding sequence is inserted,
 or where the native regulatory signals are not recognized by the host
 cell, exogenous translational control signals, including the initiation
 codon, must be provided. The initiation codon must furthermore be in phase
 with the reading frame of the protein coding sequences to ensure
 translation of the entire insert. These exogenous translational control
 signals and initiation codons can be of a variety of origins, both natural
 and synthetic.
 In addition, a host cell strain may be chosen which modulates the
 expression of the rib operon gene(s) or modifies and processes the gene
 product(s) thereof in the specific fashion desired. Expression from
 certain promoters can be elevated in the presence of certain inducers;
 thus, expression of the genetically engineered rib operon proteins may be
 controlled. In one embodiment, the regulatory regions of the operon, such
 as the promoter and the termination/anti-termination regulatory sequences,
 can be manipulated or replaced with constitutive or growth-regulated
 promoters to deregulate the rib operon and thus increase riboflavin
 production. Furthermore, appropriate cell lines or host systems can be
 chosen to ensure the desired modification and processing of the expressed
 proteins. Many manipulations are possible and within the scope of the
 present invention.
 In one specific embodiment of the invention, the 5' regulatory sequence of
 the B. subtilis rib operon can be removed and replaced with one or more of
 several B. subtilis promoters; such a construction will cause high-level
 expression of the rib biosynthetic genes. This approach would involve the
 introduction of new restriction sites within a 20-30 bp region between the
 end of the transcription terminator and the RBS sequence of the first gene
 in the operon ORF 5. Such restriction sites can be introduced by either
 site-directed mutagenesis or by deleting all regulatory sequences upstream
 from the right-most BglII (BglII.sub.R) site located within the first 30
 bp of ORF 5 (see FIGS. 3 and 4) and inserting at this site a synthetic
 oligonucleotide that finishes off the 5' end of ORF 5 (including the
 ribosomal-binding site) and contains new upstream restriction sites. Once
 these constructions are made, promoter-containing restriction fragments
 with ends compatible to the new restriction sites can be introduced,
 causing expression of the rib genes under the control of the new promoter.
 Both constitutive and growth-regulated B. subtilis promoters can be used,
 including but not limited to strong promoters from the lytic bacteriophage
 SPO1 genes, veg, amy (amylase), and apr (subtilisin).
 In another aspect of the invention, rib operon DNA fragments which have
 transcriptional regulatory activity (e.g., promoters) can be used to
 regulate the expression of heterologous gene products.
 Sequence information for such sequences can also be obtained from any known
 sequence data bank, e.g., the European Bioinformatics Institute (Hinxton
 Hall, Cambridge, GB). The DNA sequences can then be prepared on the basis
 of such sequence information using, e.g., the PCR method known in the
 state of the art and described, e.g., in the examples or other methods of
 molecular cloning known in the art.
 Once such DNA sequences are available they can be integrated for further
 manipulation into suitable vectors known in the state of the art and
 described, e.g., in the examples. Preferred are such vectors for
 integration into the chromosome of the host which is preferably Bacillus
 and more preferably a Bacillus subtilis and subsequent amplification, if
 desired. Such vectors are described, e.g., in the Examples or known in the
 state of the art. Such vectors can further carry so called transcription
 elements, like enhancers and/or promoters, like the veg-promoter and/or
 natural or synthetic ribosomal binding sites and/or terminators, like,
 e.g., the cryT-terminator which is known in the state of the art. See,
 e.g., EP 370, US 378 and US 048 the pertient portions of which are herein
 reproduced. The desired host cells can then be transformed by such vectors
 by methods described, e.g., in the Examples or which are known in the
 state of the art and grown in a suitable medium. The riboflavin secreted
 into such medium can be isolated as described in the Examples or as known
 in the state of the art.
 After the invention has been described in general hereinbefore, the
 following examples are intended to illustrate details of the invention,
 without thereby limiting it in any matter.
 EXAMPLES
 Example 1: Riboflavin-Overproducing B. subtilis Mutants
 We describe in the examples herein the production of strains of Bacillus
 subtilis which overproduce riboflavin. In order to accomplish this, we
 used classical genetics, genetic engineering, and fermentation. Classical
 genetics with selection using purine and riboflavin analogs was used to
 deregulate the pathways for purine (riboflavin precursor) and riboflavin
 biosynthesis. Riboflavin production was increased further by cloning and
 engineering the genes of the riboflavin biosynthetic pathway (the rib
 operon), allowing for constitutive, high-level production of rate-limiting
 biosynthetic enzyme(s).
 The biosynthesis of riboflavin in B. subtilis originates with GTP (FIG. 1).
 To obtain a host that overproduces riboflavin we used classical genetics
 to both increase the amount of GTP that the cell produces and to
 deregulate the riboflavin pathway. Purine overproduction in B. subtilis
 can be achieved by obtaining mutants resistant to purine analogs such as
 azaguanine and decoyinine, and other antagonists such as methionine
 sulfoxide (see e.g., Ishii and Shiio, Agric. Biol. Chem. 36(9):1511-1522,
 1972; Matsui et al., Agric. Biol. Chem. 43(8):1739-1744, 1979). The
 riboflavin pathway can be deregulated by obtaining mutants resistant to
 the riboflavin analog roseoflavin (Matsui et al., Agric. Biol. Chem.
 46(8):2003-2008, 1982). Roseoflavin-resistant strains were selected from
 several strains which had been previously mutagenized and which were
 resistant to several purine analogs. Described below are the methods used
 to produce a strain (RB50) which overproduces riboflavin.
 8-Azaguanine-Resistant Mutants
 B. subtilis is effectively killed by the purine analogue 8-azaguanine
 (Sigma Chemical Co., St. Louis, Mo.) at a concentration of 500 .mu.g/ml,
 and resistant mutants appear spontaneously at a frequency of less than 1
 in 10.sup.8. Ethyl methyl sulfonate (EMS; Sigma) at 30 .mu.g/ml was used
 as a mutagen to increase the frequency of azaguanine-resistant (Ag.sup.r)
 mutations. Mutagenesis was performed on cells from B. subtilis strain 168
 by standard procedures (Miller, 1972, Experiments in Molecular Genetics,
 Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). After plating
 4.times.10.sup.6 mutagenized cells on minimal medium (Sloma et al., J.
 Bact. 170:5557, 1988) containing 500 .mu.g/ml azaguanine and restreaking
 for single colonies, 35 Ag.sup.r colonies resulted. One mutant, RB11
 (Ag.sup.r -11), was used in the construction of RB50.
 Decoyinine-Resistant Mutants
 Decoyinine-resistant (Dc.sup.r) mutations were obtained spontaneously at a
 frequency of 1 in 10.sup.6 or after EMS mutagenesis at 1 in 10.sup.5 by
 plating cells on minimal medium containing 100 .mu.g/ml of decoyinine
 (Upjohn Co., Kalamazoo, Mich.). A Dc.sup.r mutant of RB11 was obtained by
 mutagenesis with EMS as described above. One Dc.sup.r colony, RB15
 (Ag.sup.r -11, Dc.sup.r -15), was used in the construction of RB50.
 Transfer of the Ag and Dc Mutations
 These purine analog-resistant mutations were transferred to a different
 strain background in order to isolate them from any unwanted EMS-induced
 mutations and to verify that the Ag.sup.r and Dc.sup.r mutations were due
 to single loci. Since part of the "carbon flow" from inosine monophosphate
 (IMP), a riboflavin precursor, is also used for adenine nucleotide
 biosynthesis, a host strain was selected that was blocked in the adenosine
 monophosphate (AMP) pathway via the mutation pur-60, allowing more carbon
 material to "flow" from IMP to the guanine nucleotide precursors of
 riboflavin (FIG. 2). B. subtilis strain 1A382 (hisH2, trpC2, pur-60) was
 made competent (Sloma et al., J. Bact. 170:5557 (1988)) and transformed
 (by the method of Gryczan et al., J. Bact. 134:318 (1978)) with total DNA
 prepared from the Ag.sup.r /Dc.sup.r mutant RB15. The Trp.sup.+
 (tryptophan) revertant colonies were selected, with 3.3% (10/300) of those
 also being Dc.sup.r and 2.3% (7/300) Ag.sup.r. This result was not
 unexpected since, due to "congression" (transformation of a second
 unlinked marker), a number of the Trp.sup.+ colonies should also be
 resistant to decoyinine or azaguanine.
 One Dc.sup.r colony, RB36 (his H2, pur-60, Dc.sup.r -15), one Ag.sup.r
 colony, RB40 (his H2, pur-60, Ag.sup.r -11), and one Dc.sup.r /Ag.sup.r
 colony (which was also found to be his.sup.+), RB39 (pur-60, Ag.sup.r -11,
 Dc.sup.r -15), were all selected for further study.
 Methionine Sulfoxide-Resistant Mutants
 Selection using high levels of methionine sulfoxide (MS; 10 mg/ml, Sigma)
 resulted in spontaneous mutants appearing at a sufficiently high frequency
 that mutagenesis with EMS was not necessary. The Ag.sup.r /Dc.sup.r
 mutant, RB39, was streaked onto minimal medium containing 10 .mu.g/ml MS.
 Resistant colonies were obtained and were restreaked for single resistant
 colonies. One strain, RB46 (pur-60, Ag.sup.r -11, Dc.sup.r -15, MS.sup.r
 -46) was selected for further study.
 Roseoflavin Resistant Mutants
 Although many of these Ag.sup.r, Dc.sup.r and MS.sup.r mutants were likely
 to be overproducing GTP, none of them produced levels of riboflavin
 detectable on plates. In order to deregulate the riboflavin biosynthetic
 pathway, conditions were determined to select for resistance to the
 riboflavin analog roseoflavin (Toronto Research Chemical). Maximum killing
 of cells occurred at 100 .mu.g/ml of roseoflavin in minimal or complete
 medium; increasing the concentration did not result in any additional
 killing. Mutations to roseoflavin resistance (RoF.sup.r) spontaneously
 occurred at a sufficiently high rate (approximately 5.times.10.sup.-5)
 such that mutagenesis with EMS or other chemicals was not necessary.
 Approximately 1000 RoF.sup.r colonies were obtained from each of the
 strains described above, 1A382, RB36, RB39, RB40 and RB46. RoF.sup.r
 mutants from all of these strains showed a low level of fluorescence on
 minimal media plates when exposed to long-wave UV light (366 nm),
 indicating some riboflavin production. One of the RoF.sup.r colonies
 obtained from RB46, RB46Y (pur-60, Ag.sup.r -11, Dc.sup.r -15, MS.sup.r
 -46, RoF.sup.r -46), when grown on minimal medium, produced 14 mg/l of
 riboflavin as determined by HPLC (described above).
 Of all the strains treated, only RB39 and RB46 produced a significantly
 different phenotype when RoF.sup.r colonies were selected. Approximately
 0.5% to 1.0% of the RoF.sup.r colonies of either RB39 or RB46 produced an
 intensely fluorescent, yellow colony. Of these colonies, RB51 (pur-60,
 Ag.sup.r -11, Dc.sup.r -15, RoF.sup.r -51), arising from RB39, and RB50
 (pur-60, Ag.sup.r -11, Dc.sup.r -15, MS.sup.r -46, RoF.sup.r -50), arising
 from RB46, produced a stable, fluorescent-yellow phenotype which
 correlated with a higher level of riboflavin production, as determined by
 HPLC. When grown in minimal medium, both RB50 and RB51 produced higher
 levels of riboflavin in their supernatants than the other RoF.sup.r
 strains, about 40 mg/l and 30 mg/l, respectively. The lineage of RB50 is
 depicted in FIG. 5.
 Because intensely fluorescent (and thus riboflavin overproducing) colonies
 could be obtained in non-MS.sup.r strains such as RB51, it appeared that
 this mutation in general might not be contributing significantly to the
 higher production phenotype. Both of the other mutations, Ag.sup.r and
 Dc.sup.r (Ag.sup.r -11 and Dc.sup.r -15 in RB39), appear to be necessary
 to produce high levels of riboflavin since no intensely fluorescent
 RoF.sup.r colonies could be found in strains containing only the Ag.sup.r
 -11 (from RB40) or Dc.sup.r -15 (from RB36) mutation alone.
 guaC Mutations
 Another possibly important mutation for achieving overproduction of GTP,
 and thus riboflavin, is guaC3, which prevents the conversion of GMP back
 into IMP (see FIG. 2). To construct a strain containing guaC3 that
 overproduces riboflavin, competent B. subtilis strain 62121 cells (guaC3,
 trpC2, metC7) (Endo et al., J. Bact. 15: 169, 1983) were transformed with
 RB50 DNA and selected for Dc.sup.r on plates containing 100 .mu.g/ml of
 decoyinine. Thousands of Dc.sup.r colonies resulted. Of 200 colonies which
 were patched onto Dc.sup.r plates, one was found that exhibited the
 riboflavin overproduction phenotype (based on UV fluorescence), and was
 RoF.sup.r. This colony was designated RB52 (guaC3, trpC2, metC7, Dc.sup.r
 -15, RoF.sup.r -50) and was reserved for subsequent study.
 Other Analog-Resistant Mutants
 Finally, because mutants resistant to several additional purine analogs
 also have been reported to be altered in purine metabolism, such mutations
 were assayed in order to investigate their effect on
 riboflavin-overproducing strains. It was determined that 500 g/ml of
 8-azaxanthine, 1 mg/ml of 6-thioguanine, or 2 mg/ml of sulfaguanidine
 (Sigma) effectively kills wild-type B. subtilis. The azaguanine-resistant,
 riboflavin-overproducing strains RB50::[pRF8].sub.90 and
 RB53::[pRF8].sub.90 (see below) were found to be already resistant to
 azaxanthine. Although separate azaguanine- and azaxanthine-resistant
 mutations with different properties have been described previously, in
 this case the Ag.sup.r -11 and Ag.sup.r -53 mutations appear to also
 convey azaxanthine resistance.
 HPLC Analysis of riboflavin in crude supernatants of B. subtilis
 Accumulation of riboflavin in B. subtilis cultures was quantitated by
 reverse-phase HPLC. Riboflavin standards (Sigma Chemical Co., St. Louis,
 Mo.) or cell-free supernatants from strains to be tested were fractionated
 over a 4.6 mm.times.250 mm Vydac C.sub.18 column equilibrated with 1%
 ammonium acetate (pH 6.0). At injection, the column was developed with a
 linear gradient of methanol and monitored for riboflavin at 254 nm.
 Authentic riboflavin (i.e. riboflavin "standard") elutes at the mid-point
 of the gradient.
 Example 2: Cloning B. subtilis Rib Operon
 Our general strategy to isolate a restriction fragment containing the rib
 operon was to screen a "mini" E. coli plasmid library of B. subtilis DNA
 by hybridization with a synthetic oligonucleotide probe, the DNA sequence
 of which was partially derived from the published amino acid sequence for
 the .beta. subunit of riboflavin synthase (Ludwig et al., J. Biol. Chem.
 262:1016, 1987). A summary of the protocol is presented in FIG. 6.
 A synthetic, 54-base "guess-a-mer" oligonucleotide probe was used for this
 screening based on amino acids 84-102 of the 240 amino acid riboflavin
 synthase protein, sequenced by Ludwig et al. (J. Biol. Chem.
 262:1016-1021, 1987). The third nucleotide of each codon in the probe was
 chosen according to estimates made of the most frequent codon usage of B.
 subtilis, based upon, for example, some of the sequences available in
 GenBank.RTM. (Los Alamos Nat. Lab, Los Alamos, N. Mex.). The probe
 consisted of the following sequence:
 5'-GGAGCTACAACACATTATGATTATGTTTGCAATGAAGCTGCTAAAGGAATTGCF-3' (SEQ ID NO.
 230). To test the specificity of the probe, the .sup.32 P-labelled 54-mer
 DNA was hybridized to nylon filters containing EcoRI-digested chromosomal
 DNA (Southern, J. Mol. Biol. 98:503, 1975) isolated from wild-type and the
 mutant B. subtilis strains. The probe strongly hybridized to a single 9-10
 kb fragment of EcoRI-digested B. subtilis (rib.sup.+ met.sup.-) DNA, which
 is in good agreement with the predicted size of the rib-containing
 fragment (Osina et al., FEBS. Lett. 196:75, 1986). A labelled fragment of
 the identical size was detected when the probe was hybridized to two
 mutant strains, RB46 (pur-60, Ag.sup.r -11, Dc.sup.r -15, MS.sup.r -46)
 and RB50 (pur-60, Ag.sup.r -11, Dc.sup.r -15, MS.sup.r -46, RoF.sup.r
 -50), the latter being a riboflavin overproducer. These hybridization
 experiments were repeated using HindHIII-cut chromosomal DNA, which
 resulted in the probe identifying a smaller, single fragment of
 approximately 1.8 kb; this latter result was useful in determining the
 general location of the rib biosynthetic operon within the cloned DNA.
 Isolation of Plasmids pRF1, pRF2 and pRF3,
 Containing Wild-type Rib Biosynthetic Genes
 A "mini" gene library of 9-11 kb EcoRI fragments from B. subtilis strain
 168 (rib.sup.+) DNA was prepared using pRK290, a low-copy number vector
 derived from the Pseudomonas replicon RK2 (Ditta et al., Plasmid 3:149,
 1985). EcoRI fragments (size 9-11 kb) of B. subtilis (rib.sup.+ met.sup.-)
 DNA were isolated by sucrose (10-40%) rate-zonal centrifugation. A
 four-fold excess of these fragments (0.22 .mu.g) was ligated to EcoRI-cut
 pRK290 (0.26 .mu.g), that had been dephosphorylated with calf intestinal
 alkaline phosphatase (CIAP), at a total DNA concentration of 10 .mu.g/ml.
 Approximately 10 ng of ligated DNA was transformed into E. coli DH5 (F-,
 endA1, hsdR11 [r.sub.k -, m.sub.k +], supE.sub.44, thi-1, .lambda.-,
 recA1, gyrA96, relA1), resulting in tetracycline-resistant (Tc.sup.r)
 colonies at a frequency of 7.7.times.10.sup.4 /.mu.g of DNA. To determine
 the fraction of transformants containing insert DNA of 9-11 kb, plasmid
 mini-lysates were prepared from several Tc.sup.r transformants, and their
 DNA was analyzed by restriction enzyme digestion. About 40% of the
 Tc.sup.r transformants were found to contain single EcoRI-generated
 inserts of 9-11 kb.
 Approximately 1140 of the Tc.sup.r colonies were screened with the .sup.32
 P-labelled 54-mer probe specific for the riboflavin synthase gene. One
 colony gave a positive signal. Plasmid DNA, designated pRF1, was isolated
 from this clone and tested for Rib.sup.+ -marker rescue activity by
 transforming the DNA into B. subtilis 1A210 that contains the
 riboflavin-deficient mutation rib-2, and selecting for Rib.sup.+
 prototrophic colonies. pRF1 transformed 1A210 to Rib.sup.+ prototrophy at
 a high frequency. Plasmid DNA from a randomly chosen Tc.sup.r transformant
 failed to rescue this marker.
 Restriction enzyme analysis revealed that pRF1 actually contained two
 EcoRI-fragment inserts, of 10 kb and 11 kb. To determine which fragment
 contained the rib operon, EcoRI-digested pRF1 was probed with the .sup.32
 P-labelled, 54-mer riboflavin synthase probe. The results indicated that
 only the smaller, 10 kb fragment cross-reacted with the probe. Moreover,
 when the 10 kb EcoRI fragment was recloned into the EcoRI site of pBR322,
 recombinant plasmids pRF2 and pRF3 resulted, representing the two possible
 orientations of insertion. Both plasmids were found to rescue the rib-2
 mutation of B. subtilis 1A210 to prototrophy at a high frequency.
 Isolation of Plamsids pRF6 and pRF7
 Containing Rib Biosynthetic Genes
 From RoF.sup.r -B. subtilis Strain RB50
 RB50 is one of the RoF.sup.r mutants of B. subtilis, produced as described
 above, that is deregulated for riboflavin biosynthesis. It has been
 reported that approximately 80% of RoF.sup.r mutations reside within the
 rib operon at the rib locus (Stepanov, et al., Genetika (USSR) 13:490,
 1977). Like the wild-type rib operon, rib genes in RB50 were also
 contained on a 9-10 kb EcoRI fragment; thus this fragment was cloned using
 the protocol outlined in FIG. 6, with pBR322 used as the cloning vector.
 Size-selected 9-11 kb EcoRI fragments (0.1 .mu.g) from RB50 were prepared
 as before and ligated to a two-fold excess of ends of EcoRI-cut,
 dephosphorylated pBR322 DNA (0.34 .mu.g) at a total DNA concentration of
 22 .mu.g/ml. Approximately 9 ng of ligated DNA was transformed into E.
 coli DH5, resulting in ampicillin-resistant (Ap.sup.r) colonies at a
 frequency of 3.5.times.10.sup.5 /.mu.g of DNA.
 Restriction enzyme analysis of plasmid DNA isolated from a sampling of 12
 Ap.sup.r colonies revealed that 50% contained plasmids with 9-11 kb EcoRI
 inserts. Approximately 1140 Ap.sup.r colonies were screened with the
 .sup.32 P-labelled 54-mer probe specific for the riboflavin synthase gene
 by colony hybridization. Six colonies gave positive signals. Plasmids pRF6
 and pRF7, isolated from two of these six colonies, were identified by
 restriction enzyme analysis as containing inserts with the same
 orientation as pRF2 and pRF3, respectively. In addition, both plasmids
 were able to marker-rescue the rib-2 mutation at high frequencies.
 Example 3: Introducing Rib.sup.+ DNA Into B. subtilis
 As described supra, the rib operon from both a wild-type strain and a
 RoF.sup.r mutant of B. subtilis were cloned as identical 10 kb EcoRI
 fragments into the EcoRI site of the E. coli replicon pBR322; the
 derivation of these recombinant plasmids is schematically diagrammed in
 FIG. 6. To introduce the 10 kb EcoRI fragment containing the rib operon
 into B. subtilis in multiple copies, and thus further increase riboflavin
 production, we constructed a plasmid vector which would allow integration
 into the B. subtilis chromosome. The integrated DNA was amplified by
 selecting colonies that would grow at high drug concentrations of
 chloramphenicol.
