METHOD AND MATERIALS FOR INTRODUCING DNA INTO PREVOTELLA RUMINICOLA

Abstract: A method of introducing expressible heterologous DNA into Prevotella ruminicola is provided. The method involves conjugal transfer of a shuttle vector comprising the heterologous DNA operatively linked to a promoter functional in P. ruminicola. The invention also provides shuttle vectors for use in the method and P. ruminicola produced by the method. The invention further provides a tetracycline resistance gene of the TetQ class, or fragments thereof that confer tetracycline resistance, and a protein of the TetQ class that provides resistance to tetracycline by protecting ribosomes from tetracycline, or active fragments thereof. Finally, the invention provides a promoter functional in P. ruminicola and an engineered P. ruminicola comprising expressible foreign DNA.

Description: METHOD AND MATERIALS FOR INTRODUCING DNA INTO PREVOTELLA RUMINICOLA FIELD OF THE INVENTION This invention relates generally to methods and materials for the genetic manipulation of Prevotella ruminicola. This invention also relates to a novel class of tetracycline resistance genes. BACKGROUND OF THE INVENTION A. Bacteroides and Prevotella Bacteroides is a genus of Gram negative, obligately anaerobic bacteria found in the gastro¬ intestinal tracts of humans and animals. These bacteria function in metabolizing a wide range of carbohydrates. In humans, Bacteroides account for approximately 25% of the bacteria in the colon. Prevotella ruminicola is a species of Gram negative, obligately anaerobic bacteria found in the rumen of cattle. P. ruminicola ferment carbohydrates such as hemicellulose, cellobiose, and starch and aid digestion and degradation of polysaccharides. P. ruminicola was previously classified as a member of the genus Bacteroides (Bacteroides ruminicola. because it has some characteristics associated with human colonic Bacteroides species. However, recent investigations showed that P. ruminicola shared less than 5% DNA-DNA homology with the colonic Bacteroides species. More detailed biochemical analyses also suggested that it belonged in a separate genus, Prevotella [See Shah, et al., Intl. J. Syst. Bacteriol.. 40:205-208 (1990)]. Some progress has been made in connection with genetic manipulation of obligately anaerobic Bacteroides from the human colon. For example, shuttle vectors have been developed for use with some colonic Bacteroides which contain DNA from cryptic Bacteroides plasmids which are able to replicate in a number of different Bacteroides species [See Odelson, et al., Plasmid, 17:87-109 (1987); Salyers, et al., Crit. Rev. Microbiol.. 14:49-71 (1987); Valentine, et al., J. Bacteriol.. 170:1319-1324 (1988)]. These vectors also contain sequences which allow them to replicate in E. coli and be mobilized out of E. coli by IncP plasmids. The IncP plasmids R751 and RP4 have been shown to mobilize DNA from E. coli to a variety of other species, including colonic Bacteroides species [See Salyers, et al., Crit. Rev. Microbiol.. 14:49-71 (1987); Shoemaker, et al., J. Bacteriol.. 166:959-965 (1986)]. One such E. coli-Bacteroides ' shuttle vector is pVAL-1 which contains cryptic Bacteroides plasmid pB8-51 [Valentine, et al., J. Bacteriol.. 170:1319-1324 (1988)]. Certain colonic Bacteroides strains have been found to harbor large self-transmissible elements carrying a tetracycline resistance ("Tc r ") gene which are referred to as "conjugal elements" or "Tc r elements." Some of these Tc r elements also carry a clindamycin-erythromycin resistance ("Em r ") gene and are referred to as "Tc r Em r elements." These elements are not plasmids, but are integrated into the host chromosome. The Tc r and Em r genes from a conjugal TcΕm r strain of Bacteroides. Bacteroides thetaiotaomicron DOT, have been cloned, along with regions of the element that include transfer genes [Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989)]. The Tc r Em r element from B. thetaiotaomicron DOT has been designated "TcΕm r -D0T." These conjugal elements are able to transfer themselves from one colonic Bacteroides strain to another and to mobilize co-resident plasmids, not only from Bacteroides to Bacteroides, but also from Bacteroides to E. coli [See Odelson, et al., Plasmid. 