 Construction of and Transformation with
 Integrational rib Plasmids pRF4 and pRF8
 To construct the integrational vector, the drug-resistance gene
 chloramphenicol acetyltransferase (cat), which is selectable in B.
 subtilis, was introduced into pRF2 and pRF6, the pBR322 vectors with the
 10 kb fragment from wild-type or RoF.sup.r B. subtilis strains,
 respectively. The plasmids pRF2 and pRF6 were digested with , which cuts
 the plasmids uniquely within the pBR322 sequence, and dephosphorylated
 with CIAP. The cleaved DNA was ligated to a 1.3 kb BamHI fragment
 containing the cat gene (Youngman et al., Plasmid 12: 1-9, 1984), and the
 ligated DNAs then transformed into E. coli DH5 cells (Hanahand, J. Mol.
 Biol. 166: 557, 1983). Approximately 80-90% of the Ap.sup.r transformants
 were chloramphenicol resistant (Cm.sup.r); restriction analysis of the
 isolated plasmids (Maniatis et al.) confirmed that plasmid DNA from the
 Cm.sup.r colonies contained the 1.3 kb fragment. The plasmid containing
 the wild-type riboflavin fragment and the cat gene was designated pRF4;
 the plasmid containing the cloned riboflavin fragment from the RoF.sup.r
 strain was called pRF8. (Since the RoF.sup.r mutation was subsequently
 shown to be outside the rib operon, these plasmids are presumably
 identical).
 The plasmids pRF4 and pRF8 were transformed into four different B. subtilis
 strains: the riboflavin overproducer RB50 (Ag.sup.r -11, Dc.sup.r -15,
 MS.sup.r -46, RoF.sup.r -50), the RB50 parent RB46 (Ag.sup.r -1, Dc.sup.r
 -15, MS.sup.r -46), the RB50 parent 1A382, and IS75, a common laboratory
 strain. Competent IS75 and 1A382 cells were transformed with pRF4 or pRF8;
 these same plasmids were introduced into RB46 and RB50 by transformation
 of protoplasts (Chang and Cohen, Mol. Gen. Genet 168:111-115, 1979). The
 pRF4 or pRF8 DNA integrated into each of these four strains was amplified
 by selecting for colonies that grew at higher chloramnphenicol
 concentrations. In each strain, we were able to obtain colonies that grew
 in up to 60 .mu.g/ml of chloramphenicol.
 In addition, RB52 (guaC3, trpC2, metC7 Dc.sup.r -15, RoF.sup.r -50),
 produced by transforming the guaC3 B. subtilis strain 62121 with DNA from
 RB50, was made competent and transformed with pRF8. The integrated plasmid
 in one of the many Cm.sup.r colonies that resulted was amplified using 90
 .mu.g/ml of chloramphenicol. The resulting cells, RB52::[pRF8].sub.90'
 were grown to mid-log phase and plated on minimal media containing 500
 .mu.g/ml azaguanine. Approximately 20 Ag.sup.r colonies resulted. One such
 colony seemed to produce a more intense fluorescence. The lineage of this
 strain, RB53::[pRF8].sub.90' is given in FIG. 7.
 Example 4: Riboflavin Overproduction by Strains Containing pRF4 or pRF8
 RB50 containing pRF4 or pRF8 displayed the riboflavin overproduction
 phenotype (yellow and UV-fluorescent colonies). Amplification of the
 rib.sup.+ DNA in a wild-type strain or the parent strains of RB50 did not
 yield yellow or UV-fluorescent colonies, a finding that indicates that the
 RoF.sup.r mutation (which deregulates the biosynthesis of riboflavin) is
 required for chromosomal amplification of wild-type DNA to cause
 riboflavin overproduction. A series of shake flask fermentations were
 performed in 25 ml of riboflavin minimal medium (RMM, in Table I) in a 300
 ml baffled flask (Bellco) to measure the production of riboflavin from
 RB50 that contained the integrated and amplified rib operon.
 TABLE I
 COMPOSITION OF MEDIA
 RMM g/l
 Sodium glutamate 2.0
 Casamino acids (Difco) 0.2
 Yeast extract (Difco) 0.2
 KH.sub.2 PO.sub.4 6.0
 K.sub.2 HPO.sub.4 14.0
 (NH.sub.4).sub.2 SO.sub.4 2.0
 Sodium citrate 1.0
 MgSO.sub.4.7H.sub.2 O 0.2
 Adenosine 0.05
 (adjusted to pH 7.0 and autoclaved)
 Maltose 15.0
 (added as sterile 20% solution after autoclaving)
 The fermentations were run with strrrns RB46, RB50 and RB50 containing pRF4
 amplified by selection for resistance to 30 .mu.g/ml of chloramphenicol
 (RB50::[pRF4].sub.30) and 90 .mu.g/ml of chloramphenicol
 (RB50::[pRF4].sub.90). At 24 and 48 hours, supernatant samples were
 removed and measured for riboflavin content by reverse-phase HPLC.
 As shown in Table II, RB50::[pRF4].sub.30 produced 0.3 g/l of riboflavin,
 and RB50::[pRF4].sub.90 produced 0.7 g/l of riboflavin, in 48 hours, which
 is significandy more than that produced by the strains without rib
 amplification, such as RB46 and RB50.
 TABLE II
 QUANTITATIVE ANALYSIS OF RIBOFLAVIN-
 CONTAINING SUPERNATANTS FROM B. SUBTILIS
 Culture Time Riboflavin*
 Strain (hours) (g/l)
 RB46 24 0.009
 RB50 24 0.02
 RB50::[pRF4].sub.30 24 0.1
 RB50::[pRF4].sub.90 24 0.4
 RB46 48 0.007
 RB50 48 0.05
 RB50::[pRF4].sub.30 48 0.3
 RB50::[pRF4].sub.90 48 0.7
 *Riboflavin was measured using an HPLC assay.
 The dramatic increase in riboflavin production resulting from amplification
 of rib genes in the deregulated host argues that information encoded by
 the cloned DNA is rate-limiting for riboflavin biosynthesis.
 Example 5: Mapping the RoF.sup.r -50 Mutation
 The RoF.sup.r -50 mutation in RB50 appeared to be critical to the
 riboflavin-overproduction phenotppe. To identify and possibly move the
 mutation into different strain backgrounds it was necessary to map the
 location of the RoF.sup.r -50 mutation on the B. subtilis chromosome.
 Since pRF4 and pRF8 gave very similar levels of riboflavin production in
 all strain backgrounds, it seemed unlikely that the RoF.sup.r -50 mutation
 was located on the cloned 10 kb EcoRI, rib-containing fragment. More
 likely, the RoF.sup.r -50 mutation is an unlinked repressor-type mutation,
 possibly in ribC, a repressor mutation which has been reported to map in
 the lys-aroD region of the B. subtilis chromosome (Chernk et al., Genetika
 (USSR) 15:1569, 1979). To determine whether the RoF.sup.r -50 mutation was
 linked or unlinked to the riboflavin operon, competent B. subtilis 1A210
 (rib-2) cells were transformed with RB50 DNA, selecting for rib.sup.+.
 Thousands of rib.sup.+ colonies resulted, and 200 colonies were patched
 onto tryptose blood agar base containing 100 g/ml of roseoflavin. No
 RoF.sup.r colonies resulted, and none of the colonies exhibited the
 riboflavin overproduction phenotype, confirming that the RoF.sup.r -50
 mutation is not located in the rib operon.
 Example 6: Locating rib.sup.+ Biosynthetic Genes Using CAT Insertional
 Mutagenesis
 FIG. 4 contains a restriction map of the rib-containing 10 kb EcoRI
 fragment of pRF2, prepared according to standard procedures. Restriction
 enzyme sites for XbaI, BglII, SstI, HpaI and NcoI are unique to the insert
 DNA, whereas SalI and PstI cut once in the insert and once in the vector,
 the insert does not contain any BamHI, XhoI or NheI restriction sites.
 Restriction enzyme HindIII cleaves the insert at multiple sites; the
 54-mer probe specific for the riboflavin synthase gene hybridized to an
 approximately 1.8 kb HindIII fragment, suggesting that the rib operon must
 also reside in the general area surrounding the SalI and left-most BglII
 (BglII.sub.L) sites.
 In general, to determine the boundaries of the rib operon, small
 cat-containing restriction fragments were used to construct insertions and
 deletions in the rib.sup.+ -cloned DNA fragment of pRF2.
 E. coli plasmid pEcc1 served as the primary source of restriction fragments
 bearing a cat gene which confers chloramphenicol-resistance in both E.
 coli and B. subtilis. This plasmid, a derivative of pMI1101 (Youngman et
 al., Plasmid 12:1-9, 1984) in which a non-essential region of the plasmid
 was removed by standard recombinant DNA techniques, contains a 1.3 kb
 cat-containing fragment flanked by the "polylinkers" of M13mp7, and
 therefore is capable of generating cat cassettes with either SmaI, EcoRI,
 SalI, or BamHI ends. To generate SstI or XbaI-ended fragments containing
 the cat gene, the 1.3 kb cat-containing BamHI fragments of pEcc1 was
 isolated, the ends modified with HindIII linkers, and the modified
 fragment cloned into the HindIII site within the polylinker region of
 pIC10R, generating plasmid pEcc4.
 Integrative plasmid derivatives were first constructed in E. coli and then
 transferred to the rib chromosomal locus of B. subtilis by DNA
 transformation. This was done by linearizing the plasmid by a restriction
 enzyme cut outside the cloned DNA insert, transforming competent B.
 subtilis strain 1A382 or PY79 (SP.beta..sup.c, SP.beta..sup.c rib.sup.+)
 cells with this cut DNA, and selecting for Cm.sup.r. Because the pBR322
 replicon is unable to replicate in B. subtilis, and the cat gene is
 bounded on both sides by sequences homologous to the rib.sup.+ locus, the
 cat-containing insertion or deletion can only be inserted into the
 chromosome by a double-crossover recombination event to yield Cm.sup.r
 transformants. To determine whether the insertion or deletion inactivated
 riboflavin synthesis, Cm.sup.r colonies were assessed for growth on
 minimal medium agar plates with or without the presence of riboflavin (Rib
 phenotype).
 As diagrammed in FIG. 8, cat-containing restriction fragments were inserted
 by ligation into the individual restriction sites for XbaI, SstI, SalI and
 BglII of pRF2, inserted between the pair of BglII or NcoI sites
 (generating deletions removing either a 2.0 kb BglII fragment or a 0.8 kb
 NcoI fragment) or inserted into single HaeIII and EcoRV sites of the
 approximately 1.8 kb HindIII fragment that hybridized to the rib-specific
 DNA probe, according to standard techniques. The results are shown in
 Table III.
 TABLE III
 CHARACTERIZATION OF INSERTION AND
 DELETION DERIVATIVES OF rib.sup.+ DNA
 B. subtilis.sup.b
 Insertion derivative.sup.a Riboflavin Phenotype
 A(XbaI)
 r +
 l ND
 B(SstI.sub.L)
 r +
 l ND
 C(SstI.sub.R)
 r --
 l --
 D(BglII.sub.L)
 r --
 l --
 E(SalI)
 r --
 l --
 F(BglII.sub.R)
 r --
 l --
 G(HaeIII)
 r ND
 l +
 H(EcoRV)
 r +
 l ND
 Deletion derivative
 Bgl
 r --
 l --
 Nco
 r +
 l +
 .sup.a "r" (right) and "l" (left) identify the transcriptional orientation
 of the inserted cat gene relative to the restriction map in FIG. 8.
 .sup.b B. subtilis strain 1A382 (rib.sup.+, trpC2, pur-60, hisH2) or PY79
 (SP .beta..sup.c, rib.sup.+)
 As summarized in FIG. 8 and Table III, insertions into the SalI, either
 BglII, or the "right most" SstI (SstI.sub.R) sites, or deletion of the 2.0
 kb BglII fragment, all generated Cm.sup.r colonies that could not produce
 riboflavin (Rib.sup.-), indicating that the rib operon was centrally
 located within the cloned DNA. Significantly, removal of the 0.8 kb NcoI
 fragment apparently had no effect on riboflavin production (Rib.sup.+),
 suggesting that one end of the rib gene cluster was located to the left of
 the "left most" NcoI (NcoI.sub.L) site. The other end of the rib operon
 was initially determined to map within the approximately 1.8 kb HindIII
 fragment because the two insertions at sites within the fragment, EcoRV
 and HaeIII, as well as sites distal to the fragment, XbaI and SstI.sub.L,
 all generated Cm.sup.r colonies that produced riboflavin.
 Example 7: Nucleotide Sequence of the Rib Operon
 Based on the cat-insertional mutagenesis of the cloned 10 kb DNA fragment,
 the entire rib operon was localized within a 6.0 kb region bounded by the
 SstI.sub.L and NcoI.sub.L sites.
 This 6.0 kb region of pRF2 containing the rib operon and flanking regions
 was sequenced by the dideoxy method of Sanger et al. (Proc. Natl. Acad.
 Sci. USA 74:5463, 1977). Briefly, M13 clones for sequencing were prepared
 either by subcloning specific restriction fragments into M13, by using the
 exonuclease activity of T4 DNA polymerase to generate a series of
 overlapping deletions (Dale et al., Plasmid 13:31, 1985), or by "shot-gun"
 cloning random fragments, from sonicated restriction fragments, into M13.
 In some cases, the nucleotide sequence across a restriction site juncture
 of adjacent fragments was also determined by primer extension sequencing.
 Approximately 5500 bp were sequenced on both strands and analyzed for
 sequences resembling typical open reading frames with gram
 positive-bacteria ribosome binding sites, gram-positive promoters and
 rho-independent transcription termination sites.
 Analysis revealed six complete, non-overlapping open reading frames (FIG.
 3): ORF 2 (124 amino acids), the gene coding for the .beta. subunit of
 riboflavin synthase (154 amino acids), ORF 3 (398 amino acids), ORF 4 (215
 amino acids), ORF 5 (361 amino acids) and ORF 6 (105 amino acids). Each
 ORF was preceded by a strong Bacillus ribosome binding site (RBS) with
 calculated thermostability ranging from .DELTA.G=-16 to -22 kcal/mol, and
 all of them were oriented in the same transcriptional direction. In
 addition, within the coding region of ORF 5, a second RBS site and ATG
 start codon were identified, potentially encoding a smaller protein of 248
 amino acids. However, based on S-30 in vitro coupled
 transcription/translation reactions (see below), ORF 5 appears to encode
 only a 361 amino acid protein. Finally, part of another coding region, ORF
 1, encoding the last 170 amino acids of a protein and oriented in the
 opposite direction, was also identified.
 Based on the following observations, riboflavin biosynthesis in Bacillus is
 controlled by a single operon containing 5 genes: the .beta. riboflavin
 synthase gene, ORF 2, ORF 3, ORF 4, and ORF 5, of which at least four, the
 .beta.-riboflavin synthase gene, ORF 3, ORF 4 and ORF 5, unambiguously
 encode biosynthetic enzymes, with the remaining one, ORF 2, possibly
 encoding a biosynthetic enzyme.
 1. ORF 3, ORF 4 and ORF 5 overlap restriction enzyme sites where insertion
 cat-containing restriction fragments caused inactivation of riboflavin
 production in B. subtilis (FIGS. 4 and 8).
 2. ORF 1 overlaps a restriction enzyme site(s) where insertion of
 cat-containing restriction fragments did not cause inactivation of
 riboflavin production in a rib.sup.+ B. subtilis strain (Table III and
 FIG. 8), nor did it cause reduction of riboflavin production in the
 deregulated, RoF.sup.r B. subtilis strain RB52.
 3. ORF 2 also overlaps a restriction enzyme site, EcoRV, where insertion of
 cat-containing restriction fragments did not cause inactivation of
 riboflavin production in a rib.sup.+ B. subtilis strain (Table III and
 FIG. 8). However, such an insertion did cause a detectable reduction of
 riboflavin production in the deregulated, RoF.sup.r B. subtilis strain
 RB52, indicating that the mutated ORF 2 gene product was partially
 inactive for riboflavin production. The results suggest that ORF 2 does
 encode a rib-specific enzyme.
 4. Two DNA sequences capable of forming stem-loop structures indicative of
 rho-independent transcriptional termination sites were identified within
 the intercistronic gaps between ORF 1 and ORF 2 and between ORF 5 and ORF
 6 (FIGS. 4 and 9). Removal of structures between ORF5 and ORF 6 enhances
 expression of riboflavin. The structures impart riboflavin sensitivity to
 lacZ-fusion constructs; thus, they can be used to impart such sensitivity
 to any other gene to which they are fused at the 5' end upstream of the
 promoter.
 5. A DNA sequence, TTGCGT-(17 bp)-TATAAT, (SEQ ID NO. 231) resembling the
 promoter recognized by the .sigma..sup.A (vegetative form) of B. subtilis
 RNA polymerase was identified approximately 290 bp upsteam from ORF 5,
 oriented in the same transcriptional direction as ORF 5 (FIG. 4). A
 transcriptional fusion of this promoter (P.sub.1, on a 1.1 kb BglII-NcoI
 restriction fragment) to a promoterless E. coli lacZ gene (P.sub.1 -lacZ)
 displayed riboflavin-regulated expression of .beta.-galactosidase activity
 in a rib.sup.+, B. subtilis strain (62121) and high-level, constitive
 (unregulated) expression of .beta.-galactosidase activity in a rib.sup.+,
 RoF.sup.r B. subtilis strain (RB52) only when the promoter was oriented in
 the same transcriptional direction as the gene, as shown in Table IV.
 Primer extension analysis was used to confirm the start site.
 Transcriptional and Northern analyses were used to show a polycistronic
 RNA of 4.2 kb encompasses the entire rib operon.
 TABLE IV
 RIBOFLAVIN-REGULATED EXPRESSION OF
 P.sub.1 -LacZ TRANSCRIPTIONAL FUSIONS
 .beta.-Galactosidase Specific
 Activity (Miller Units)
 + Riboflavin
 Strain (integrated plasmid) (2 .mu.g/ml) - Riboflavin
 B. subtilis 62121 (P.sub.1 -lacZ.sup.a) 1.3 4.2
 B. subtilis RB52 (P.sub.1 -lacZ.sup.a) 31 38
 B. subtilis 62121 (P.sub.i -lacZ.sup.b) &lt;0.1 &lt;0.1
 B. subtilis 62121 &lt;0.1 &lt;0.1
 .sup.a P.sub.1 and lacZ oriented in the same direction
 .sup.b P.sub.1 and lacZ oriented in opposite directions
 Based on these results, this .sigma..sup.A promoter, P.sub.1, is a primary
 promoter for transcription of ORF 5, ORF 4, ORF 3, .beta.-riboflavin
 synthase gene and ORF 2.
 6. A second DNA sequence, TTGAAG-(17 bp)-TACTAT, (SEQ ID NO. 232)
 resembling a promoter recognized by the .sigma..sup.A (vegetative form) of
 B. subtilis RNA polymerase was identified within the 3' end of ORF 4,
 approximately 295 bp upstream from ORF 3 and oriented in the same
 transcriptional direction as ORF 3 (FIG. 4). Integration into B. subtilis
 by a Campbell-type recombination event of an E. coli plasmid containing
 this promoter sequence on a 0.7 kb SalI-BglII restriction fragment did not
 cause inactivation of riboflavin production in B. subtilis, results which
 indicated that this second sequence (P.sub.2) has promoter activity and
 thus may actually control transcription (in addition to the .sigma..sup.A
 P.sub.1 promoter) of ORF 3, the .beta. subunit riboflavin synthase gene
 and ORF 2. LacZ fusions and Northern analysis confirmed the existence of
 this promoter.
 7. A third DNA sequence, TTGAAT-(18 bp)-TAAAAA, (SEQ ID NO. 233) possibly
 resembling the promoter recognized by the .sigma..sup.A (vegetative form)
 of B. subtilis RNA polymerase was identified within the intercistronic
 region between the .beta. subunit of the riboflavin synthase gene and ORF
 2, approximately 83 bp upstream of ORF 2 and oriented in the same
 transcriptional direction (FIG. 4). This .sigma..sup.A promoter, P.sub.3,
 may also control transcription of ORF 2, in addition to P.sub.1 and
 P.sub.2.
 8. In vitro-coupled transcription/translation analysis of S-30 reactions of
 the cloned DNA confirmed that ORF 2, ORF 3, ORF 4, and ORF 5 all actually
 encoded proteins of the size predicted from their respective sequences.
 9. Three of the five presumed enzymatic steps in riboflavin biosynthesis
 were assigned to specific coding regions by comparing predicted amino acid
 sequences or molecular weights of their products to published protein
 sequences, using GenBank.RTM., or known protein sizes.
 a. The putative protein encoded by the open reading frame between ORF 2 and
 ORF 3 almost identically matched the published 154 amino acid sequence of
 the .beta. subunit for the riboflavin synthase enzyme (Ludwig et al., J.
 Biol. Chem. 262:1016, 1987). Only one amino acid difference was detected:
 lysine was substituted for glycine at residue 65. This enzyme is reported
 to catalyze the formation of 6,7-dimethyl-8-ribityllumazine from
 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione-5'-phosphate (FIG. 1,
 structures 5 and 4, respectively) and 3,4-dihydroxybutanone-4-phosphate.
 b. A 39% identity in an 88-amino acid overlap was identified between the
 putative product of ORF5 and deoxycytidylate deaminase, a 188 amino acid
 protein encoded by the E. coli bacteriophage T.sub.2 (Maley et al., J.
 Biol. Chem. 258:8290, 1983). Based on this result, ORF 5 most likely
 encodes the rib-specific deaminase that catalyzes the formation of
 5-amino-6-(ribosylamino)-2,4(1H,3H-3H)-pyrimidinedione-5'-phosphate from
 2,5-diamino-6-(ribosylamino)-4(3H)-pyridinone-5-phosphate (FIG. 1,
 structures 3 and 2, respectively).
 c. The predicted molecular weight of the ORF 4 gene product (26,000 Da) was
 in good agreement with the molecular weight of the .alpha.-subunit for
 riboflavin synthase (23,000 Da; Bacher et al., J. Biol. Chem. 255:632,
 1980). Based on this result, ORF 4 encodes the .alpha.-subunit for
 riboflavin synthase, which catalyzes the final step of the biosynthetic
 pathway: dismutation of 6,7-dimethyl-8-ribityllumazine to riboflavin (FIG.
 1, structures 5 and 6, respectively) and
 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.
 10. The remaining enzymatic steps in riboflavin synthesis were tentatively
 assigned to coding regions by aligning the position of ORFs to a physical
 map of rib mutations in the operon (Morozov et al., Mol. Genet. Mik.
 Virusol. no. 7:42 (1984)). Mutations for defective GTP cyclohydrolase were
 reported to map to the 0.5 kb HindIII fragment. Since ORF 3 encompasses
 this restriction fragment, we concluded that ORF 3, at least in part,
 encodes this enzymatic function, which catalyzes the formation of
 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone-5'-phosphate from GTP
 (FIG. 1, structures 2 and 1, respectively). In addition, the biosynthetic
 gene encoding a rib-specific reductase was reported to be contained
 entirely within the approximately 1.8 kb HindIII fragment. Since this
 fragment contains only two complete coding regions, the .beta. subunit of
 the riboflavin synthase gene and ORF 2, we speculate that ORF 2 encodes
 the reductase, which catalyzes the formation of
 5-amino-6-(ribitylamino)-2,4(1H,3E)-pyrimidinedione-5'-phosphate from
 5-amino-6-(ribosylamino)-2,4(1H,3H)pyrimidinedione-5'-phosphate (FIG. 1,
 structures 4 and 3, respectively).
 In addition, a similar rho-independent transcription termination site was
 detected in the apparent leader region of the operon, downstream of the
 putative .sigma..sup.A P.sub.1 promoter but just upstream of the first
 coding region of the operon, ORF 5 (FIGS. 4 and 9). This potential
 terminator structure may be involved in regulation of the rib operon by a
 termination/anti-termination mechanism. In addition, a
 roseoflavin-resistant (R.sub.O F.sup.R) dependent regulatory region is
 present on a 0.7 kb SalI-BglII restriction fragment of ORF3.
 Assignment of rib ORFs to Protein Products
 One method for confirming whether the rib-specific ORFs encode proteins is
 to "visualize" the size and number of proteins synthesized from the cloned
 DNA in an S-30 in vitro coupled transcription/translation reaction using
 pRF2 and its various derivatives as templates. The S-30 fraction kit (New
 England Nuclear, used according to manufacturer's specifications) is
 especially efficient in translating B. subtilis genes due to the presence
 of their strong ribosome binding sites.
 Using the cloned 10 kb EcoRI fragment of pRF2 or pRF4 as templates, we
 expected to detect five putative rib-specific proteins: .beta. riboflavin
 synthase, 14.7 kilodaltons (kd) (Ludwig et al., J. Biol. Chem. 262:1016,
 1987); and the proteins from ORF 2, 13.6 kd; ORF 3, 43.7 kd; ORF 4, 23 kd;
 and ORF 5, 39.7 kd. We also expected to detect at least two other
 proteins, encoded by ORF 6 (11.6 kd) and ORF 1 (at least 18.7 kd), as well
 as any additional proteins encoded by genes present in the unsequenced
 regions of the 10 kb cloned DNA fragment. In addition, vector-associated
 proteins, including the bla and cat antibiotic resistance gene products,
 were also expected (the tet gene is not strongly expressed in S-30
 reactions).
 Excluding the bla- and cat-specific proteins (32 kd and 18 kd,
 respectively) and other vector-associated proteins, a total of six major
 .sup.35 S-labelled proteins were detected, with molecular weights of 47
 kd, 44 kd, 38 kd, 26 kd, 20 kd and 15 kd, on a 15%-SDS polyacrylamide gel
 of the S-30 reactions with pRF2 or pRF4. To assign these protein products
 to their corresponding rib-specific ORFs, S-30 reactions were repeated
 using various available deletion derivatives, cat-insertion derivatives,
 and subcloned fragments of the 10 kb EcoRI cloned DNA (FIG. 10). The
 results are shown in Table V.
 TABLE V
 RIB-SPECIFIC PROTEINS OBSERVED IN S-30 REACTIONS
 47,000 44,000 26,000 15,000
 Daltons Daltons Daltons Daltons
 Plasmid (ORF 3) (ORF 5) (ORF 4) (ORF 2)
 pRF2 + + + +
 pRF4 + + + +
 pRF21 - - + -
 pRF5 - - - +
 pRF29 - - - -
 pRF12 + - + +
 pRF10 - - - -
 pRF38 - - - -
 pRF24/pRF20 - + + +
 pRP23 + - + +
 Based on these results, protein products were assigned to ORF 3 (47 kd);
 ORF 5 (44 kd); ORF 4 (26 kd); and ORF 2 (15 kd), with molecular weights in
 close agreement with the predicted sizes.
 The assignment of products to ORF 2 and the .beta. riboflavin syntnmase
 gene were less straightforward than the assignments to the other ORFs.
 Since the S-30 reaction of pRF2 produce a 15 kd protein which was close to
 the predicted size of the proteins encoded by either gene, it was first
 assumed that this protein band actually contained both protein species.
 However, the cat insertion into ORF 2 in plasmid pRF38 completely removed
 this protein band, replacing it with a much smaller protein of 6 kd, which
 is in close agreement with the predicted size of the truncated ORF 2.
 Based on these results, the 15 kd protein appears to be generated only by
 ORF 2. It is not clear why the .beta. riboflavin synthase protein is not
 visualized on the gels of the S-30 reactions. Taken in total, however, the
 results confirmed the existence of five rib-specific coding regions: ORF
 5, ORF 4, ORF 3, ORF 2 and the .beta. riboflavin synthase gene.
 In addition, ORF 1 appeared to encode a 38 kd protein, while no product was
 identified for ORF 6.
 Regulatory Mechanisms of the Rib Operon
 In B. subtilis, a recurring pattern of gene organization and regulation for
 biosynthetic pathways has been observed by several investigators. The
 nucleotide sequences of the tryptophan biosynthetic pathway (Henner et
 al., Gene 34:169, 1984) and the de novo purine nucleotide pathway (Ebbole
 and Zallin, J. Biol. Chem. 262:8274, 1987) of B. subtilis both contain
 clustered, overlapping genes transcribed as a polycistronic message and
 regulated at least in part by a novel transcription
 termination/anti-termination mechanism, involving a repressor protein
 which can be encoded by a gene unlinked to the biosynthetic operon (Zalkin
 and Ebbole, J. Biol. Chem. 263:1595, 1988). Since we found that the
 organization of the rib biosynthetic and regulatory genes is strikingly
 similar to those of the B. subtilis trp and pur pathways, we hypothesized
 that the rib operon might be regulated, at least in part, in a similar
 manner.
 Briefly, the key characteristics of the transcription
 termination/anti-termination model include (Shimotsu et al., J. Bacteriol.
 166:461, 1986): (i) the presence of a long 5' leader sequence that
 precedes the first gene in the operon; (ii) the presence in the RNA leader
 of two or more overlapping dyad symmetries that have the potential to form
 mutually exclusive RNA stem-loops, one structure functioning as a
 rho-independent transcription terminator and the other as an
 "anti-terminator" (blocking the formation of the rho-independent
 transcription terminator); (iii) under repressive conditions, the
 repressor protein, activated by the end product of the pathway, binds to
 the nascent mRNA at a site which prevents formation of the
 anti-terminator, thus allowing formation of the terminator which
 terminates transcription; (iv) under depressive conditions, binding of the
 unactivated repressor is precluded, resulting in the formation of the
 anti-terminator causing read-through transcription into the coding region
 of the operon.
 As discussed above, the most likely site for initiation of transcription in
 the rib operon is a .sigma..sup.A promoter, P.sub.1, located about 290 bp
 upstream from the first gene in the operon. Preliminary analysis of the
 RNA leader sequence indicated that it contained most, if not all, of the
 structures required for regulation by the termination/anti-termination
 model. Within this region, a stem-loop structure followed by a string of
 thymidines resembling a rho-independent transcription terminator was
 identified approximately 50 bp upstream of ORF 5; this sequence has the
 potential to form a hairpin with a .DELTA.G of -26 kcal/mol (FIG. 9). In
 addition, several potential stem-loop structures with .DELTA.G's ranging
 from -13 to -16 kcal/mol were located within the rib 5' leader that could
 possibly qualify as the anti-terminator sequence.
 In addition to the primary site for the initiation of transcription,
 usually located upstream from the first gene in the operon, there exist in
 some biosynthetic pathways secondary promoter sites located within the
 internal regions of the operon. The possibility of there being a second
 promoter site within the rib locus was also suggested by previous R-loop
 heteroduplex studies of the rib operon (Osina et al., FEBS Letters
 196:75-78, 1986), showing two or more sites for the initiation of mRNA
 synthesis. Our preliminary analysis of the intercistronic gaps of the rib
 operon did not detect such secondary promoter sites. However, when this
 analysis was extended to all of the sequences within the operon, another
 .sigma..sup.A promoter, P.sub.2, was identified within the 3' end of ORF
 4, just downstream from the SalI restriction site (FIG. 4) Thus it is
 possible that the expression of ORF 2, ORF 3, and the .beta.-subunit for
 riboflavin synthase is also under the control of this secondary promoter.
 In addition, a possible third .sigma..sup.A promoter, P.sub.3, was
 identified just upstream of ORF 2. Therefore ORF 2 is possibly also under
 the control of this additional promoter.
 The location of putative coding regions, promoters and transcription
 termination sites on the DNA sequence of the 5.5 kb B. subtilis
 rib-specific region is shown in Table VI.
 TABLE VI
 CODING REGIONS, PROMOTER, AND
 TRANSCRIPTION TERMINATION SITES
 OF THE B. SUBTILIS RIB OPERON
 bp Number.sup.a
 Coding Regions ORF 6 364-678
 ORF 5 1101-2183
 ORF 4 2197-2841
 ORF 3 2859-4052
 .beta.riboflavin- 4088-4549
 synthase gene
 ORF 2 4665-5036
 ORF 1 5567-5057.sup.b
 .sigma..sup.A Promoters P.sub.1 771-799
 P.sub.2 2528-2556
 P.sub.3 4545-4574
 rho-Independent Upstream from 708-748
 Termination Sites 5' promoter 1034-1067
 Within 5'
 leader RNA 5038-5090
 At 3' end of
 rib operon
 .sup.a of FIG. 3.
 .sup.b Coding region oriented in opposite direction.
 Example 8: Construction of Vectors Containing a Modified Rib Operon
 The above functional analysis of the rib operon of Bacillus subtilis for
 the first time delimiting the regulatory regions and open reading frames
 in the nucleodde sequence permits construction of new vectors which are
 useful for increasing the yield of riboflavin production. The knowledge of
 the location of the specific genes required for riboflavin biosynthesis,
 of the location of transcriptional control regions, and other relevant
 regions (e.g., RBS) in those genes allows changes in such regions to be
 made. There follow a few examples of such manipulations.
 Construction of an integration plasmid with a rib operon on a smaller DNA
 fragment
 The integrating vector used to construct the riboflavin overproducing
 strain RB50::[pRF8] contains a 10 kb EcoRI fragment including the rib
 operon. Since the rib operon appears to occupy less than 6 kb of DNA a new
 integration vector was constructed (pRF40) containing the, rib operon on a
 smaller DNA fragment. The smaller size of this clone allows higher
 amplification of rib genes resulting in higher yields of riboflavin.
 Referring to FIG. 12, pRF40 was constructed from pRF36 which is a plasmid
 in which the 0.8 kb NcoI fragment of pRF2 is replaced with a cat gene. The
 rib operon is contained on a 6.5 kb XbaI-EcoRI fragment. This fragment was
 isolated and ligated to pUC19 (Yanisch-Perron et al., 33 Gene 103, 1985;
 available from New England Biolabs, Boston, Mass., and Bethesda Research
 Laboratories, Maryland) digested with XbaI and EcoRI. The ligated DNA was
 transformed into DH5.alpha.E. coli and plated onto LB plates containing 40
 .mu.g/ml X-gal and 50 .mu.g/ml ampicillin. Analysis of miniprep DNA
 prepared from white colonies indicated that pRF39 contained the 6.5 kb
 Xbal-EcoRI fragment.
 pRF39 was digested with EcoRI, treated with CIAP, and then ligated to a 1.6
 kb EcoRI fragment containing the cat gene. The ligated DNA was then
 transformed into DH5.alpha.E. coli and appropriate colonies selected for
 plating onto LB+10 .mu.g/ml chloramphenicol; two colonies were
 chloramphenicol-resistant. Analysis of miniprep DNA prepared from these
 colonies confirmed the presence of the cat gene. One of these plasmids is
 pRF40 (FIG. 14).
 Construction of plasmids containing transcriptionally modified rib operon
 As described above, it is useful to replace the promoter and operator
 regions of the riboflavin operon with promoters allowing constitutive
 expression of the riboflavin biosynthetic genes. Plasmids containing such
 constructs can then be used to produce bacterial strains which will
 produce increased levels of riboflavin. A few examples, not limiting in
 the invention, are provided below.
 Referring to FIG. 13, the riboflavin promoter and regulatory region were
 removed and replaced with an SPO1 promoter. We took advantage of the BglII
 site located at position 1130 at the start of ORF3. Oligonucleotides were
 synthesized (RB5 and RB6, see FIG. 18) that recreated the DNA sequence 5'
 to the BglII site (the first few amino acids of ORF5 and the SD sequence)
 up to position 1058. Reconstruction of the 5'-end of the operon stopped
 before any of the proposed DNA regulatory structures (FIG. 13). At their
 5' ends the oligonucleotides contained BamHI, NsiI, and EcoRI restriction
 sites, allowing for placement of various promoters 5' to the rib operon.
 Because of the various restrictions sites in the rib operon it was;
 necessary to construct the operon with the new promoters in several steps,
 as follows.
 A 1.4 kb SalI-BglII fragment was isolated from pRF36 (FIG. 13). This
 fragment was ligated with the two oligonucleotides and EcoRI-SalI-digested
 pUC19. The ligated mixture was then transformed into E. coli DH5.alpha.
 cells and plated onto LB containing 50 .mu.g/ml ampicillin and 40 .mu.g/ml
 X-gal. Minipreps were prepared from Ap.sup.r white colonies; one plasmid
 having the desired structure is pRF46 (FIG. 13).
 pRF46 was digested with BamHI and SalI and the 1.4 kb fragment isolated.
 This fragment was then ligated with the 400 bp EcoRl-BamHI fragment of
 pNH202 (pUC8 containing the SPO1-15 promoter, Lee and Pero, J. Mol. Biol.,
 152:247-265, 1981) and pUC19 cut with SalI and EcoRI. The ligated DNA was
 then transformed in DH5.alpha.E. coli, which were plated onto
 LB+ampicillin+X-gal. Miniprep DNA was prepared from white colonies; and
 pRF48 had the desired structure (FIG. 13).
 pRF48 was digested with EcoRI and SalI and the 1.8 kb fragment isolated.
 This fragment was ligated with the 4.0 kb XbaI-SalI fragment (containing
 the rest of the rib operon) from pRF2 and XbaI, EcoRI-cut pUC19. The
 ligated mixture was then transformed into E. coli DH5.alpha. cells which
 were plated on LB+ampicillin+X-gal. Miniprep DNA was prepared from white
 colonies; pRF49 had the desired structure, and supernatants from culture
 containing this plasmid was yellow, indicating riboflavin production (FIG.
 13).
 To place the cat gene in pRF49, to allow selection in B. subtilis, the
 plasmid was digested with XbaI and ligated to a 1.3 kb cat-containing Xbal
 fragment from pEcc4. The ligated DNA was transformed in E. coli DH5 cells.
 Hundreds of Ap.sup.r colonies resulted, and the colonies were patched onto
 plates containing LB+10 .mu.g/ml chloromphenicol. Approximately 10% of the
 colonies grew on the chloramphenicol plates, indicating the presence of
 the cat gene. One cat-containing plasmid is called pRF50 (FIG. 14).
 The above example shows placement of a new promoter upstream of ORF5. We
 found that it is also useful to place a promoter after P.sub.2 between
 ORF3 and ORF4 in order to further increase riboflavin production. An
 example of such construction now follows.
 Referring to FIGS. 14 and 15, to place a copy of the SPO1-15 promoter
 upstream of ORF3 we made use of the restriction sites adjacent to the
 ORF4-ORF3 junction. The ClaI site at position 2767 is located at the end
 of ORF4 and is unique in the rib operon. Another useful restriction site
 near the beginning of ORF3 is the DraI site at position 2892.
 Oligonucleotides were synthesized that recreated the sequence from the
 above-mentioned DraI site past the start of ORF3 and placed a unique BamHI
 site before the beginning of ORF3 (linkers P2-A and P2-B, FIG. 18).
 Another set of oligonucleotides recreated the sequence from the ClaI site
 past the end of ORF4 and placed an EcoRI site at that location (linkers
 P2-CII and P2-DII, FIG. 18). The SPO1-15 promoter, located on a
 EcoRI-BamHI fragment, was then be placed between the BamHI and EcoRI sites
 created by the oligonucleotides. The entire operon was put together with
 this additional SPO1-15 promoter as follows.
 Referring to FIG. 15, the 750 bp SalI-BglII fragment containing the
 ORF4-ORF3 function was subcloned to pIC2OR (Marsh et al., Gene 32:481-485,
 1984). The resulting plasmid, pRF57, was then digested with DraI and
 BglII, and the predicted 270 DraI-BglII fragment was isolated. This
 fragment and linkers P2-A and P2-B were ligated to pIC2OR cut with SalI
 and BglII. The linkers placed BamHI and SalI sites upstream of the 5' end
 of ORF3. (The SalI site was chosen for convenience since BglII and BamHI
 sites are compatible and will be removed later.) The ligation was
 transformed into E. coli DH5.alpha. cells. Plating onto LB medium+Amp and
 X-gal resulted in white colonies; pRF58 had the desired structure. The 330
 bp BglII-SalI fragment from pRF58 was isolated and ligated with 3.3 kb
 BglII-XbaI fragment containing the 3'-end of the rib operon from pRF36
 (FIG. 12) and pUC19 cut with XbaI and SalI. The ligated DNA was then
 transformed into E. coli DH5.alpha. cells, resulting in white colonies;
 pRF62 (FIG. 15) had the desired structure. For convenience, the 3.6 kb
 BamHI-XbaI fragment was isolated from pRF62 and subcloned into BamHI-,
 XbaI-cut pUC19 (pRF64, FIG. 15). This plasmid now contained the 3.6 kb
 3'-end of the rib operon with an engineered BamHI site preceding ORF3.
 To place the SPO1-15 promoter in front of the 3'-half of the rib operon
 containing the last three open reading frames, we digested pRF64 with
 EcoRI and BamHI and ligated it to a 400 bp EcoRI-BamHI fragment containing
 the SPO1-15 promoter. The ligated DNA was transformed into E. coli DH5
 cells and miniprep DNA was prepared; pRF65 has the desired structure.
 The SPO1-15 promoter was than engineered to place a ClaI site upstream of
 the promoter to reconstruct the end of ORF4. The EcoRI-BamHI fragment from
 pNH202 containing the SPO1-15 promoter was ligated with linkers P2-CII and
 P2-DII and pCI2OR-digested with BamHI and ClaI. The ligated DNA was then
 transformed into E. coli DH5.alpha. cells. White colonies resulted and
 miniprep analyses indicated that pRF63 had the desired structure. The 470
 bp CarI- BamHI fragment was isolated then from pRF63 and ligated to the 2
 kb EcoRI-ClaI fragment from pRF49 containing the SPO1-15 promoter and the
 5'-end of the rib operon and pRF64 (FIG. 15), containing the SPO1 promoter
 and the 3'-end of the operon, digested with EcoRI and BamHI. The ligated
 DNA was then transformed into E. coli DH5.alpha. cells. Miniprep DNA was
 prepared; pRF66 had the desired structure. In addition, E. coli containing
 pRF66 produced small amounts of riboflavin on LB medium+ampicillin plates,
 confirming that the operon was still intact
 The last step was to ligate the cat gene into the unique XbaI sites of
 pRF66 as described above. The resulting plasmid, pRF69 (FIG. 15) contained
 the cat gene in the same direction as the rib operon.
 To construct a plasmid containing the entire operon with the natural or
 wild-type ribP.sub.1 promoter and the SPO1-15 promoter after ribP.sup.2,
 the 6.3 kb EcoRI-BamHI fragment of pRF64, the 2.75 kb EcoRI-ClaI fragment
 of pRF36, and the 470 bp ClaI-BamHI fragment of pRF63 were ligated and
 tranformed into E. coli DH5.alpha. cells. About 50% of the Ap.sup.r
 colonies were yellow, indicating ribflavin production. Miniprep DNA was
 prepared from yellow colonies and pRF68 had the desired structure (FIG.
 16). A cat gene was added to pRF68 at the XbaI site, as discussed above,
 to generate pRF71 (FIG. 16). This plasmid contained the cat gene in the
 same direction as the rib operon.
 As another example of the construction of useful plasmids in this
 invention, there now follows an example in which one or more promoters can
 be introduced within the riboflavin operon without prior removal of
 existing DNA sequences.
 As an example, a prototype modified operon was constructed in pRF78, which
 contains a single copy of the SPO1-15 promoter inserted within a 30 bp
 non-essential region located between ribP.sub.1 and a putative
 rho-independent transcriptional termination site (FIG. 14), an inactivated
 ribP.sub.1 promoter to prevent possible transcriptional interference of
 the SPO1-15 promoter, an active ribP.sup.2 promoter, the five structural
 genes encoding rib biosynthetic enzymes, and approximately 1.5 kb of
 flanning DNA nucleotide sequences downstream from the end of the
 riboflavin operon.