17:87-109 (1987); Salyers, et al., Crit. Rev. Microbiol. , 14:49-71 (1987); Thomson, et al., FEMS Microbiol. Letters. 61:101-104 (1989); Stevens, et al., J. Bacteriol. , 172:4271-4279 (1990)]. Thus, the Tc r and Tc r Em r conjugal elements found in the colonic Bacteroides strains appear to be able to mediate mating pair formation between diverse genera of bacteria. The conjugal element, TcΕm r 12256, has been found to mobilize co-resident plasmids at high frequencies [See Valentine, et al., J. Bacteriol.. 170:1319-1324 (1988)]. Furthermore, the TcΕm r 12256 element appears to exhibit constitutive transfer, as opposed to other Tc r and TcΕm r elements which require pre-exposure to tetracycline to obtain maximum transfer frequencies. Plasmid DNA has been introduced into some colonic Bacteroides using transformation techniques [See Salyers, et al., CRC Clinical Reviews in Microbiology. 14:49-71 (1987); Odelson, et al., Plasmid. 17:87-109 (1987); Smith, J. Bacteriol.. 164:294-301 (1985)]. For instance, one colonic Bacteroides species has been transformed by electroporation [Thomson, et al., FEMS Microbiol. Letters. 61:101-104 (1989)]. An E. coli- colonic Bacteroides shuttle vector, pDPl, was isolated from Bacteroides uniformis and electroporated into B. uniformis at a frequency of 10 6 transporants per microgram of DNA. However, the same plasmid, when isolated from E. coli EM24, gave only 10 3 transporants per microgram of DNA. Standard methods, however, appear to be inadequate in several respects for the transformation of the colonic Bacteroides. For example, large plasmids are difficult to introduce into these species by trans- formation techniques. Best results are obtained when the plasmid DNA is less that 5 kbp in size. Also, to obtain good rates of transformation, the donor plasmid must be isolated from the same strain used as the recipient. The difficulties encountered in crossing species lines are believed to be due to the presence of restriction barriers. Also, successful transformation of many species of colonic Bacteroides has been sporadic [See Odelson, et al., Plasmid. 17:102 (1987)]. Clearly, much improvement is needed in transformation methods for colonic Bacteroides. Despite progress in understanding the genetics of colonic Bacteroides. P. ruminicola is not well under¬ stood genetically. There have been some biochemical studies of polysaccharide utilization by P. ruminicola. and a xylanase gene from P. ruminicola has been cloned and expressed in E. coli [See Whitehead, et al., Appl. Environ. Microbiol.. 55:893-896 (1989)]. Recently, a naturally-occurring plasmid carrying a gene coding for tetracycline resistance has been identified ("pRRI4") in P. ruminicola 223/M2/7. The pRRI4 plasmid was shown to transfer from P. ruminicola 223/M2/7 into P. ruminicola FlOl, but not into P. ruminicola 23, by conjugation [Flint, et al., Appl. Environ. Microbiol. f 54:855-860 (1988)]. It has also been reported that the pRRI4 plasmid can be introduced into P. ruminicola FlOl by electro- poration, but not into P. ruminicola 118B, M384, GA33 by this method [Thomson and Flint, FEMS Microbiol. Letters f 61:101-104 (1989)]. This article also reports that pRRI4 isolated from P. ruminicola could not be introduced into B. uniformis. a colonic Bacteroides. by electroporation. Thomson and Flint also discloses that the E. coli-colonic Bacteroides shuttle vector pDPl could not be introduced into P. ruminicola by electroporation. This was true whether pDPl was extracted from B. uniformis or E. coli. From the above discussion, it is clear that, prior to the present invention, the genetic manipulation of P. ruminicola was not possible. Little was known about the genetics of P. ruminicola. making the use of vectors that could be manipulated and amplified in a known host, such as E. coli. highly desirable. However, no shuttle vectors were known that could be used in P. ruminicola. Transformation and conjugal transfer of pRRI4 was possible, but pRRI4 cannot be used as a shuttle vector due to its relatively large size (19.5 kbp) and its inability to replicate in E. coli. B. Tetracycline