 Referring to FIG. 14, the 1.7 kb NcoI-PstI fragment of pRF2, a fragment
 that contains the 5' promoter region of the rib operon and flanking
 regions, was first subcloned into mp19, a derivative of the E. coli
 bacteriophage vector M13 (United States Biochemical Catalog, 60-61, 1987;
 available from New England Biolabs, Massachusetts). One recombinant phage,
 M1.7, was recovered and standard DNA sequence analysis of the promoter
 region revealed a spontaneous mutation of the -10 region of the ribP.sub.1
 promoter, a TA-to-CT change, which mays inactivate the promoter Single
 stranded DNA was prepared and annealed to a synthetically-generated 55 bp
 DNA oligomer (see FIG. 17), containing a combination of restriction
 enzymes sites, 5'-EcoRI-SmaI-BamHI-3', flanked on either side by
 additional sequences homologous to the DNA region upstream from
 ribP.sub.1. Double-stranded DNA molecules were synthesized using standard
 site-directed mutagenesis (SDM) protocols. These DNA molecules were
 introduced into the E. coli host TG-1 (available from Amersham Corp.
 Illinois) by transfection to generate recombinant phage plaques. One
 recombinant phage was found to contain the desired modified DNA sequence,
 as determined by standard DNA sequence analysis.
 The modified rib promoter region was then rejoined to the rib structural
 genes of the operon using a pair of unique NsiI restriction enzymes sites
 750 bp apart that flank the ribP.sub.1 region and surrounding sequences.
 Double-stranded DNA molecules of the phage recombinant were prepared,
 digested with NsiI, the 750 bp fragment isolated, and the fragment ligated
 to dephosphorylated, 8.7 kb NsiI fragment of pRF39.DELTA.R1 (a plasmid
 derived from pRF39, FIG. 12, that contains the wild-type rib operon). The
 ligated DNA molecules were introduced into E. coli DH5.alpha. cells by
 transformation, selecting for ampicillin-resistance, which resulted in the
 recovery of an Ap.sup.r colony harboring the desired recombinant plasmid,
 pRF75.
 The SPO1-15 promoter was next inserted upstream from ribP.sub.1 by
 digesting pRF75 with a combination of EcoRI and BamHI enzymes, ligating
 the cut DNA to purified 400 bp EcoRI-BamHI SPO1-15-containing restriction
 fragment, and introducing the ligated DNA into E. coli DH5.alpha. cells by
 transformation, selecting for ampicillin-resistance. One Ap.sup.r colony
 was found to harbor the recombinant plasmid, pRF77, containing the desired
 SPO1-15-modified rib operon. A chlorarnphenicol-resistance gene, cat, on a
 1.6 kb XbaI restriction fragment, was subsequently introduced into pRF77
 at the unique XbaI site, generating plasmid pRF78 (FIG. 14).
 This prototype operon was further modified to contain an active ribP.sup.1,
 promoter, and/or a second copy of the SPO1-15 promoter introduced
 downstream from ribP.sub.2 within a intercistronic region between the rib
 coding regions ORF3 and ORF4, as described above. For example, plasmid
 pRF88, containing a derivative of the modified rib operon in pRF78 with an
 active ribP.sub.1 promoter (FIG. 14) was constructed by the same procedure
 described above, using a recombinant phage containing the wild-type
 ribP.sub.1 promoter. In other examples, a second copy of the SPO1-15
 promoter, located downstream from ribP.sub.2, was inserted into the
 existing modified rib operon-containing plasmids pRF78 and pRF88 by
 removing the 2.0 kb BglII fragment of either plasmid DNA and inserting the
 2.4 kb BglII fragment of pRF66, generating plasmids pRF81 and pRF89
 respectively (FIG. 14).
 Construction of Ade.sup.+ RB50 strains
 It is important to use strains of bacteria that require as few components
 to be added to a fermentation medium as possible. Such strains are cheaper
 to ferment in order to produce riboflavin. To this end, adenine revertants
 which contained amplified modified rib operons were constructed. These
 revertants may not be true revertants of pur-60, but rather include
 mutations at another site which suppresses the requirement for adenine. As
 discussed below they produce about 25% more riboflavin than the
 non-reverted strains. Examples of such constructions are now described.
 Plasmids pRF8, pRF40, pRF50, pRF69, pRF71, pRF78, pRF81, pRF88 and pRF89
 were each transformed into RB50 (a RoF.sup.r, deregulated B. subtilis
 strain) selecting for chloramphonicol resistance (Cm.sup.r). A resistant
 colony was chosen for each strain. Ade.sup.+ revertants of each strain was
 isolated by growing bacteria in RMM1 broth containing 10 .mu.g/ml
 adenosine, and plating samples of the cultures onto minimal agar plates.
 One colony from each Ade.sup.+ strain was selected and the vector DNA was
 amplified by selecting colonies that grow on increasingly higher levels of
 chloramphenicol, to a maximum level of 60 .mu.g/ml.
 Second site Integration
 As described above, it is important to amplify an engineered rib operon in
 the B. subtilis chromosome to achieve high titers of riboflavin. It is
 also important to ensure that the number of DNA copies of the rib operon
 within a chromosome are not limiting to riboflavin production. Further
 amplification of the rib operon can be achieved by integrating and
 amplifying copies of the rib operon at more than one site in the B.
 subtilis chromosome to further increase riboflavin yield. One example of
 how such second site integration can be achieved is described below.
 The above described vectors have all relied upon the cat gene to select for
 integration at the site of the rib operon. In order to insert the rib
 genes at a second site, it is preferable to have a different antibiotic
 resistance gene for use at that second site. For example, a
 tetracycline-resistance (tet) from B. subtilis can be used (Perkins and
 Youngman, J. Bacteriol., 155:607-615, 1983). Such tet genes are well known
 to those of ordinary skill in the art and are readily available to such
 persons. In one such construction, for example, pRF78 (FIG. 14), which
 contains a modified version of the rib operon, the plasmid can be cut with
 XbaI and ligated to a 2.4 XbaI fragment containing the tet gene. The
 resulting plasmid contains the tet gene at the XbaI site and is called
 pRF85 as shown in FIG. 16.
 A strain which is deleted for the entire rib operon and which has a tet
 gene integrated at a second site is required to cause integration of pRF85
 at that site. One such site is the bpr gene encoding bacillopeptidase F, a
 minor non-essentiall extracellular protease. An E. coli plasmid containing
 the bpr gene, pKT2, (Sloma et al., J. Bacteriol., 172:1470-1477, 1990) was
 digested with EcoRV. This EcoRV site is in the coding region of bpr. The
 DNA was then ligated to a 2.4 kb EcoRI fragment containing the tet gene
 that had been blunt-ended. The resulting plasmid (containing the tet gene
 at the EcoRV site of bpr) was called pKT2-tet. This DNA was linearized
 with EcoRI and then transformed into RB52, a strain deregulated for
 riboflavin synthesis. Tet.sup.r colonies resulted and one such colony was
 called RB54. The integrated tet gene at bpr will function as a homologous
 sequence for the integration of pRF85.
 To ensure that the cloned riboflavin operon of pRF85 would be inserted at
 the second chromosomal site containing the tetracycline-resistance gene, a
 region containing the original riboflavin operon and flanking DNA,
 equalling that contained in pRF85, was deleted from the chromosome of RB54
 by in vitro methods. Briefly, this involves first generating an E. coli
 recombinant plasmid where the cloned riboflavin operon and flanking
 regions between the NcoI and XbaI restriction sites are removed and
 replaced by a chloramphenicol-resistance gene, cat that is expressed in B.
 subtilis bacteria. This plasmid is then used to delete the chromosomal
 riboflavin operon by transforming RF54 with linearized plasmid molecules
 and selecting for chloramphenicol resistant (Cm.sup.r) bacteria. Cm.sup.r
 bacteria result from a recombinant event (marker-replacment) which
 replaces the wild-type rib genes with the deleted copy containing the cat
 gene.
 Specifically, plasmid pRF34 (see example 6) was used to generate an E. coli
 plasmid containing an in vitro-generated riboflavin operon deletion. This
 plasmid is derived from pRF2 where the riboflavin operon is flanked on
 either end by two unique XbaI sites (one site located upstream from the
 5'-end of the rib operon next to the deleted 0.8 kb NcoI fragment and the
 second site located approximately 1.6 kb downstream from the end of the
 operon) and a cat gene is inserted outside of this region. By digesting
 pRF34 with XbaI and ligating the cut DNA molecules under dilute DNA
 concentrations, a recombinant plasmid, pRF82, was recovered where a 7.2 kb
 region containing the riboflavin operon is removed and essentially
 replaced with the cat gene. Plasmid pRF82 was linearized by restriction
 enzyme digestion and the cut DNA used to remove the chromosomal riboflavin
 operon of RB54 by DNA transformation, selecting for Cm.sup.r bacteria,
 resulting in marker replacement. Cm.sup.r colonies were screened for
 riboflavin auxotrophy and one Rib.sup.- Cm.sup.r colony, RB55, was
 recovered for further investigation.
 Plasmid pRF85 was transformed into strain RB55, selecting for Rib.sup.+.
 One Rib.sup.+ transformant was chosen and called RB58. This strain has the
 rib operon integrated at bpr by homologous recombination between the
 tet.sup.r genes in the plasmid and the chromosome. A transducing lysate of
 RB58 was prepared using standard techniques, and it was used to transduce
 RB50::[pRF69], selecting for Tet.sup.r. These resistant colonies were
 found to have the modified rib operon integrated at the site of the rib
 operon and at bpr. One such Tc.sup.r colony RB50::[pRF69].sub.60
 ::[pRF85].sub.120 Ade.sup.+ was recovered for further study. The rib
 operon integrated at rib was amplified by selecting for colonies that grow
 in the presence of increasing levels of chloramphenicol as described
 above, and the second copy of the rib operon was amplified by selecting
 colonies that grow on increasing levels of tetracycline to 120 .mu.g/ml.
 Example 9: Fermentative Production of Riboflavin
 Evaluation of riboflavin-overproducing strains was conducted in Chemap
 14-liter vessels in carbon-limited fed-batch fermentations, with
 riboflavin content measured by HPLC. Since enzymes encoded by the genes
 for riboflavin synthesis are rate-limiting, the rib genes, which were
 amplified, were maintained at high-copy number by the inclusion of 60
 .mu.g/ml chloramphenicol in the inoculum seed train, but not in the
 fermentor.
 A culture of a riboflavin-overproducing strain such as B. subtilis
 RB50::[pRF69].sub.60 Ade.sup.+ was grown on Tryptose Blood Agar Base (TBAB
 Difco) containing 60 .lambda.g/ml of chloramphenicol (CAM). Colonies were
 transferred to 300 ml baffled flasks containing 25 ml of riboflavin
 minimal medium (RMM; containing sodium glutamate 2.0 g/l, Casamino acids
 (Difco) 0.2 g/l, Yeast extract (Difco) 0.2 g/l, KH2PO.sub.4 6.0 g/l,
 K.sub.2 HPO.sub.4 14.0 g/l, (NH.sub.4).sub.2 SO.sub.4 2.0 g/l, sodium
 citrate 1.0 g/l, MgSO.sub.4.7H.sub.2 O 0.2 g/l, glucose 15.0 g/l, pH 7.0)
 with 60 .mu.g/ml CAM. The inoculated flasks were incubated by shaking at
 250 rpm and 37.degree. C. After 8 hours, sterile glycerol was added to a
 final concentration of 15% and 1 ml aliquots were stored at -80.degree. C.
 In order to initiate a fermentation a frozen vial of the appropriate
 strain, e.g., RB50::[pRF69].sub.60 Ade.sup.+ was thawed at 37.degree. C.
 and transferred into a 300 ml baffled flask with 25 ml of RMM with 60
 .mu.g/ml CAM and shaken at 250 rpm and 37.degree. C. After 8 hours, 6 ml
 of the growing culture was used to inoculate 300 ml of fermentation medium
 (see Table VII below) in a series of 2 liter transfer flasks. Each flask
 contained 300 ml of fermentation medium to which had been added 90 ml of
 15% glucose. Chloramphemnicol was added to a final concentration of 60
 .mu.g/ml. After incubation for 12 hours at 200 rpm on an shaker with a 2"
 diameter orbit at 37.degree. C., the contents of each flask was
 transferred to 7 liters of fermentation medium in a 14 liter fermentation
 vessel.
 During fermentation, the broth was continually monitored for pH and
 dissolved oxygen (DO.sub.2). Off gas was continuously analyzed by
 quadrapole mass spectrometry and carbon dioxide evolution (CER) and oxygen
 uptake rates were recorded.
 A comparison of several fermentations demonstrated the reproducibility of
 the control systems. The initial carbohydrate was exhausted from
 fermentation with RB50::[pRF8].sub.60 after 4 hours of growth, causing a
 rise in pH and a fall in CER. At that point, carbohydrate feeding was
 initiated and logarithmic growth resumed until DO.sub.2 became limiting at
 6 hours. The rate of carbohydrate feeding was computer-controlled to
 maintain the DO.sub.2 between 10-20% of saturation throughout the
 remaining fermentation time.
 Excess carbohydrate in the fermentors does lead to oxygen starvation and
 reduced riboflavin production. Oxygen transfer limitations determine the
 duration of logarithmic growth, final cell density and the riboflavin
 production rate. To increase the oxygen transfer rate, Chemap fermentors
 were run at 1000 rpm with a head pressure of 0.6 atmospheres.
 Supplementation of the medium carbohydrate feed with yeast extract led to
 an increase in riboflavin production as compared to media without
 supplementation (FIG. 11, open squares: RBF-14; Table VII). However,
 because of its high cost, the amount of yeast extract was systematically
 reduced by substituting less expensive, inorganic ingredients.
 Substitution of ammonium hydroxide for sodium hydroxide in pH control
 allowed a reduction of yeast extract in the feed and resulted in an
 increase in both cell mass and riboflavin titer (FIG. 11, closed squares:
 RBF-22; Table VII). Fermentation times were also reduced. In other
 fermentations, moreover, yeast extract was completely eliminated from the
 feed and replaced with a combination of inorganic salts of ammonium and
 phosphate, resulting in a further increase in riboflavin production and a
 reduction of process time (FIG. 11, open circles: RBF-23; Table VII).
 The original RB50::[pRF8].sub.60 was auxotrophic for adenine because of its
 pur-60 mutation. When experiments were conducted to determine the minimum
 amount of adenosine required by the strain, in order to minimize its
 inhibition of earlier biosynthetic enzymes involved in the pathway leading
 to the riboflavin-precursor IMP (FIG. 2), RB50::[pRF8].sub.60 (and, in
 general, RB50 strains with a rib operon amplified within their chromosome)
 was found to be unstable in its adenosine requirement and prototrophic
 revertants (Ade.sup.+) were produced at a fairly high frequency. In shake
 flasks, the Ade.sup.+ revertants appeared to grow and produce riboflavin
 at least as well as the RB50::[pRF.sup.8 ].sub.60 parent. When evaluated
 in fermentors, the revertant, RB50::[pRF8].sub.60 (Ade.sup.+), did not
 require adenosine in the media formulation. More importantly, the
 revertant grew at a faster rate and produced 25% more riboflavin than its
 parent strain in less time. A titer of 5.4 g/l riboflavin was produced in
 49 hours (FIG. 11, closed circles: RBF-29; Table VII). In additional
 fermentations, moreover, Hy Soy T was removed from the initial charge or
 medium and replaced with corn steep liquor, resulting in a further
 increase in riboflavin production to 6.3 g/l in 48 hours. (RBF-42, Table
 VII).
 Under these fermentation conditions, further significant increases in
 riboflavin production were demonstrated using bacterial strains that
 contained engineered riboflavin operon DNA. Strains containing the
 wild-type riboflavin operon on a 6.5 kb EcoRI-XbaI restriction fragment,
 RB50::[pRF40].sub.60 (Ade.sup.+), produced 7.4 g/l of riboflavin in 48
 hours. Moreover, strains containing a transcriptionally-modified rib
 operon where the ribP.sub.1 promoter and regulatory region were replaced
 by the constitutive SPO1-15 promoter, RB50::[pRF50].sub.60 (Ade.sup.+),
 produced 9.0 g/l of riboflavin in 48 hours. These results demonstrate that
 modification of the riboflavin operon through the removal of regulatory
 regions and/or through the introduction of stronger, constitutive
 exogenous promoters leads to increases in riboflavin titer.
 TABLE VII
 Component RBF-14 RBF-22 RBF-23 RBF-29 RBF-42
 Initial Charge (g/l)
 Glucose 10.00 15.00 15.00 15.00 15.00
 Corn step liquor -- -- -- -- 10.00
 Hy Soy T 15.00 15.00 15.00 10.00 --
 Sodium glutamate -- -- -- 5.00 5.00
 Amberex 500 15.00 15.00 20.00 20.00 20.00
 KH.sub.2 PO.sub.4 5.00 5.00 7.50 7.50 7.50
 M.sub.g Cl.sub.2.6H.sub.2 O 0.5 0.5 1.50 1.50 1.50
 MnSO.sub.4 0.05 0.05 0.05 0.05 0.05
 Adenosine 0.05 0.05 0.05 -- --
 MAZU DF37 2.50 2.50 2.50 2.50 2.50
 FeCl.sub.3 -- -- 0.025 0.02 0.02
 CaCl.sub.2 -- -- 0.50 0.50 0.50
 ZnSO.sub.4 -- -- 0.0005 -- --
 CuCl.sub.2 -- -- 0.001 -- --
 CoCl.sub.2 -- -- 0.0013 -- --
 Nutrient Feed (g/l)
 Amberex 500 160.00 120.00 -- -- --
 NH.sub.4 Cl -- -- 7.50 7.50 7.50
 (NH.sub.4).sub.2 SO.sub.4 -- -- 7.50 7.50 7.50
 KH.sub.2 PO.sub.4 -- -- 15.00 15.00 15.50
 MgSO.sub.4.7H.sub.2 O -- -- 2.50 2.50 2.50
 DL-70 syrup 600.00 600.00 600.00 660.00 600.00
 (as DS)
 pH Control Range
 6.6 H.sub.2 SO.sub.4 H.sub.2 SO.sub.4 H.sub.2 SO.sub.4
 H.sub.2 SO.sub.4 H.sub.2 SO.sub.4
 6.5 NaOH NH.sub.4 OH NH.sub.4 OH NH.sub.4 OH NH.sub.3
 Conditions
 Air (vvm) 1.0 1.5 1.5-2.0 1.5 1.50
 RPM 1000.0 1000.0 1000.0 1000.0 1000.0
 Temp .degree. C. 37.0 37.0 37.0 37.0 37.0
 Pressure (bar) 0.5 0.5 0.5-0.75 0.6 0.6
 Riboflavin (g/l) 3.4 4.1 4.3 5.4 6.3
 (64 hrs) (56 hrs) (53 hrs) (49 hrs) (48 hrs)
 Dry Weight (g/l) 33.6 36.0 36.8 ND 44.6
 The kinetics of riboflavin production in the various fermentations were
 analyzed using the Luedeking-Piret model. In all cases, the specific
 productivity declined from the conclusion of the exponential growth phase
 to the end of fermentation. Also, it was clear that riboflavin production
 was growth-associated under the fermentation conditions used.
 We have discovered that the yield of riboflavin can be increased by
 changing the fermentation components and conditions. The yield of
 riboflavin can be increased compared to those conditions described above
 using those fermentation components and conditions shown in Table VIII.
 TABLE VIII
 RBF 150 RBF 184
 (g/liter) (g/liter)
 Initial Batch
 Yeast Extract 20 20
 Glucose 25 25
 KH.sub.2 PO.sub.4 7.5 7.5
 MgCl.sub.2.H.sub.2 O 1.5 1.5
 CaCl.sub.2.2H.sub.2 O 1.0 1.0
 MnSO.sub.4 0.05 0.05
 FeCl.sub.3.6H.sub.2 O 0.025 0.025
 Mazu DF37C 2.5 2.5
 Corn Steep Liquor 10 --
 Sodium Glutamate 5 5
 (NH.sub.4).sub.2 SO.sub.4 -- 0.3
 Feed Medium (3 liters total used)
 Glucose 583.3 --
 NaCitrate 6.67 6.67
 KH.sub.2 PO.sub.4 15 15
 Succinic Acid 1.67 1.67
 MgSO.sub.4.7H.sub.2 O 1.67 1.67
 Corn Syrup Solids -- 833
 Briefly, in one such fermentation the startng material is 6.65 liters of
 batch medium and 0.35 liters of bacterial (RB50::[pRF50].sub.60 Ade.sup.+)
 inoculant. Oxygen levels are monitored with a Chemap polarographic
 dissolved oxygen electrode. Dissolved oxygen levels are maintained at
 15%.+-.5% by means of computer regulated addition of the feed medium.
 Total feed added is about 3.0 liters in 48-56 hours. Fermentation pH is
 maintained at 6.5.+-.0.1 (using 1N H.sub.2 SO.sub.4 and NH.sub.3 gas), and
 fermenter pressure is maintained at 0.6 bars, temperature at 37.degree.
 C., and air flow at 10.5 liters/min. Under these conditions, strain
 RB50::[pRF50].sub.60 (Ade.sup.+) produced 11.0 g/l riboflavin in 48 hours,
 which represents an improvement in production of approximately 20%
 compared to the previous fermentation conditions. An increase in
 riboflavin production was demonstrated (RBF150, Table VIII) using the
 bacterial strains RB50::[pRF69].sub.60 (Ade.sup.+) containing a
 transcriptionally-modified riboflavin operon containing two SPO1-15
 promoters, one replacing ribP.sub.1 and regulatory sequences, and a second
 inserted between ORF3 and ORF4. This strain produced 13.0-14.0 g/l
 riboflavin in 48 hours, and 15 g/l in 56 hours, demonstrating that
 increased transcription of the riboflavin operon using two strong
 exogeneous promoters increases production levels of riboflavin. Finally, a
 further increase in riboflavin production was demonstrated using the
 bacterial strain RB50::[pRF69].sub.60 ::[pRF85].sub.120 Ade.sup.+
 containing two amplifiable rib loci as in Example 8. This strain was grown
 at pH 6.8 and 39.degree. C. using the modified fermentation medium shown
 in Table VIII (RBF 184) and riboflavin was isolated.
 Example 10
 Construction of B. subtilis Strain RB50::[pFR69]::[pRF93]
 The riboflavin producing strain RB50::[pRF69] is a genetically modified
 strain of B. subtilis. RB50 refers to the host strain of B. subtilis,
 which contains several mutations introduced to improve production of
 nucleotides and riboflavin. pRF69 refers to a rib operon modified by the
 introduction of strong phage promoters which was introduced at the rib
 locus of pRF50. The modified operon pRF69 can be amplified to high copy
 numbers. A detailed description of the strain RB50::[pRF69] is presented
 above. To further increase the number of rib operons, a second plasmid
 with a modified rib operon and a tetracycline resistance gene was
 introduced at the bpr locus (essentially as described in example 8, second
 site integration). This second plasmid, pRF93, was constructed from pRF89
 (FIG. 14) by replacing the chloramphenicol resistance gene by a
 tetracyclin resistance gene (Example 8, second site integration). The
 resulting strain is RB 50::[pRF69].sub.n ::[pRF93].sub.m (n and m refer to
 the copy numbers of the modified operons).
 Example 11
 Cloning Vectors pDSNdeHis, pXI12 and pXI16
 NdeI is a restriction enzyme with a 6 bp recognition site ending with ATG.
 It is therefore the enzyme of choice for the cloning of open reading
 frames (ORFs) which generally begin withl the translation start codon ATG.
 The vectors pDSNdeHis, pXI12 and pXI16 all contain an NdeI cloning site.
 In the pXI vectors, the ATG of the NdeI site was positioned such that it
 corresponds to the translational start codon, whereas in pDSNdeHis, a
 histidine-tag is added at the amino-terminus of the expressed protein (see
 below).
 Construction of pDSNdeHis The expression vector pDS/RBSII, 6.times.His(-2)
 (Stuiber, D., Matile, H. and Garotta, G., 1990. System for high-level
 production in Escherichia coli and rapid purification of recombinant
 proteins: application to epitope mapping, preparation of antibodies and
 structure-function analysis. Immunol. Meth. vol. IV, p. 121-152) was used
 as parent plasmid for the construction of pDSNdeHis. pDS/RBSII,
 6.times.His(-2) is identical to pDS/RBSII, 6 His (Stulber et al. 1990; see
 also Accession No. DSM 5298) except for an additional G nucleotide in
 front of the BamHI site. An existing NdeI site in a non-essential region
 of the plasmid was eliminated by cutting, filling-in of the sticky ends
 and religating. The resulting plasmid was cut with BamHI and HindIII and a
 polylinker carrying sites for the restriction endonucleases ClaI, NdeI,
 SalI, BamHI and HindIII was introduced (FIG. 19). The proteins expressed
 from this vector possess an N-terminal extension containing six
 consecutive histidine residues which permit a one step purification
 (Stiber et al., 1990).
 Construction of pXI12 (FIG. 20)
 The backbone of the vector is the EagI-AatII fragment of pBR322 containing
 the ampicillin resistance gene, the origin of replication and the rop gene
 (Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heynecker,
 H. L., Boyer, H. W., Crosa, J. H. and Falkow, S., 1977. Construction and
 characterization of new cloning vehicles. II. A multipurpose cloning
 system. Gene 2, 95-113). The pBR322 sequence is flanked by two sequences
 derived from the levansucrase gene (sacB) of B. subtilis which were
 obtained by polymerase chain reaction (PCR). The recognition sites for the
 restriction endonucleases AatII, EagI and NheI (FIG. 22) were introduced
 through the PCR primers. The sacB-5' sequence starts at position 729 of
 the levansucrase sequence (database accession number X02730 of the
 Genebank database or the database of the European Bioinformatics
 Institute, Hinxton Hall, Cambridge, GB) and ends at position 1266. The
 sacB-3' fragment includes the sequence between positions 1336 and 1794. As
 a selectable marker the erythromycine resistance gene ermAM from the
 plasmid pAM.beta.1 (accession number Y00116 of the Genebank database or
 the database of the European Bioinformatics Institute, Hinxton Hall,
 Cambridge, GB) was introduced. The sequence, also obtained by PCR, starts
 at position 107 of the sequence with the accession number Y00116 and ends
 at position 1091. The flanking AatII and PmeI sites originate from the PCR
 primers. The promoter driving the transcription of the cloned gene is the
 medium strength, constitutive vegI promoter from B. subtilis and
 corresponds to positions 30 to 101 of the published sequence (accession
 number J01552 of the Genebank database or the database of the European
 Bioinformatics Institute, Hinxton Hall, Cambridge, GB). The restriction
 sites EagI and XhoI which flank the promoter originate from the PCR
 primers. The cryT transcriptional terminator is from B. thuringiensis and
 corresponds to positions 268 to 380 of the sequence in accession number
 M13201 of the Genebank database or the database of the European
 Bioinformatics Institute, Hinxton Hall, Cambridge, GB. Again, the flanking
 PmeI and SmaI sites are primer derived. The ribosome binding site
 (underlined) and the polylinker stretch including the translational start
 site within the NdeI site (bold) was introduced as synthetic DNA with the
 sequence CTCGAGAATTAAAGGAGGGTTTCATATGAATTCGGATCCCGGG. (SEQ ID NO. 234) The
 sequences at the joints of the various elements are shown in table IX. The
 sequence shown in table IX column 1, line 1 is SEQ ID NO. 235. The
 sequence shown in table IX column 1, line 2 is SEQ ID NO. 236. The
 sequence shown in table IX column 1, line 3 is SEQ ID NO. 237. The
 sequence shown in table IX column 1, line 4 is SEQ ID NO. 238. The
 sequence shown in table IX column 1, line 5 is SEQ ID NO. 239. The
 sequence shown in table IX column 1, line 6 is SEQ ID NO. 240. The
 sequence shown in table IX column 1, line 7 is SEQ ID NO. 241. The
 sequence shown in table DC column 2, line 5 is SEQ ID NO. 242. The
 sequence shown in table IX column 2, lne 6 is SEQ ID NO. 243. The sequence
 shown in table IX column 3, fine 1 is SEQ ID NO. 244. The sequence shown
 in table IX column 3, line 2 is SEQ ID NO. 245. The sequence shown in
 table IX column 3, line 3 is SEQ ID NO. 246. The sequence shown in table
 IX column 3, line 4 is SEQ ID NO. 247. The sequence shown in table IX
 column 3, line 5 is SEQ ID NO. 248. The sequence shown in table I column
 3, line 6 is SEQ ID NO. 249. The sequence shown in table IX column 3, fine
 7 is SEQ ID NO. 250.
 Construction of pXI16
 The expression of the genes cloned in pXI12 might not be tolerated in E.
 coli. To avoid potential problems, a variant of pXI12 was created in which
 the veg promoter is interrupted by a short sequence. To construct this
 modified vector, named pXI16, a point mutation was introduced between the
 -35 and the -10 regions of the veg promoter to create an ApaI site and a
 30 bp polylinker was then introduced (FIG. 20).
 Example 12
 Cloning of the ribA gene
 The ribA gene was isolated from plasmid pRF2 (FIG. 6) with polymerase chain
 reaction (PCR) using the primers ribA5 and ribA3. This approach allowed
 the introduction of an NdeI restriction endonuclease site site at the
 start codon of the gene and a BambI site after the stop codon. The primers
 had the following sequence:
 ribA5 5'GAAGATTcatATGTTTCATC (SEQ ID NO.251)
 ribA3 5'TATggaTccTTAGAAATGAA (SEQ ID NO.252)
 The underlined sequences correspond to the restriction endonuclease sites
 NdeI (CATATG) and BamHI (GGATCC), small caps indicate changed nucleotides
 as compared to the sequence of the B. subtilis rib operon and bold
 sequences mark the translational start codon (ATG) and the reverse
 complement of the stop codon (TTA).
 The GeneAmp DNA amplification reagent kit from Perkin Elmer Cetus was used
 for PCR reactions following the instructions of the manufacturer. Reaction
 conditions were: 1 min 94.degree. C., 1 min 45.degree. C. and 2 min
 72.degree. C. for 25 cycles. The concentration of each primer was 1 .mu.M
 and the template DNA was added at a concentration of about 1 pM. The PCR
 reaction product was separated from the primers by agarose gel
 electrophoresis and the PCR fragment containing ribA was isolated (Heery,
 D. M., Gannon, F. and Powell, R., 1990. A simple method for subcloning DNA
 fragments from gel slices. Trends in Genetics 6, 173; Vaux, D. L., 1992.
 Rapid recovery of DNA from agarose gels. Trends in Genetics 8, 81), cut
 with NdeI and BamHI and cloned into the vector pDSNdeHis, resulting in the
 plasmid pDSribA.
 Example 13
 Construction of the Strain VB2XL1
 The ribA gene was excised from pDSribA with NdeI and BamHI and subcloned
 into the NdeI-BamHI cleaved pXI16. After isolation of the plasmid from E.
 coli the DNA was cut with ApaI, the 30 bp insert was deleted and the veg
 promoter was reconstituted by ligation. The plasmid was then linearised by
 cutting with FspI which cleaves only in the pBR322 derived sequence (FIG.
 20) and the cloned gene was introduced into transformation-competent B.
 subtilis cells following the described two-step procedure (Cutting, S. M.
 and Vander Horn, P. B., 1990. In: Molecular biological methods for
 Bacillus (Harwood and Cutting, eds.), p. 67-71, John Wiley & Sons Ltd.,
 Chichester, England). Since the production strain RB50::[pRF69]::[pRF93]
 cannot be made competent for transformation B. subtilis strain 1012
 (Saito, H., Shibata, T. and Ando, T., 1979. Mapping of genes determining
 nonpermissiveness and host-specific restriction to bacteriophages in
 Bacillus subtilis Marburg. Mol. Gen. Genet. 170, 117-122) was chosen as an
 intermediate host. Erythromycin resistant clones had the ribA gene and the
 erythromycin-resistance gene erMAM, inserted in the sacB locus by double
 cross-over in the homology regions sacB-3' and sacB-5' (FIG. 21). The
 modified sacB locus was then introduced into RB50::[pRF69]::[pRF93] by
 transduction with the phage PBS1 according to the liquid culture method as
 described (Cutting and Vander Horn, 1990).
 Example 14
 Construction of RB50::[pFR69]::[pRF93] Strains With ribA-M or ribA-C in the
 sacB Locus
 RibA encodes two enzymatic activities, the GTP cyclohydrolase II and the
 mutase (3,4-dihydroxy-2-butanone 4-phosphate synthase), which are encoded
 by two separate genes in E. coli (Richter, G., Ritz, H., Katzenmeier, G.,
 Volk, R., Kohnle, A., Lottspeich, F., Allendorf, D. and Bacher, A., 1993.
 Biosynthesis of riboflavin: cloning, sequencing, mapping and expression of
 the gene coding for GTP cyclohydrolase II of Escherichia coli. J. Bact.
 175, 4045-4051). The mutase activity is located in the amninoterminal half
 of the B. subtilis ribA and the GTP cyclohydrolase II activity resides in
 the carboxyterminal half. The mutase encoding portion of ribA, named
 ribA-M, was excised from pDSribA with NdeI and HincII and cloned into the
 NdeI and SmaI cleaved pXI16 vector. The single HincII site is conveniently
 located in the middle of ribA and cleavage occurs between two codons
 separating the two activities encoding halfs of the gene. The introduction
 of ribA-M into the sacB locus of RB50::[pRF69]::[pRF93] was done as
 described above. The GTP cyclohydrolase encoding portion of ribA, named
 ribA-C, was excised from pDSribA with HincII and BamHI and introduced into
 the HincII-BamHI cleaved pDSNdeHis. Note that the single HincII
 recognition site in pDSNdeHis is the same as the SalI site (GTCGAC, FIG.
 19) and HincII cleavage results in a blunt end cut through the middle of
 the recognition site. The resulting plasmid was then cut with NdeI and
 BamHI and ribA-C was cloned into the NdeI BamHI cut pXI12. The
 introduction of ribA-C into the sacB locus of RB50:: [pRF69]::[pRF93] was
 done as described above except that the ApaI cleavage and religation step
 was omitted, since pXI12 with an uninterrupted veg promoter was used (FIG.
 20).
 Example 15
 Amplification of the Modified Operons pRF69 and pRF93
 Amplification of the modified operons is important for high riboflavin
 titers. The following amplification scheme was used: 30 .mu.l of an
 overnight culture grown in VY (25 g/L veal infusion broth (Difco), 5 g/L
 yeast extract, 15 g/L glucose) with 20 4.mu.g/ml tetracycline and 10
 .mu.g/ml chloramphenicol were used to inoculate three tubes containing 3
 ml VY and 30, 60 or 75 .mu.g/ml tetracycline. After 8-9 hours at
 37.degree. C. and 200 rpm, 30-50 .mu.l of the culture with the highest
 tetracycline concentration still resulting in good growth was used to
 inoculate tubes containing 3 ml VY and 30, 45 or 60 .mu.g/ml
 chloramphenicol. After overnight incubation at 37.degree. C. and 200 rpm,
 30-50 .mu.l of the culture with the highest chloramphenicol concentration
 still resulting in good growth was used to inoculate tubes containing 3 ml
 VY and 60, 75, 90 or 120 .mu.g/ml tetracycline. In the same manner growth
 continued in VY with 45, 60 or 80 .mu.g/ml chloramphenicol, VY with 75,
 90, 120 or 140 .mu.g/ml tetracycline and VY with 60 or 80 .mu.g/ml
 chloramphenicol. The last transfer was with 200 .mu.l into two tubes
 containing 15 ml VY and 120 or 140 .mu.g/ml tetracycline. The cells were
 grown to an OD.sub.600 of about 0.8 and then distributed into 1 ml
 aliquots. Glycerol was added to a final concentration of 15% and the cells
 were stored at -70.degree. C. The strain VB2XL1 reached the maximal
 antibiotic concentrations of 140 .mu.g/ml tetracycline and 80 .mu.g/ml
 chloramphenicol.
 Fermentative Production of Riboflavin
 Fermentative production of riboflavin was done by using the trans-formed
 B.subtilis strains as described above in glucose limited feedbatch as
 described hereinbelow. In general, bacteria that are prototrophic for
 riboflavin survive on minimal medium in the absence of riboflavin.
 Production of riboflavin can be detected and quantified by various
 methods. In a preferred embodiment, overproduction of riboflavin is
 readily observed when overproducing bacteria are exposed to UV light at
 366 nm, as described infra, producing an observable, yellow fluorescence.
 For example, many of the engineered plasmids of the present invention are
 produced in E. coli. For some of these plasmids, overproduction of
 riboflavin has been confirmed by this method. The amount of riboflavin
 produced can be quantitated, e.g., with reverse-phase high performance
 liquid chromatography (HPLC). Cell-free supernatants from bacteria can be
 fractionated over an HPLC column, as described infra, and monitored for
 riboflavin at 254 nm. By extrapolation from a standard curve, the
 concentration of riboflavin can be determined by the area of the peak on
 the chromatogram.
 Riboflavin can also be quantitated by fluorescence spectrophotometry. For
 example, samples containing riboflavin can be read in a fluorescence
 spectrophometer set at an emission wavelength of 525 nm and an excitation
 wavelength of 450 nm.
 In addition, other methods known in the art are available to detect or
 quantitate riboflavin based on its physical and biological properties.
 Fermentation
 Riboflavin overproducing bacteria can be grown in vessels ranging from
 shake flasks to large "batch" fermentors, by methods known in the art (see
 below). In a preferred embodiment, nutrient feed can be manipulated to
 maximize riboflavin production at the minimum cost by varying the
 nutrients in the medium.
 In a specific embodiment, amplified bcontaining genes can be maintained at
 high-copy number in the bacterial chromosome by the inclusion of about 60
 .mu.g/ml chloramphenicol in the inoculum seed strain (but not necessarily
 in the fermentor). Chemap 14-liter fermentors can be used at 1000 rpm with
 a head pressure of 0.6 atmospheres.
 The cells (especially recombinant bacteria as specifically mentioned
 herein) are grown under suitable growth conditions. Such suitable growth
 conditions are characterized by limiting the availability of a component
 of the growth medium and/or feed medium in such a way that aerobic
 conditions for the growth of said recombinant bacterium are maintained.
 Such conditions can be also characterized e.g. by maintaining a level of
 dissolved oxygen at a concentration between about 5% to 30%. One skilled
 in the art is familiar with the fact that such levels of dissolved oxygen
 can vary dependent on the specific technical equipment used for growing
 said recombinant bacteria and for measuring said dissolved oxygen
 concentration. Under anaerobic conditions the synthesis of riboflavin is
 reduced. In some embodiments, the limiting component is chosen from a
 carbon source, nitrogen source, or a component required by the cells
 (e.g., in the feed medium). For example, if the cells are auxotrophic, for
 example, for methionine, a limiting level of methionine may be provided in
 the growth medium. In another example, such component could be glucose or
 a carbonic acid, e.g. a citric acid cycle acid, such as citric acid or
 succinic acid, or an amino acid.
 Example 16
 Fourth Site Integration
 The strain VB2XL3 is generically identical to VB2XL1 except that an
 additional ribA gene was introduced in the amyE locus to further increase
 the ribA level. VB2XL3 was constructed basically as described above for
 VB2XL1. In brief, the erythromycin marker in pXI6 (FIG. 20) was replaced
 by a neomycin resistance gene and the sacB homology regions were replaced
 by amyE homology sequences resulting in the vector pXI191. The neomycin
 resistance gene was obtained from the plasmid pBEST501 (M. Itaya et al.,
 Nucl. Acids Res., 1989, p. 4410) and the inherent ApaI site was eliminated
 by a silent point mutation. The amyE homology regions were amplified with
 PCR from the B. subtilis genome with primers which were designed according
 to the known sequence (Accession number X02150). The ribA gene was then
 cloned in pXI191 and the resulting plasmid was used as a vehicle to
 introduce the ribA gene into the amyE locus of VB2XL1 essentially as
 described above. The new strain was named VB2XL3.
 TABLE IX
 Sequence in pXI12 at the joints of the various elements.
 synthetic
 sequence function and origin linker sequence function and
 origin
 Pveg(Ac# JO1552)* pos. 101 XhoI RBS snd polylinker
 (synthetic)
 .vertline.
 AAATGTAGTG CTCGAG AATTAAAGGA
 RBS and polylinker (synthetic) SmaI pos. 268 cryT (Ac#
 M13201)
 CCCGGG .vertline.
 GAATTCGGAT TCCAAGAGCA
 cryT (Ac# M13201) pos.380 PmeI pos. 107 ermAM (Ac#
 YO0116)
 .vertline. GGTTTAAAC .vertline.
 GTAATACATA AGCAAAGAAT
 ermAM (Ac# YO0116) pos. 1091 AatII pos. 1266 aacB-5'
 (Ac#XO273O)
 .vertline. GACGTC .vertline.
 GCTTCCAAGG TCAGTTCCAG
 sacB-5'(Ac#XO2730) pos. 729 NheI + AatII pos. 4283
 pBR322(Ac#JO1749)
 .vertline. GCTAGCGACGTC .vertline.
 TTTTGTAATG AGGTGGCACT
 pBR322 (Ac#JO1749) pos. 945 EagI + NheI pos. 1794
 sacB-3'(Ac#XO2730)
 .vertline. CGGCCGCTAGC .vertline.
 CCCAGCGCGT GCAAACGTTG
 sacB-3'(Ac#XO2730) pos. 1336 EagI pos. 30 Pveg (Ac#JO1552)
 .vertline. CGGCCG .vertline.
 ACTTTCTTGA GTCTTATTAA
 *Ac#: GenBank (release 93.0) or EMBL database (release 46.0) accession
 numbers
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 252
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 5567
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 1
 ctgcaggtcg actctagagg atcccccatg gacagccgta acggccttgg cctcttcacg 60
 aaaaaacaaa ttgcgggtac gtcaaagttt gttttctacc cgtttaacga aatgcgcaaa 120
 acaaattagg atcaagcagc ttcccattgg ggctgctttt tttatatctt ttttacggtc 180
 atcccctaaa aacagaacat aaattcgtat atctatagaa aagaaatttt tgcagaaatg 240
 tgaaacatat tcccgttatg catcgttata ttaataattt acgagaattt acggtttttt 300
 attcatgaaa aaaaggaata actcatatga atgaatagat tcatattggc tggaggttta 360
 gaaatgggaa gaataaaaac caagattacc attctgttag tgcttttgct tttacttgca 420
 ggcggttata tgtacataaa tgatattgag ctgaaggatg ttccgacagc aattggacaa 480
 accttgtcct cggaagaaga ggaatacacc atccaggaat ataaagtgac gaaaattgac 540
 ggctcagagt atcatggagt agcagaaaac ggaacgaaaa tcatcttcaa cggaaaaaaa 600
 ttaaatcagg atttatctga tataaaagaa ggtgacaaga ttaaggctta cttcagcaaa 660
 tcaaagcgga tcgacggtta atcaaggttg caaaagtgaa tgattaaaaa acatcacctt 720
 tcggatcgaa gggtgatgtt ttgtttttct caaattgtaa gtttatttca ttgcgtactt 780
 taaaaaggat cgctataata accaataagg acaaatgaat aaagattgta tccttcgggg 840
 cagggtggaa atcccgaccg gcggtagtaa agcacatttg ctttagagcc cgtgacccgt 900
 gtgcataagc acgcggtgga ttcagtttaa gctgaagccg acagtgaaag tctggatggg 960
 agaaggatga tgagccgcta tgcaaaatgt ttaaaaatgc atagtgttat ttcctattgc 1020
 gtaaaatacc taaagccccg aattttttat aaattcgggg cttttttgac ggtaaataac 1080
 aaaagagggg agggaaacaa atggaagagt attatatgaa gctggcctta gatcttgcga 1140
 agcagggcga aggacagacc gaatccaatc cgctcgtcgg cgctgttgtc gtaaaggacg 1200
 gacaaattgt cggaatgggc gcccatttaa aatatggtga agctcatgca gaagttcatg 1260
 ccatccatat ggctggagca catgcagagg gtgccgacat ttacgttaca ctcgaaccgt 1320
 gcagccatta cggaaaaaca ccgccatgtg cagaattgat tatcaactct ggtatcaaaa 1380
 gagtgttcgt ggcgatgaga gatcctaatc cgcttgtggc tggaagaggg atcagcatga 1440
 tgaaagaagc tggcattgag gtaagggaag gcatcctggc agaccaggcg gagaggctga 1500
 atgaaaaatt tctgcacttt atgaggacag gccttccgta cgtcacgcta aaagcggctg 1560
 ccagccttga cggcaagata gctaccagca cgggtgacag caaatggatc acgtcagagg 1620
 ctgcaagaca ggatgctcag caatacagga aaacacacca aagcatttta gtcggagttg 1680
 gcacagtgaa agccgacaat ccgagcttaa cctgcagact gccgaatgta acaaaacagc 1740
 cggttcgggt catacttgat accgtactct cgattcctga ggacgctaaa gtgatttgcg 1800
 atcaaatagc gccgacatgg atttttacga cggcacgcgc agacgaggaa aagaaaaaac 1860
 ggctttcagc tttcggagtg aacatattta cacttgaaac cgagcgcatt caaattcctg 1920
 atgttttgaa gatcctagcg gaagaaggca tcatgtcggt gtatgtggaa ggcggttcag 1980
 ctgttcacgg aagctttgtc aaagaaggct gttttcaaga aatcatcttc tattttgccc 2040
 ctaaactaat cggaggaacg catgctccca gcttaatctc cggtgaaggt tttcaatcaa 2100
 tgaaagatgt ccccttatta caattcactg atataaccca aatcggccgt gatatcaaac 2160
 tgacggcaaa accgacaaag gaataggatg gtgaccatgt ttacaggaat tatcgaagaa 2220
 acaggcacaa tcgaatccat gaaaaaagca gggcatgcaa tggccttaac tattaaatgc 2280
 tcaaagattt tagaggatgt tcatcttggc gacagcattg cagtgaacgg catttgtctg 2340
 actgtcactg attttacaaa aaatcaattc acagtggatg ttatgcctga aacagtcaaa 2400
 gctacgtcac tgaatgattt aacaaaagga agcaaagtaa atctggaaag agcgatggcg 2460
 gcaaacggcc gtttcggagg ccatttcgtc tcaggccatg tcgacggaac tgcggaaatc 2520
 acacgaattg aagagaaaag caacgcagtt tactatgatt taaaaatgga cccgtcatta 2580
 acaaaaacat tggttttaaa gggatcaatt actgtggatg gcgtgagctt aaccatattc 2640
 ggcctgacag aagacacagt gacgatctcc ttaataccgc atacgatcag cgaaacgatc 2700
 ttttcagaaa aaacgatcgg ctctaaagtg aatatcgaat gcgatatgat cggaaaatat 2760
 atgtatcgat ttttgcataa agccaatgaa aataagaccc aacaaaccat tacaaaagcc 2820
 ttcttaagcg aaaacggctt ttagagagga agatttgcat gtttcatccg atagaagaag 2880
 cactggacgc tttaaaaaaa ggcgaagtca tcatcgttgt agatgatgaa gacagagaaa 2940
 atgaaggaga ctttgtggct cttgccgagc atgcaacgcc ggaagtcatt aactttatgg 3000
 cgacacatgg gagaggactg atctgcacgc cgctcagtga ggaaatcgca gacaggcttg 3060
 atcttcaccc tatggttgag cataatacag actctcacca cactgcattt accgtaagca 3120
 tagaccatcg tgaaacgaag acaggtatca gcgctcaaga aagatctttt accgttcaag 3180
 cattgctgga cagcaaatcc gtgccatctg attttcagcg tccggggcac atttttccac 3240
 tgattgcgaa aaaaggaggt gtcctgaaaa gcgcgggcca tacagaagct gctgttgatc 3300
 ttgctgaagc ttgcggatct ccaggagccg gcgtcatttg tgaaattatg aatgaagacg 3360
 gaacgatggc gagagtgcct gagctcattg aaattgcgaa aaagcatcaa ttaaaaatga 3420
 tcaccattaa ggatttgatt caataccgtt acaatctgac aacacttgtc gagcgtgaag 3480
 ttgacattac gctgcctact gattttggga catttaaggt ttatggatac acaaatgagg 3540
 tagatggaaa agagcatgtc gcatttgtga tgggagatgt gccgttcgga gaagaaccgg 3600
 tattggtccg ggtgcattca gaatgtctca caggtgacgt gtttggctct catcgctgtg 3660
 attgcggacc gcagctgcac gccgcgctga accaaattgc cgcagaaggc cgtggagtgc 3720
 tcctgtactt gcgccaagaa ggacgaggca tcggtttaat caataaatta aaagcttata 3780
 agcttcagga acaaggctat gacaccgtag aagccaatga ggcgcttgga ttcttgccgg 3840
 atcttcgcaa ctatggcatc ggagcacaaa ttttacgcga cctcggtgtc cggaatatga 3900
 agcttttgac gaataatccg cgaaaaatcg caggccttga aggctacgga ctcagtattt 3960
 cagaaagagt gccgcttcaa atggaggcga aagaacacaa taaaaaatat ttgcaaacca 4020
 aaatgaacaa gctaggtcat ttacttcatt tctaatcaca aatatcacaa aaaaggatgg 4080
 gaatcatatg aatatcatac aaggaaattt agttggtaca ggtcttaaaa tcggaatcgt 4140
 agtaggaaga tttaatgatt ttattacgag caagctgctg agcggagcag aagatgcgct 4200
 gctcagacat ggcgtagaca caaatgacat tgatgtggct tgggttccag gcgcatttga 4260
 aataccgttt gctgcgaaaa aaatggcgga aacaaaaaaa tatgatgcta ttatcacatt 4320
 gggcactgtc atcagaggcg caacgacaca ttacgattat gtctgcaatg aagctgcaaa 4380
 aggcatcgcg caagcagcaa acactactgg tgtacctgtc atctttggaa ttgtaacaac 4440
 tgaaaacatc gaacaggcta tcgagcgtgc cggcacaaaa gcgggcaaca aaggtgtaga 4500
 ttgtgctgtt tctgccattg aaatggcaaa tttaaaccgc tcatttgaat aatttgctga 4560
 aaacagttta aaaatatggc gaaaatgata taatgtgaga aaacggatca cctattcgta 4620
 tccgttaata gcagactgga cattttggat atagaggggt ttttatgtta attcgttata 4680
 aaaaatcgtt tgaaaagatt gcgatggggc ttctttcgtt tatgccgaat gaaaaagacc 4740
 ttaagcagct tcagcagaca attaaggact acgaaacgga tacagaccgc cagctctttc 4800
 tttggaaaga ggacgaggat atcgtcggag caatcggagt cgaaaaaaag gattctgagg 4860
 ttgagatccg gcatatcagt gtgaatcctt ctcatcgcca tcaaggaatc ggaaaacaga 4920
 tgatggatgc tttaaagcat ttattcaaaa cgcaagtact ggttccaaat gaattaacgc 4980
 agagcttttt cgaacgttgt caaggtcagc aggatcaaga catttcatac aataattaag 5040
 cagaggctgt gatcagtctc tgcttttttt tctgcgttct atttcttttt cacgttcacg 5100
 gatgacgtca gtccgatccc gcaaacggtg tttgtcgata agaaatatgt tgctgagtgc 5160
 actgggctgc ccccatgtat actttttttt cctgcattcg atcctgcatg cttcctccag 5220
 tttctcatct ttgattggca gtataatgct tttataggca gagacggttt cgatttgttc 5280
 gtaaaccgat tgcataagtt cgagcaaacg gccatgatca agccctaagt cttcgactgc 5340
 ccggtgttct gcttgaagaa tccggatgct gttcgccatc agtctttttg ccccggctgt 5400
 attctgcctt ctgtgatgat ataaagccac tgcaagctga ataaagccca cccaatagcg 5460
 ttttcgtttc tttggcggat cttccttcca atattcttct aatatttcat ggcattcaaa 5520
 ataatcccgt gtcgcatgaa actcaacgag ataatctata taagctt 5567
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 42
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 2
 Leu Gln Val Asp Ser Arg Gly Ser Pro Met Asp Ser Arg Asn Gly Leu
 1 5 10 15
 Gly Leu Phe Thr Lys Lys Gln Ile Ala Gly Thr Ser Lys Phe Val Phe
 20 25 30
 Tyr Pro Phe Asn Glu Met Arg Lys Thr Asn
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 19
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 3
 Asp Gln Ala Ala Ser His Trp Gly Cys Phe Phe Tyr Ile Phe Phe Thr
 1 5 10 15
 Val Ile Pro
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 17
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 4
 Lys Gln Asn Ile Asn Ser Tyr Ile Tyr Arg Lys Glu Ile Phe Ala Glu
 1 5 10 15
 Met
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 5
 Asn Ile Phe Pro Leu Cys Ile Val Ile Leu Ile Ile Tyr Glu Asn Leu
 1 5 10 15
 Arg Phe Phe Ile His Glu Lys Lys Glu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 116
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 6
 Met Asn Arg Phe Ile Leu Ala Gly Gly Leu Glu Met Gly Arg Ile Lys
 1 5 10 15
 Thr Lys Ile Thr Ile Leu Leu Val Leu Leu Leu Leu Leu Ala Gly Gly
 20 25 30
 Tyr Met Tyr Ile Asn Asp Ile Glu Leu Lys Asp Val Pro Thr Ala Ile
 35 40 45
 Gly Gln Thr Leu Ser Ser Glu Glu Glu Glu Tyr Thr Ile Gln Glu Tyr
 50 55 60
 Lys Val Thr Lys Ile Asp Gly Ser Glu Tyr His Gly Val Ala Glu Asn
 65 70 75 80
 Gly Thr Lys Ile Ile Phe Asn Gly Lys Lys Leu Asn Gln Asp Leu Ser
 85 90 95
 Asp Ile Lys Glu Gly Asp Lys Ile Lys Ala Tyr Phe Ser Lys Ser Lys
 100 105 110
 Arg Ile Asp Gly
 115
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 7
 Ser Arg Leu Gln Lys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 27
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 8
 Met Ile Lys Lys His His Leu Ser Asp Arg Arg Val Met Phe Cys Phe
 1 5 10 15
 Ser Gln Ile Val Ser Leu Phe His Cys Val Leu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 9
 Lys Gly Ser Leu
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 10
 Pro Ile Arg Thr Asn Glu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 14
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 11
 Arg Leu Tyr Pro Ser Gly Gln Gly Gly Asn Pro Asp Arg Arg
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 19
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 12
 Ser Thr Phe Ala Leu Glu Pro Val Thr Arg Val His Lys His Ala Val
 1 5 10 15
 Asp Ser Val
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 13
 &lt;211&gt; LENGTH: 62
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 13
 Ala Glu Ala Asp Ser Glu Ser Leu Asp Gly Arg Arg Met Met Ser Arg
 1 5 10 15
 Tyr Ala Lys Cys Leu Lys Met His Ser Val Ile Ser Tyr Cys Val Lys
 20 25 30
 Tyr Leu Lys Pro Arg Ile Phe Tyr Lys Phe Gly Ala Phe Leu Thr Val
 35 40 45
 Asn Asn Lys Arg Gly Glu Gly Asn Lys Trp Lys Ser Ile Ile
 50 55 60
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 14
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 14
 Ile Leu Arg Ser Arg Ala Lys Asp Arg Pro Asn Pro Ile Arg Ser Ser
 1 5 10 15
 Ala Leu Leu Ser
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 15
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 15
 Arg Thr Asp Lys Leu Ser Glu Trp Ala Pro Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 16
 &lt;211&gt; LENGTH: 42
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 16
 Asn Met Val Lys Leu Met Gln Lys Phe Met Pro Ser Ile Trp Leu Glu
 1 5 10 15
 His Met Gln Arg Val Pro Thr Phe Thr Leu His Ser Asn Arg Ala Ala
 20 25 30
 Ile Thr Glu Lys His Arg His Val Gln Asn
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 17
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 17
 Leu Ser Thr Leu Val Ser Lys Glu Cys Ser Trp Arg
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 18
 &lt;211&gt; LENGTH: 13
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 18
 Glu Ile Leu Ile Arg Leu Trp Leu Glu Glu Gly Ser Ala
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 19
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 19
 Lys Lys Leu Ala Leu Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 20
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 20
 Gly Lys Ala Ser Trp Gln Thr Arg Arg Arg Gly
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 21
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 21
 Met Lys Asn Phe Cys Thr Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 22
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 22
 Gly Gln Ala Phe Arg Thr Ser Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 23
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 23
 Lys Arg Leu Pro Ala Leu Thr Ala Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 24
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 24
 Leu Pro Ala Arg Val Thr Ala Asn Gly Ser Arg Gln Arg Leu Gln Asp
 1 5 10 15
 Arg Met Leu Ser Asn Thr Gly Lys His Thr Lys Ala Phe
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 25
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 25
 Ser Glu Leu Ala Gln
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 26
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 26
 Lys Pro Thr Ile Arg Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 27
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 27
 Pro Ala Asp Cys Arg Met
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 28
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 28
 Gln Asn Ser Arg Phe Gly Ser Tyr Leu Ile Pro Tyr Ser Arg Phe Leu
 1 5 10 15
 Arg Thr Leu Lys
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 29
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 29
 Phe Ala Ile Lys
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 30
 &lt;211&gt; LENGTH: 23
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 30
 Arg Arg His Gly Phe Leu Arg Arg His Ala Gln Thr Arg Lys Arg Lys
 1 5 10 15
 Asn Gly Phe Gln Leu Ser Glu
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 31
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 31
 Thr Tyr Leu His Leu Lys Pro Ser Ala Phe Lys Phe Leu Met Phe
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 32
 &lt;211&gt; LENGTH: 36
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 32
 Arg Lys Lys Ala Ser Cys Arg Cys Met Trp Lys Ala Val Gln Leu Phe
 1 5 10 15
 Thr Glu Ala Leu Ser Lys Lys Ala Val Phe Lys Lys Ser Ser Ser Ile
 20 25 30
 Leu Pro Leu Asn
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 33
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 33
 Ser Glu Glu Arg Met Leu Pro Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 34
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 34
 Ser Pro Val Lys Val Phe Asn Gln
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 35
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 35
 Lys Met Ser Pro Tyr Tyr Asn Ser Leu Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 36
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 36
 Pro Lys Ser Ala Val Ile Ser Asn
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 37
 &lt;211&gt; LENGTH: 226
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 37
 Arg Gln Asn Arg Gln Arg Asn Arg Met Val Thr Met Phe Thr Gly Ile
 1 5 10 15
 Ile Glu Glu Thr Gly Thr Ile Glu Ser Met Lys Lys Ala Gly His Ala
 20 25 30
 Met Ala Leu Thr Ile Lys Cys Ser Lys Ile Leu Glu Asp Val His Leu
 35 40 45
 Gly Asp Ser Ile Ala Val Asn Gly Ile Cys Leu Thr Val Thr Asp Phe
 50 55 60
 Thr Lys Asn Gln Phe Thr Val Asp Val Met Pro Glu Thr Val Lys Ala
 65 70 75 80
 Thr Ser Leu Asn Asp Leu Thr Lys Gly Ser Lys Val Asn Leu Glu Arg
 85 90 95
 Ala Met Ala Ala Asn Gly Arg Phe Gly Gly His Phe Val Ser Gly His
 100 105 110
 Val Asp Gly Thr Ala Glu Ile Thr Arg Ile Glu Glu Lys Ser Asn Ala
 115 120 125
 Val Tyr Tyr Asp Leu Lys Met Asp Pro Ser Leu Thr Lys Thr Leu Val
 130 135 140
 Leu Lys Gly Ser Ile Thr Val Asp Gly Val Ser Leu Thr Ile Phe Gly
 145 150 155 160
 Leu Thr Glu Asp Thr Val Thr Ile Ser Leu Ile Pro His Thr Ile Ser
 165 170 175
 Glu Thr Ile Phe Ser Glu Lys Thr Ile Gly Ser Lys Val Asn Ile Glu
 180 185 190
 Cys Asp Met Ile Gly Lys Tyr Met Tyr Arg Phe Leu His Lys Ala Asn
 195 200 205
 Glu Asn Lys Thr Gln Gln Thr Ile Thr Lys Ala Phe Leu Ser Glu Asn
 210 215 220
 Gly Phe
 225
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 38
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 38
 Arg Gly Arg Phe Ala Cys Phe Ile Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 39
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 39
 Lys Lys His Trp Thr Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 40
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 40
 Lys Lys Ala Lys Ser Ser Ser Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 41
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 41
 Met Met Lys Thr Glu Lys Met Lys Glu Thr Leu Trp Leu Leu Pro Ser
 1 5 10 15
 Met Gln Arg Arg Lys Ser Leu Thr Leu Trp Arg His Met Gly Glu Asp
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 42
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 42
 Ser Ala Arg Arg Ser Val Arg Lys Ser Gln Thr Gly Leu Ile Phe Thr
 1 5 10 15
 Leu Trp Leu Ser Ile Ile Gln Thr Leu Thr Thr Leu His Leu Pro
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 43
 &lt;211&gt; LENGTH: 39
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 43
 Thr Ile Val Lys Arg Arg Gln Val Ser Ala Leu Lys Lys Asp Leu Leu
 1 5 10 15
 Pro Phe Lys His Cys Trp Thr Ala Asn Pro Cys His Leu Ile Phe Ser
 20 25 30
 Val Arg Gly Thr Phe Phe His
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 44
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 44
 Leu Arg Lys Lys Glu Val Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 45
 &lt;211&gt; LENGTH: 27
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 45
 Lys Ala Arg Ala Ile Gln Lys Leu Leu Leu Ile Leu Leu Lys Leu Ala
 1 5 10 15
 Asp Leu Gln Glu Pro Ala Ser Phe Val Lys Leu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 46
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 46
 Met Lys Thr Glu Arg Trp Arg Glu Cys Leu Ser Ser Leu Lys Leu Arg
 1 5 10 15
 Lys Ser Ile Asn
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 47
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 47
 Ser Pro Leu Arg Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 48
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 48
 Phe Asn Thr Val Thr Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 49
 &lt;211&gt; LENGTH: 27
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 49
 Gln His Leu Ser Ser Val Lys Leu Thr Leu Arg Cys Leu Leu Ile Leu
 1 5 10 15
 Gly His Leu Arg Phe Met Asp Thr Gln Met Arg
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 50
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 50
 Met Glu Lys Ser Met Ser His Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 51
 &lt;211&gt; LENGTH: 39
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 51
 Trp Glu Met Cys Arg Ser Glu Lys Asn Arg Tyr Trp Ser Gly Cys Ile
 1 5 10 15
 Gln Asn Val Ser Gln Val Thr Cys Leu Ala Leu Ile Ala Val Ile Ala
 20 25 30
 Asp Arg Ser Cys Thr Pro Arg
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 52
 &lt;211&gt; LENGTH: 22
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 52
 Thr Lys Leu Pro Gln Lys Ala Val Glu Cys Ser Cys Thr Cys Ala Lys
 1 5 10 15
 Lys Asp Glu Ala Ser Val
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 53
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 53
 Lys Leu Ile Ser Phe Arg Asn Lys Ala Met Thr Pro
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 54
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 54
 Lys Pro Met Arg Arg Leu Asp Ser Cys Arg Ile Phe Ala Thr Met Ala
 1 5 10 15
 Ser Glu His Lys Phe Tyr Ala Thr Ser Val Ser Gly Ile
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 55
 &lt;211&gt; LENGTH: 38
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 55
 Arg Ile Ile Arg Glu Lys Ser Gln Ala Leu Lys Ala Thr Asp Ser Val
 1 5 10 15
 Phe Gln Lys Glu Cys Arg Phe Lys Trp Arg Arg Lys Asn Thr Ile Lys
 20 25 30
 Asn Ile Cys Lys Pro Lys
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 56
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 56
 Val Ile Tyr Phe Ile Ser Asn His Lys Tyr His Lys Lys Gly Trp Glu
 1 5 10 15
 Ser Tyr Glu Tyr His Thr Arg Lys Phe Ser Trp Tyr Arg Ser
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 57
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 57
 Asn Arg Asn Arg Ser Arg Lys Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 58
 &lt;211&gt; LENGTH: 22
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 58
 Phe Tyr Tyr Glu Gln Ala Ala Glu Arg Ser Arg Arg Cys Ala Ala Gln
 1 5 10 15
 Thr Trp Arg Arg His Lys
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 59
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 59
 Cys Gly Leu Gly Ser Arg Arg Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 60
 &lt;211&gt; LENGTH: 14
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 60
 Asn Thr Val Cys Cys Glu Lys Asn Gly Gly Asn Lys Lys Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 61
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 61
 Cys Tyr Tyr His Ile Gly His Cys His Gln Arg Arg Asn Asp Thr Leu
 1 5 10 15
 Arg Leu Cys Leu Gln
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 62
 &lt;211&gt; LENGTH: 23
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 62
 Ser Cys Lys Arg His Arg Ala Ser Ser Lys His Tyr Trp Cys Thr Cys
 1 5 10 15
 His Leu Trp Asn Cys Asn Asn
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 63
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 63
 Lys His Arg Thr Gly Tyr Arg Ala Cys Arg His Lys Ser Gly Gln Gln
 1 5 10 15
 Arg Cys Arg Leu Cys Cys Phe Cys His
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 64
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 64
 Asn Gly Lys Phe Lys Pro Leu Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 65
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 65
 Lys Gln Phe Lys Asn Met Ala Lys Met Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 66
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 66
 Cys Glu Lys Thr Asp His Leu Phe Val Ser Val Asn Ser Arg Leu Asp
 1 5 10 15
 Ile Leu Asp Ile Glu Gly Phe Leu Cys
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 67
 &lt;211&gt; LENGTH: 70
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 67
 Phe Val Ile Lys Asn Arg Leu Lys Arg Leu Arg Trp Gly Phe Phe Arg
 1 5 10 15
 Leu Cys Arg Met Lys Lys Thr Leu Ser Ser Phe Ser Arg Gln Leu Arg
 20 25 30
 Thr Thr Lys Arg Ile Gln Thr Ala Ser Ser Phe Phe Gly Lys Arg Thr
 35 40 45
 Arg Ile Ser Ser Glu Gln Ser Glu Ser Lys Lys Arg Ile Leu Arg Leu
 50 55 60
 Arg Ser Gly Ile Ser Val
 65 70
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 68
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 68
 Ile Leu Leu Ile Ala Ile Lys Glu Ser Glu Asn Arg
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 69
 &lt;211&gt; LENGTH: 13
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 69
 Ser Ile Tyr Ser Lys Arg Lys Tyr Trp Phe Gln Met Asn
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 70
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 70
 Arg Arg Ala Phe Ser Asn Val Val Lys Val Ser Arg Ile Lys Thr Phe
 1 5 10 15
 His Thr Ile Ile Lys Gln Arg Leu
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 71
 &lt;211&gt; LENGTH: 91
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 71
 Ser Val Ser Ala Phe Phe Ser Ala Phe Tyr Phe Phe Phe Thr Phe Thr
 1 5 10 15
 Asp Asp Val Ser Pro Ile Pro Gln Thr Val Phe Val Asp Lys Lys Tyr
 20 25 30
 Val Ala Glu Cys Thr Gly Leu Pro Pro Cys Ile Leu Phe Phe Pro Ala
 35 40 45
 Phe Asp Pro Ala Cys Phe Leu Gln Phe Leu Ile Phe Asp Trp Gln Tyr
 50 55 60
 Asn Ala Phe Ile Gly Arg Asp Gly Phe Asp Leu Phe Val Asn Arg Leu
 65 70 75 80
 His Lys Phe Glu Gln Thr Ala Met Ile Lys Pro
 85 90
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 72
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 72
 Val Phe Asp Cys Pro Val Phe Cys Leu Lys Asn Pro Asp Ala Val Arg
 1 5 10 15
 His Gln Ser Phe Cys Pro Gly Cys Ile Leu Pro Ser Val Met Ile
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 73
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 73
 Ser His Cys Lys Leu Asn Lys Ala His Pro Ile Ala Phe Ser Phe Leu
 1 5 10 15
 Trp Arg Ile Phe Leu Pro Ile Phe Phe
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 74
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 74
 Tyr Phe Met Ala Phe Lys Ile Ile Pro Cys Arg Met Lys Leu Asn Glu
 1 5 10 15
 Ile Ile Tyr Ile Ser
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 75
 &lt;211&gt; LENGTH: 66
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 75
 Cys Arg Ser Thr Leu Glu Asp Pro Pro Trp Thr Ala Val Thr Ala Leu
 1 5 10 15
 Ala Ser Ser Arg Lys Asn Lys Leu Arg Val Arg Gln Ser Leu Phe Ser
 20 25 30
 Thr Arg Leu Thr Lys Cys Ala Lys Gln Ile Arg Ile Lys Gln Leu Pro
 35 40 45
 Ile Gly Ala Ala Phe Phe Ile Ser Phe Leu Arg Ser Ser Pro Lys Asn
 50 55 60
 Arg Thr
 65
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 76
 &lt;211&gt; LENGTH: 23
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 76
 Ile Arg Ile Ser Ile Glu Lys Lys Phe Leu Gln Lys Cys Glu Thr Tyr
 1 5 10 15
 Ser Arg Tyr Ala Ser Leu Tyr
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 77
 &lt;211&gt; LENGTH: 18
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 77
 Phe Thr Arg Ile Tyr Gly Phe Leu Phe Met Lys Lys Arg Asn Asn Ser
 1 5 10 15
 Tyr Glu
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 78
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 78
 Ile Asp Ser Tyr Trp Leu Glu Val
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 79
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 79
 Lys Trp Glu Glu
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 80
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 80
 Lys Pro Arg Leu Pro Phe Cys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 81
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 81
 Cys Phe Cys Phe Tyr Leu Gln Ala Val Ile Cys Thr
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 82
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 82
 Met Ile Leu Ser
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 83
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 83
 Arg Met Phe Arg Gln Gln Leu Asp Lys Pro Cys Pro Arg Lys Lys Arg
 1 5 10 15
 Asn Thr Pro Ser Arg Asn Ile Lys
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 84
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 84
 Arg Lys Leu Thr Ala Gln Ser Ile Met Glu
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 85
 &lt;211&gt; LENGTH: 13
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 85
 Gln Lys Thr Glu Arg Lys Ser Ser Ser Thr Glu Lys Asn
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 86
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 86
 Ile Arg Ile Tyr Leu Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 87
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 87
 Lys Lys Val Thr Arg Leu Arg Leu Thr Ser Ala Asn Gln Ser Gly Ser
 1 5 10 15
 Thr Val Asn Gln Gly Cys Lys Ser Glu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 88
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 88
 Leu Lys Asn Ile Thr Phe Arg Ile Glu Gly
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 89
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 89
 Cys Phe Val Phe Leu Lys Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 90
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 90
 Val Tyr Phe Ile Ala Tyr Phe Lys Lys Asp Arg Tyr Asn Asn Gln
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 91
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 91
 Gly Gln Met Asn Lys Asp Cys Ile Leu Arg Gly Arg Val Glu Ile Pro
 1 5 10 15
 Thr Gly Gly Ser Lys Ala His Leu Leu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 92
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 92
 Pro Val Cys Ile Ser Thr Arg Trp Ile Gln Phe Lys Leu Lys Pro Thr
 1 5 10 15
 Val Lys Val Trp Met Gly Glu Gly
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 93
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 93
 Ala Ala Met Gln Asn Val
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 94
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 94
 Lys Cys Ile Val Leu Phe Pro Ile Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 95
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 95
 Ser Pro Glu Phe Phe Ile Asn Ser Gly Leu Phe
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 96
 &lt;211&gt; LENGTH: 54
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 96
 Ile Thr Lys Glu Gly Arg Glu Thr Asn Gly Arg Val Leu Tyr Glu Ala
 1 5 10 15
 Gly Leu Arg Ser Cys Glu Ala Gly Arg Arg Thr Asp Arg Ile Gln Ser
 20 25 30
 Ala Arg Arg Arg Cys Cys Arg Lys Gly Arg Thr Asn Cys Arg Asn Gly
 35 40 45
 Arg Pro Phe Lys Ile Trp
 50
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 97
 &lt;211&gt; LENGTH: 55
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 97
 Ser Ser Cys Arg Ser Ser Cys His Pro Tyr Gly Trp Ser Thr Cys Arg
 1 5 10 15
 Gly Cys Arg His Leu Arg Tyr Thr Arg Thr Val Gln Pro Leu Arg Lys
 20 25 30
 Asn Thr Ala Met Cys Arg Ile Asp Tyr Gln Leu Trp Tyr Gln Lys Ser
 35 40 45
 Val Arg Gly Asp Glu Arg Ser
 50 55
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 98
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 98
 Ser Ala Cys Gly Trp Lys Arg Asp Gln His Asp Glu Arg Ser Trp His
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 99
 &lt;211&gt; LENGTH: 14
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 99
 Gly Lys Gly Arg His Pro Gly Arg Pro Gly Gly Glu Ala Glu
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 100
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 100
 Lys Ile Ser Ala Leu Tyr Glu Asp Arg Pro Ser Val Arg His Ala Lys
 1 5 10 15
 Ser Gly Cys Gln Pro
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 101
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 101
 Arg Gln Asp Ser Tyr Gln His Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 102
 &lt;211&gt; LENGTH: 53
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 102
 Gln Gln Met Asp His Val Arg Gly Cys Lys Thr Gly Cys Ser Ala Ile
 1 5 10 15
 Gln Glu Asn Thr Pro Lys His Phe Ser Arg Ser Trp His Ser Glu Ser
 20 25 30
 Arg Gln Ser Glu Leu Asn Leu Gln Thr Ala Glu Cys Asn Lys Thr Ala
 35 40 45
 Gly Ser Gly His Thr
 50
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 103
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 103
 Tyr Arg Thr Leu Asp Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 104
 &lt;211&gt; LENGTH: 35
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 104
 Ser Asp Leu Arg Ser Asn Ser Ala Asp Met Asp Phe Tyr Asp Gly Thr
 1 5 10 15
 Arg Arg Arg Gly Lys Glu Lys Thr Ala Phe Ser Phe Arg Ser Glu His
 20 25 30
 Ile Tyr Thr
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 105
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 105
 Asn Arg Ala His Ser Asn Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 106
 &lt;211&gt; LENGTH: 40
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 106
 Cys Phe Glu Asp Pro Ser Gly Arg Arg His His Val Gly Val Cys Gly
 1 5 10 15
 Arg Arg Phe Ser Cys Ser Arg Lys Leu Cys Gln Arg Arg Leu Phe Ser
 20 25 30
 Arg Asn His Leu Leu Phe Cys Pro
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 107
 &lt;211&gt; LENGTH: 13
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 107
 Thr Asn Arg Arg Asn Ala Cys Ser Gln Leu Asn Leu Arg
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 108
 &lt;211&gt; LENGTH: 14
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 108
 Arg Phe Ser Ile Asn Glu Arg Cys Pro Leu Ile Thr Ile His
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 109
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 109
 Tyr Asn Pro Asn Arg Pro
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 110
 &lt;211&gt; LENGTH: 13
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 110
 Tyr Gln Thr Asp Gly Lys Thr Asp Lys Gly Ile Gly Trp
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 111
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 111
 Pro Cys Leu Gln Glu Leu Ser Lys Lys Gln Ala Gln Ser Asn Pro
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 112
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 112
 Lys Lys Gln Gly Met Gln Trp Pro
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 113
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 113
 Leu Leu Asn Ala Gln Arg Phe
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 114
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 114
 Arg Met Phe Ile Leu Ala Thr Ala Leu Gln
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 115
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 115
 Thr Ala Phe Val
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 116
 &lt;211&gt; LENGTH: 23
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 116
 Leu Ser Leu Ile Leu Gln Lys Ile Asn Ser Gln Trp Met Leu Cys Leu
 1 5 10 15
 Lys Gln Ser Lys Leu Arg His
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 117
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 117
 Gln Lys Glu Ala Lys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 118
 &lt;211&gt; LENGTH: 40
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 118
 Ile Trp Lys Glu Arg Trp Arg Gln Thr Ala Val Ser Glu Ala Ile Ser
 1 5 10 15
 Ser Gln Ala Met Ser Thr Glu Leu Arg Lys Ser His Glu Leu Lys Arg
 20 25 30
 Lys Ala Thr Gln Phe Thr Met Ile
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 119
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 119
 Lys Trp Thr Arg His
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 120
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 120
 Gln Lys His Trp Phe
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 121
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 121
 Arg Asp Gln Leu Leu Trp Met Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 122
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 122
 Pro Tyr Ser Ala
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 123
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 123
 Gln Lys Thr Gln
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 124
 &lt;211&gt; LENGTH: 18
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 124
 Tyr Arg Ile Arg Ser Ala Lys Arg Ser Phe Gln Lys Lys Arg Ser Ala
 1 5 10 15
 Leu Lys
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 125
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 125
 Ile Ser Asn Ala Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 126
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 126
 Ser Glu Asn Ile Cys Ile Asp Phe Cys Ile Lys Pro Met Lys Ile Arg
 1 5 10 15
 Pro Asn Lys Pro Leu Gln Lys Pro Ser
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 127
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 127
 Ala Lys Thr Ala Phe Arg Glu Glu Asp Leu His Val Ser Ser Asp Arg
 1 5 10 15
 Arg Ser Thr Gly Arg Phe Lys Lys Arg Arg Ser His His Arg Cys Arg
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 128
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 128
 Arg Gln Arg Lys
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 129
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 129
 Arg Arg Leu Cys Gly Ser Cys Arg Ala Cys Asn Ala Gly Ser His
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 130
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 130
 Leu Tyr Gly Asp Thr Trp Glu Arg Thr Asp Leu His Ala Ala Gln
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 131
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 131
 Gly Asn Arg Arg Gln Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 132
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 132
 Ser Ser Pro Tyr Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 133
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 133
 Tyr Arg Leu Ser Pro His Cys Ile Tyr Arg Lys His Arg Pro Ser
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 134
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 134
 Asn Glu Asp Arg Tyr Gln Arg Ser Arg Lys Ile Phe Tyr Arg Ser Ser
 1 5 10 15
 Ile Ala Gly Gln Gln Ile Arg Ala Ile
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 135
 &lt;211&gt; LENGTH: 28
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 135
 Phe Ser Ala Ser Gly Ala His Phe Ser Thr Asp Cys Glu Lys Arg Arg
 1 5 10 15
 Cys Pro Glu Lys Arg Gly Pro Tyr Arg Ser Cys Cys
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 136
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 136
 Ser Leu Arg Ile Ser Arg Ser Arg Arg His Leu
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 137
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 137
 Arg Arg Asn Asp Gly Glu Ser Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 138
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 138
 Asn Cys Glu Lys Ala Ser Ile Lys Asn Asp His His
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 139
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 139
 Gly Phe Asp Ser Ile Pro Leu Gln Ser Asp Asn Thr Cys Arg Ala
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 140
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 140
 His Tyr Ala Ala Tyr
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 141
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 141
 Phe Trp Asp Ile
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 142
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 142
 Gly Leu Trp Ile His Lys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 143
 &lt;211&gt; LENGTH: 32
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 143
 Gly Arg Trp Lys Arg Ala Cys Arg Ile Cys Asp Gly Arg Cys Ala Val
 1 5 10 15
 Arg Arg Arg Thr Gly Ile Gly Pro Gly Ala Phe Arg Met Ser His Arg
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 144
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 144
 Arg Val Trp Leu Ser Ser Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 145
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 145
 Leu Arg Thr Ala Ala Ala Arg Arg Ala Glu Pro Asn Cys Arg Arg Arg
 1 5 10 15
 Pro Trp Ser Ala Pro Val Leu Ala Pro Arg Arg Thr Arg His Arg Phe
 20 25 30
 Asn Gln
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 146
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 146
 Ile Lys Ser Leu
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 147
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 147
 Ala Ser Gly Thr Arg Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 148
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 148
 His Arg Arg Ser Gln
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 149
 &lt;211&gt; LENGTH: 31
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 149
 Gly Ala Trp Ile Leu Ala Gly Ser Ser Gln Leu Trp His Arg Ser Thr
 1 5 10 15
 Asn Phe Thr Arg Pro Arg Cys Pro Glu Tyr Glu Ala Phe Asp Glu
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 150
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 150
 Ser Ala Lys Asn Arg Arg Pro
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 151
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 151
 Arg Leu Arg Thr Gln Tyr Phe Arg Lys Ser Ala Ala Ser Asn Gly Gly
 1 5 10 15
 Glu Arg Thr Gln
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 152
 &lt;211&gt; LENGTH: 182
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 152
 Lys Ile Phe Ala Asn Gln Asn Glu Gln Ala Arg Ser Phe Thr Ser Phe
 1 5 10 15
 Leu Ile Thr Asn Ile Thr Lys Lys Asp Gly Asn His Met Asn Ile Ile
 20 25 30
 Gln Gly Asn Leu Val Gly Thr Gly Leu Lys Ile Gly Ile Val Val Gly
 35 40 45
 Arg Phe Asn Asp Phe Ile Thr Ser Lys Leu Leu Ser Gly Ala Glu Asp
 50 55 60
 Ala Leu Leu Arg His Gly Val Asp Thr Asn Asp Ile Asp Val Ala Trp
 65 70 75 80
 Val Pro Gly Ala Phe Glu Ile Pro Phe Ala Ala Lys Lys Met Ala Glu
 85 90 95
 Thr Lys Lys Tyr Asp Ala Ile Ile Thr Leu Gly Thr Val Ile Arg Gly
 100 105 110
 Ala Thr Thr His Tyr Asp Tyr Val Cys Asn Glu Ala Ala Lys Gly Ile
 115 120 125
 Ala Gln Ala Ala Asn Thr Thr Gly Val Pro Val Ile Phe Gly Ile Val
 130 135 140
 Thr Thr Glu Asn Ile Glu Gln Ala Ile Glu Arg Ala Gly Thr Lys Ala
 145 150 155 160
 Gly Asn Lys Gly Val Asp Cys Ala Val Ser Ala Ile Glu Met Ala Asn
 165 170 175
 Leu Asn Arg Ser Phe Glu
 180
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 153
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 153
 Phe Ala Glu Asn Ser Leu Lys Ile Trp Arg Lys
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 154
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 154
 Tyr Asn Val Arg Lys Arg Ile Thr Tyr Ser Tyr Pro Leu Ile Ala Asp
 1 5 10 15
 Trp Thr Phe Trp Ile
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 155
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 155
 Arg Gly Phe Tyr Val Asn Ser Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 156
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 156
 Lys Asp Cys Asp Gly Ala Ser Phe Val Tyr Ala Glu
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 157
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 157
 Ala Ala Ser Ala Asp Asn
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 158
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 158
 Gly Leu Arg Asn Gly Tyr Arg Pro Pro Ala Leu Ser Leu Glu Arg Gly
 1 5 10 15
 Arg Gly Tyr Arg Arg Ser Asn Arg Ser Arg Lys Lys Gly Phe
 20 25 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 159
 &lt;211&gt; LENGTH: 35
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 159
 Asp Pro Ala Tyr Gln Cys Glu Ser Phe Ser Ser Pro Ser Arg Asn Arg
 1 5 10 15
 Lys Thr Asp Asp Gly Cys Phe Lys Ala Phe Ile Gln Asn Ala Ser Thr
 20 25 30
 Gly Ser Lys
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 160
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 160
 Ile Asn Ala Glu Leu Phe Arg Thr Leu Ser Arg Ser Ala Gly Ser Arg
 1 5 10 15
 His Phe Ile Gln
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 161
 &lt;211&gt; LENGTH: 73
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 161
 Leu Ser Arg Gly Cys Asp Gln Ser Leu Leu Phe Phe Leu Arg Ser Ile
 1 5 10 15
 Ser Phe Ser Arg Ser Arg Met Thr Ser Val Arg Ser Arg Lys Arg Cys
 20 25 30
 Leu Ser Ile Arg Asn Met Leu Leu Ser Ala Leu Gly Cys Pro His Val
 35 40 45
 Tyr Phe Phe Phe Leu His Ser Ile Leu His Ala Ser Ser Ser Phe Ser
 50 55 60
 Ser Leu Ile Gly Ser Ile Met Leu Leu
 65 70
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 162
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 162
 Ala Glu Thr Val Ser Ile Cys Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 163
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 163
 Thr Asp Cys Ile Ser Ser Ser Lys Arg Pro
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 164
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 164
 Ser Ser Pro Lys Ser Ser Thr Ala Arg Cys Ser Ala
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 165
 &lt;211&gt; LENGTH: 19
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 165
 Arg Ile Arg Met Leu Phe Ala Ile Ser Leu Phe Ala Pro Ala Val Phe
 1 5 10 15
 Cys Leu Leu
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 166
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 166
 Tyr Lys Ala Thr Ala Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 167
 &lt;211&gt; LENGTH: 5
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 167
 Ile Lys Pro Thr Gln
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 168
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 168
 Arg Phe Arg Phe Phe Gly Gly Ser Ser Phe Gln Tyr Ser Ser Asn Ile
 1 5 10 15
 Ser Trp His Ser Lys
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 169
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 169
 Ser Arg Val Ala
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 170
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 170
 Asn Ser Thr Arg
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 171
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 171
 Ala Gly Arg Leu
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 172
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 172
 Arg Ile Pro His Gly Gln Pro
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 173
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 173
 Arg Pro Trp Pro Leu His Glu Lys Thr Asn Cys Gly Tyr Val Lys Val
 1 5 10 15
 Cys Phe Leu Pro Val
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 174
 &lt;211&gt; LENGTH: 36
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 174
 Arg Asn Ala Gln Asn Lys Leu Gly Ser Ser Ser Phe Pro Leu Gly Leu
 1 5 10 15
 Leu Phe Leu Tyr Leu Phe Tyr Gly His Pro Leu Lys Thr Glu His Lys
 20 25 30
 Phe Val Tyr Leu
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 175
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 175
 Lys Arg Asn Phe Cys Arg Asn Val Lys His Ile Pro Val Met His Arg
 1 5 10 15
 Tyr Ile Asn Asn Leu Arg Glu Phe Thr Val Phe Tyr Ser
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 176
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 176
 Lys Lys Gly Ile Thr His Met Asn Glu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 177
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 177
 Ile His Ile Gly Trp Arg Phe Arg Asn Gly Lys Asn Lys Asn Gln Asp
 1 5 10 15
 Tyr His Ser Val Ser Ala Phe Ala Phe Thr Cys Arg Arg Leu Tyr Val
 20 25 30
 His Lys
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 178
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 178
 Ala Glu Gly Cys Ser Asp Ser Asn Trp Thr Asn Leu Val Leu Gly Arg
 1 5 10 15
 Arg Gly Ile His His Pro Gly Ile
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 179
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 179
 Ser Asp Glu Asn
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 180
 &lt;211&gt; LENGTH: 26
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 180
 Arg Leu Arg Val Ser Trp Ser Ser Arg Lys Arg Asn Glu Asn His Leu
 1 5 10 15
 Gln Arg Lys Lys Ile Lys Ser Gly Phe Ile
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 181
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 181
 Tyr Lys Arg Arg
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 182
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 182
 Gly Leu Leu Gln Gln Ile Lys Ala Asp Arg Arg Leu Ile Lys Val Ala
 1 5 10 15
 Lys Val Asn Asp
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 183
 &lt;211&gt; LENGTH: 36
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 183
 Lys Thr Ser Pro Phe Gly Ser Lys Gly Asp Val Leu Phe Phe Ser Asn
 1 5 10 15
 Cys Lys Phe Ile Ser Leu Arg Thr Leu Lys Arg Ile Ala Ile Ile Thr
 20 25 30
 Asn Lys Asp Lys
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 184
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 184
 Ile Lys Ile Val Ser Phe Gly Ala Gly Trp Lys Ser Arg Pro Ala Val
 1 5 10 15
 Val Lys His Ile Cys Phe Arg Ala Arg Asp Pro Cys Ala
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 185
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 185
 Ala Arg Gly Gly Phe Ser Leu Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 186
 &lt;211&gt; LENGTH: 18
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 186
 Lys Ser Gly Trp Glu Lys Asp Asp Glu Pro Leu Cys Lys Met Phe Lys
 1 5 10 15
 Asn Ala
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 187
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 187
 Cys Tyr Phe Leu Leu Arg Lys Ile Pro Lys Ala Pro Asn Phe Leu
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 188
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 188
 Ile Arg Gly Phe Phe Asp Gly Lys
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 189
 &lt;211&gt; LENGTH: 368
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 189
 Gln Lys Arg Gly Gly Lys Gln Met Glu Glu Tyr Tyr Met Lys Leu Ala
 1 5 10 15
 Leu Asp Leu Ala Lys Gln Gly Glu Gly Gln Thr Glu Ser Asn Pro Leu
 20 25 30
 Val Gly Ala Val Val Val Lys Asp Gly Gln Ile Val Gly Met Gly Ala
 35 40 45
 His Leu Lys Tyr Gly Glu Ala His Ala Glu Val His Ala Ile His Met
 50 55 60
 Ala Gly Ala His Ala Glu Gly Ala Asp Ile Tyr Val Thr Leu Glu Pro
 65 70 75 80
 Cys Ser His Tyr Gly Lys Thr Pro Pro Cys Ala Glu Leu Ile Ile Asn
 85 90 95
 Ser Gly Ile Lys Arg Val Phe Val Ala Met Arg Asp Pro Asn Pro Leu
 100 105 110
 Val Ala Gly Arg Gly Ile Ser Met Met Lys Glu Ala Gly Ile Glu Val
 115 120 125
 Arg Glu Gly Ile Leu Ala Asp Gln Ala Glu Arg Leu Asn Glu Lys Phe
 130 135 140
 Leu His Phe Met Arg Thr Gly Leu Pro Tyr Val Thr Leu Lys Ala Ala
 145 150 155 160
 Ala Ser Leu Asp Gly Lys Ile Ala Thr Ser Thr Gly Asp Ser Lys Trp
 165 170 175
 Ile Thr Ser Glu Ala Ala Arg Gln Asp Ala Gln Gln Tyr Arg Lys Thr
 180 185 190
 His Gln Ser Ile Leu Val Gly Val Gly Thr Val Lys Ala Asp Asn Pro
 195 200 205
 Ser Leu Thr Cys Arg Leu Pro Asn Val Thr Lys Gln Pro Val Arg Val
 210 215 220
 Ile Leu Asp Thr Val Leu Ser Ile Pro Glu Asp Ala Lys Val Ile Cys
 225 230 235 240
 Asp Gln Ile Ala Pro Thr Trp Ile Phe Thr Thr Ala Arg Ala Asp Glu
 245 250 255
 Glu Lys Lys Lys Arg Leu Ser Ala Phe Gly Val Asn Ile Phe Thr Leu
 260 265 270
 Glu Thr Glu Arg Ile Gln Ile Pro Asp Val Leu Lys Ile Leu Ala Glu
 275 280 285
 Glu Gly Ile Met Ser Val Tyr Val Glu Gly Gly Ser Ala Val His Gly
 290 295 300
 Ser Phe Val Lys Glu Gly Cys Phe Gln Glu Ile Ile Phe Tyr Phe Ala
 305 310 315 320
 Pro Lys Leu Ile Gly Gly Thr His Ala Pro Ser Leu Ile Ser Gly Glu
 325 330 335
 Gly Phe Gln Ser Met Lys Asp Val Pro Leu Leu Gln Phe Thr Asp Ile
 340 345 350
 Thr Gln Ile Gly Arg Asp Ile Lys Leu Thr Ala Lys Pro Thr Lys Glu
 355 360 365
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 190
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 190
 Asp Gly Asp His Val Tyr Arg Asn Tyr Arg Arg Asn Arg His Asn Arg
 1 5 10 15
 Ile His Glu Lys Ser Arg Ala Cys Asn Gly Leu Asn Tyr
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 191
 &lt;211&gt; LENGTH: 24
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 191
 Met Leu Lys Asp Phe Arg Gly Cys Ser Ser Trp Arg Gln His Cys Ser
 1 5 10 15
 Glu Arg His Leu Ser Asp Cys His
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 192
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 192
 Phe Tyr Lys Lys Ser Ile His Ser Gly Cys Tyr Ala
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 193
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 193
 Asn Ser Gln Ser Tyr Val Thr Glu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 194
 &lt;211&gt; LENGTH: 37
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 194
 Phe Asn Lys Arg Lys Gln Ser Lys Ser Gly Lys Ser Asp Gly Gly Lys
 1 5 10 15
 Arg Pro Phe Arg Arg Pro Phe Arg Leu Arg Pro Cys Arg Arg Asn Cys
 20 25 30
 Gly Asn His Thr Asn
 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 195
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 195
 Arg Glu Lys Gln Arg Ser Leu Leu
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 196
 &lt;211&gt; LENGTH: 55
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 196
 Phe Lys Asn Gly Pro Val Ile Asn Lys Asn Ile Gly Phe Lys Gly Ile
 1 5 10 15
 Asn Tyr Cys Gly Trp Arg Glu Leu Asn His Ile Arg Pro Asp Arg Arg
 20 25 30
 His Ser Asp Asp Leu Leu Asn Thr Ala Tyr Asp Gln Arg Asn Asp Leu
 35 40 45
 Phe Arg Lys Asn Asp Arg Leu
 50 55
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 197
 &lt;211&gt; LENGTH: 17
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 197
 Ser Glu Tyr Arg Met Arg Tyr Asp Arg Lys Ile Tyr Val Ser Ile Phe
 1 5 10 15
 Ala
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 198
 &lt;211&gt; LENGTH: 419
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 198
 Asp Pro Thr Asn His Tyr Lys Ser Leu Leu Lys Arg Lys Arg Leu Leu
 1 5 10 15
 Glu Arg Lys Ile Cys Met Phe His Pro Ile Glu Glu Ala Leu Asp Ala
 20 25 30
 Leu Lys Lys Gly Glu Val Ile Ile Val Val Asp Asp Glu Asp Arg Glu
 35 40 45
 Asn Glu Gly Asp Phe Val Ala Leu Ala Glu His Ala Thr Pro Glu Val
 50 55 60
 Ile Asn Phe Met Ala Thr His Gly Arg Gly Leu Ile Cys Thr Pro Leu
 65 70 75 80
 Ser Glu Glu Ile Ala Asp Arg Leu Asp Leu His Pro Met Val Glu His
 85 90 95
 Asn Thr Asp Ser His His Thr Ala Phe Thr Val Ser Ile Asp His Arg
 100 105 110
 Glu Thr Lys Thr Gly Ile Ser Ala Gln Glu Arg Ser Phe Thr Val Gln
 115 120 125
 Ala Leu Leu Asp Ser Lys Ser Val Pro Ser Asp Phe Gln Arg Pro Gly
 130 135 140
 His Ile Phe Pro Leu Ile Ala Lys Lys Gly Gly Val Leu Lys Ser Ala
 145 150 155 160
 Gly His Thr Glu Ala Ala Val Asp Leu Ala Glu Ala Cys Gly Ser Pro
 165 170 175
 Gly Ala Gly Val Ile Cys Glu Ile Met Asn Glu Asp Gly Thr Met Ala
 180 185 190
 Arg Val Pro Glu Leu Ile Glu Ile Ala Lys Lys His Gln Leu Lys Met
 195 200 205
 Ile Thr Ile Lys Asp Leu Ile Gln Tyr Arg Tyr Asn Leu Thr Thr Leu
 210 215 220
 Val Glu Arg Glu Val Asp Ile Thr Leu Pro Thr Asp Phe Gly Thr Phe
 225 230 235 240
 Lys Val Tyr Gly Tyr Thr Asn Glu Val Asp Gly Lys Glu His Val Ala
 245 250 255
 Phe Val Met Gly Asp Val Pro Phe Gly Glu Glu Pro Val Leu Val Arg
 260 265 270
 Val His Ser Glu Cys Leu Thr Gly Asp Val Phe Gly Ser His Arg Cys
 275 280 285
 Asp Cys Gly Pro Gln Leu His Ala Ala Leu Asn Gln Ile Ala Ala Glu
 290 295 300
 Gly Arg Gly Val Leu Leu Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly
 305 310 315 320
 Leu Ile Asn Lys Leu Lys Ala Tyr Lys Leu Gln Glu Gln Gly Tyr Asp
 325 330 335
 Thr Val Glu Ala Asn Glu Ala Leu Gly Phe Leu Pro Asp Leu Arg Asn
 340 345 350
 Tyr Gly Ile Gly Ala Gln Ile Leu Arg Asp Leu Gly Val Arg Asn Met
 355 360 365
 Lys Leu Leu Thr Asn Asn Pro Arg Lys Ile Ala Gly Leu Glu Gly Tyr
 370 375 380
 Gly Leu Ser Ile Ser Glu Arg Val Pro Leu Gln Met Glu Ala Lys Glu
 385 390 395 400
 His Asn Lys Lys Tyr Leu Gln Thr Lys Met Asn Lys Leu Gly His Leu
 405 410 415
 Leu His Phe
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 199
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 199
 Ser Gln Ile Ser Gln Lys Arg Met Gly Ile Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 200
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 200
 Ile Ser Tyr Lys Glu Ile
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 201
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 201
 Leu Val Gln Val Leu Lys Ser Glu Ser
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 202
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 202
 Glu Asp Leu Met Ile Leu Leu Arg Ala Ser Cys
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 203
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 203
 Ala Glu Gln Lys Met Arg Cys Ser Asp Met Ala
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 204
 &lt;211&gt; LENGTH: 72
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 204
 Thr Gln Met Thr Leu Met Trp Leu Gly Phe Gln Ala His Leu Lys Tyr
 1 5 10 15
 Arg Leu Leu Arg Lys Lys Trp Arg Lys Gln Lys Asn Met Met Leu Leu
 20 25 30
 Ser His Trp Ala Leu Ser Ser Glu Ala Gln Arg His Ile Thr Ile Met
 35 40 45
 Ser Ala Met Lys Leu Gln Lys Ala Ser Arg Lys Gln Gln Thr Leu Leu
 50 55 60
 Val Tyr Leu Ser Ser Leu Glu Leu
 65 70
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 205
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 205
 Gln Leu Lys Thr Ser Asn Arg Leu Ser Ser Val Pro Ala Gln Lys Arg
 1 5 10 15
 Ala Thr Lys Val
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 206
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 206
 Ile Val Leu Phe Leu Pro Leu Lys Trp Gln Ile
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 207
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 207
 Thr Ala His Leu Asn Asn Leu Leu Lys Thr Val
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 208
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 208
 Lys Tyr Gly Glu Asn Asp Ile Met
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 209
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 209
 Glu Asn Gly Ser Pro Ile Arg Ile Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 210
 &lt;211&gt; LENGTH: 135
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 210
 Gln Thr Gly His Phe Gly Tyr Arg Gly Val Phe Met Leu Ile Arg Tyr
 1 5 10 15
 Lys Lys Ser Phe Glu Lys Ile Ala Met Gly Leu Leu Ser Phe Met Pro
 20 25 30
 Asn Glu Lys Asp Leu Lys Gln Leu Gln Gln Thr Ile Lys Asp Tyr Glu
 35 40 45
 Thr Asp Thr Asp Arg Gln Leu Phe Leu Trp Lys Glu Asp Glu Asp Ile
 50 55 60
 Val Gly Ala Ile Gly Val Glu Lys Lys Asp Ser Glu Val Glu Ile Arg
 65 70 75 80
 His Ile Ser Val Asn Pro Ser His Arg His Gln Gly Ile Gly Lys Gln
 85 90 95
 Met Met Asp Ala Leu Lys His Leu Phe Lys Thr Gln Val Leu Val Pro
 100 105 110
 Asn Glu Leu Thr Gln Ser Phe Phe Glu Arg Cys Gln Gly Gln Gln Asp
 115 120 125
 Gln Asp Ile Ser Tyr Asn Asn
 130 135
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 211
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 211
 Ala Glu Ala Val Ile Ser Leu Cys Phe Phe Phe Cys Val Leu Phe Leu
 1 5 10 15
 Phe His Val His Gly
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 212
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 212
 Arg Gln Ser Asp Pro Ala Asn Gly Val Cys Arg
 1 5 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 213
 &lt;211&gt; LENGTH: 4
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 213
 Glu Ile Cys Cys
 1
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 214
 &lt;211&gt; LENGTH: 25
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 214
 Val His Trp Ala Ala Pro Met Tyr Thr Phe Phe Ser Cys Ile Arg Ser
 1 5 10 15
 Cys Met Leu Pro Pro Val Ser His Leu
 20 25
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 215
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 215
 Cys Phe Tyr Arg Gln Arg Arg Phe Arg Phe Val Arg Lys Pro Ile Ala
 1 5 10 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 216
 &lt;211&gt; LENGTH: 48
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 216
 Val Arg Ala Asn Gly His Asp Gln Ala Leu Ser Leu Arg Leu Pro Gly
 1 5 10 15
 Val Leu Leu Glu Glu Ser Gly Cys Cys Ser Pro Ser Val Phe Leu Pro
 20 25 30
 Arg Leu Tyr Ser Ala Phe Cys Asp Asp Ile Lys Pro Leu Gln Ala Glu
 35 40 45
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 217
 &lt;211&gt; LENGTH: 41
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 217
 Ser Pro Pro Asn Ser Val Phe Val Ser Leu Ala Asp Leu Pro Ser Asn
 1 5 10 15
 Ile Leu Leu Ile Phe His Gly Ile Gln Asn Asn Pro Val Ser His Glu
 20 25 30
 Thr Gln Arg Asp Asn Leu Tyr Lys Leu
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 218
 &lt;211&gt; LENGTH: 42
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 218
 aaaaacatca cctttcggat cgaagggtga tgttttgttt tt 42
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 219
 &lt;211&gt; LENGTH: 36
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 219
 aaagccccga attttttata aattcggggc tttttt 36
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 220
 &lt;211&gt; LENGTH: 35
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 220
 taagcagagg ctgtgatcag tctctgcttt ttttt 35
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 221
 &lt;211&gt; LENGTH: 116
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence A
 synthetically-generated DNA oligomer containing a
 combination of restriction enzymes sites,
 5'-EcoRI-SmaI-BamHI-3', flanked on either side by
 additional sequence
 &lt;400&gt; SEQUENCE: 221
 tgattaaaaa acatcacctt tcggatcgaa ggggtgatgt tttgtttttc tcgaattccc 60
 gggatccaaa ttgtaagttt atttcattgc gtactttaaa aaggatcgct ataata 116
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 222
 &lt;211&gt; LENGTH: 80
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Recreation
 of the DNA sequence 5' to the BglII site
 &lt;400&gt; SEQUENCE: 222
 aattcatgca tggatccgac ggtaaataac aaaagagggg agggaaacaa atggaagagt 60
 attatatgaa gctggcctta 80
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 223
 &lt;211&gt; LENGTH: 80
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceRecreation
 of the DNA sequence 5' to the BglII site
 &lt;400&gt; SEQUENCE: 223
 gatctaaggc cagcttcata taatactctt ccatttgttt ccctcccctc ttttgttatt 60
 taccgtcgga tccatgcatg 80
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 224
 &lt;211&gt; LENGTH: 66
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Recreation
 of the sequence from the above-mentioned DraI site
 past the start of ORF3
 &lt;400&gt; SEQUENCE: 224
 tcgacggatc cttttagaga ggaagatttg catgtttcat ccgatagaag aagcactgga 60
 cgcttt 66
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 225
 &lt;211&gt; LENGTH: 62
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Recreation
 of the sequence from the above-mentioned DraI site
 past the start of ORF3
 &lt;400&gt; SEQUENCE: 225
 aaagcgtcca gtgcttcttc tatcggatga aacatgcaaa tcttcctctc taaaaggatc 60
 cg 62
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 226
 &lt;211&gt; LENGTH: 78
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Recreation
 of the sequence from the ClaI site past the end of ORF4
 &lt;400&gt; SEQUENCE: 226
 cgatttttgc ataaagccaa tgaaaataag acccaacaaa ccattacaaa agccttctta 60
 agcgaaaacg gcttttag 78
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 227
 &lt;211&gt; LENGTH: 80
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 Recreation
 of the sequence from the ClaI site past the end of ORF4
 &lt;400&gt; SEQUENCE: 227
 aattctaaaa gccgttttcg cttaagaagg cttttgtaat ggtttgttgg gtcttatttt 60
 cattggcttt atgcaaaaat 80
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 228
 &lt;211&gt; LENGTH: 83
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceSynthetic
 polylinker containing a variety of restriction
 sites.
 &lt;400&gt; SEQUENCE: 228
 aggagaaatt aactatgaga ggatctcatc accatcacca tcacgggatc gatcatatgg 60
 tcgacggatc caagcttaat tag 83
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 229
 &lt;211&gt; LENGTH: 68
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence The
 promoter interrupting sequence of pXI16 and the introduced
 restriction sites
 &lt;400&gt; SEQUENCE: 229
 aattttattt gacaaaaatg ggaagcttga tatcgagctc gtcgaccccg tgttgtacaa 60
 taaatgta 68
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 230
 &lt;211&gt; LENGTH: 54
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence A
 synthetic, 54-base oligonucleotide probe used for screening
 &lt;400&gt; SEQUENCE: 230
 ggagctacaa cacattatga ttatgtttgc aatgaagctg ctaaaggaat tgct 54
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 231
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -35_signal
 &lt;222&gt; LOCATION: (1) . .(6)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -10_signal
 &lt;222&gt; LOCATION: (24) . .(29)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: variation
 &lt;222&gt; LOCATION: (7) . .(23)
 &lt;223&gt; OTHER INFORMATION: Region can be variable.
 &lt;400&gt; SEQUENCE: 231
 ttgcgtnnnn nnnnnnnnnn nnntataat 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 232
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -35_signal
 &lt;222&gt; LOCATION: (1) . .(6)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -10_signal
 &lt;222&gt; LOCATION: (24) . .(29)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: variation
 &lt;222&gt; LOCATION: (7) . .(23)
 &lt;223&gt; OTHER INFORMATION: Region can be variable.
 &lt;400&gt; SEQUENCE: 232
 ttgaagnnnn nnnnnnnnnn nnntactat 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 233
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -35_signal
 &lt;222&gt; LOCATION: (1) . .(6)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: -10_signal
 &lt;222&gt; LOCATION: (25) . .(30)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: variation
 &lt;222&gt; LOCATION: (7) . .(24)
 &lt;223&gt; OTHER INFORMATION: Region can be variable.
 &lt;400&gt; SEQUENCE: 233
 ttgaatnnnn nnnnnnnnnn nnnntaaaaa 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 234
 &lt;211&gt; LENGTH: 43
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence Ribosome
 binding site and the polylinker stretch including
 the translational start site within the NdeI site
 &lt;400&gt; SEQUENCE: 234
 ctcgagaatt aaaggagggt ttcatatgaa ttcggatccc ggg 43
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 235
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# J01522
 &lt;400&gt; SEQUENCE: 235
 aaatgtagtg 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 236
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceRBS and
 polylinker
 &lt;400&gt; SEQUENCE: 236
 gaattcggat 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 237
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# M13201
 &lt;400&gt; SEQUENCE: 237
 gtaatacata 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 238
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# Y00116
 &lt;400&gt; SEQUENCE: 238
 gcttccaagg 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 239
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# X02730
 &lt;400&gt; SEQUENCE: 239
 ttttgtaatg 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 240
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# J01749
 &lt;400&gt; SEQUENCE: 240
 cccagcgcgt 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 241
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# X02730
 &lt;400&gt; SEQUENCE: 241
 actttcttga 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 242
 &lt;211&gt; LENGTH: 12
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceSynthetic
 Linker
 &lt;400&gt; SEQUENCE: 242
 gctagcgacg tc 12
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 243
 &lt;211&gt; LENGTH: 11
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceSynthetic
 Linker
 &lt;400&gt; SEQUENCE: 243
 cggccgctag c 11
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 244
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceSynthetic
 polylinker
 &lt;400&gt; SEQUENCE: 244
 aattaaagga 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 245
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# M13201
 &lt;400&gt; SEQUENCE: 245
 tccaagagca 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 246
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# Y00116
 &lt;400&gt; SEQUENCE: 246
 agcaaagaat 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 247
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# X02730
 &lt;400&gt; SEQUENCE: 247
 tcagttccag 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 248
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# J01749
 &lt;400&gt; SEQUENCE: 248
 aggtggcact 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 249
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# 1360836
 &lt;400&gt; SEQUENCE: 249
 gcaaacgttg 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 250
 &lt;211&gt; LENGTH: 10
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Ac# J01552
 &lt;400&gt; SEQUENCE: 250
 gtcttattaa 10
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 251
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 251
 gaagattcat atgtttcatc 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 252
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 252
 tatggatcct tagaaatgaa 20