Resistance Many bacteria, including strains of Bacteroides and Prevotella. possess tetracycline resistance genes. Three types of tetracycline resistance have been described and subdivided into classes defined by DNA-DNA hybridization. The first type, tetracycline efflux, is mediated by a 40-50 kDa membrane protein which transports tetracycline out of the cell. Examples of this mode of resistance have been found in Gram-negative enterics [classes TetA-G; Aoki, Micro. Sci.. 5:219-223 (1988); Levy, ASM News. 54:418-421 (1988)] and some Gram-positive bacteria [classes TetK and TetL; Lacks, et al., J. Mol. Biol.. 192:753-765 (1986); McMurry, et al., Antimicrob. Agents Che other.. 32:1646-1650 (1987)]. The second type of tetracycline resistance, ribosome protection, is mediated by a 72-75 kDa cytoplasmic protein which interacts with ribosomes and prevents inhibitory binding of tetracycline. Examples of this mode of resistance have been found in many Gram- positive and some Gram-negative bacteria [classes TetM and TetO; Burdett, J. Bacteriol.. 165:564-569 (1986); Manavathu, et al., Gene- 62:17-26 (1988); Sougakoff, et al., FEMS Microbiol. Lett.. 44:153-159 (1987)]. The third type of resistance, tetracycline modification, is mediated by a 44 kDa cytoplasmic protein which chemically inactivates tetracycline. The only known representative of this mode of resistance, class TetX, was orginally found in B. fragilis [Speer and Salyers, J. Bacteriol.. 170:1423-1429 (1988)]. Two other Tc r genes are known. TetN is an unsequenced streptococcal Tc r which is reported to confer ribosome protection type resistance [Burdett, J. Bacteriol.. 165:564-569 (1986)]. TetP is an uncharacterized Tc r determinant from Clostridium prefringens [Abraham, et al., Plasmid. 19:113-120 (1988)]. SUMMARY OF THE INVENTION The invention provides for the first time a method for the genetic manipulation of Prevotella ruminicola. In particular, the present invention provides a method for introducing heterologous DNA into P. ruminicola. The method comprises transforming E. coli with a shuttle vector comprising: a mobilization region which permits transfer of the shuttle vector from E. coli to a colonic Bacteroides species; a mobilization region which permits transfer of the shuttle vector from the colonic Bacteroides species to a P. ruminicola; and heterologous DNA operatively linked to a promoter functional in P. ruminicola. After transformation of the E. coli with the shuttle vector, the E. coli is contacted 'with the colonic Bacteroides species under conditions sufficient so that the shuttle vector is transferred from the E. coli to the colonic Bacteroides species. Finally, the colonic Bacteroides species containing the shuttle vector is contacted with the P. ruminicola under conditions sufficient so that the shuttle vector is transferred from the colonic Bacteroides species to the P. ruminicola. The invention also comprises P. ruminicola produced by this method and a shuttle vector useful for transferring heterologous DNA to P. ruminicola by conjugation. The shuttle vector comprises: a mobilization region which permits transfer of the shuttle vector from E. coli to a colonic Bacteroides species; a mobilization region which permits transfer of the shuttle vector from the colonic Bacteroides species to a P. ruminicola; and heterologous DNA operatively linked to a promoter functional in the P. ruminicola. These shuttle vectors are particularly advantageous because they can be amplified and manipulated in E. coli before they are used to introduce heterologous DNA into P. ruminicola. The invention further provides a tetracycline resistance gene of the TetQ class, or fragments thereof, that confer tetracycline resistance. The TetQ class is a new class of tetracycline resistance genes which confers tetracycline resistance by ribosome protection. The complete DNA sequence of one such gene has been determined and is presented below. The invention also comprises proteins of the TetQ class, or active fragments thereof, that provide tetracycline resistance by ribosome protection. Finally, the invention provides a promoter functional in P. ruminicola and an engineered P. ruminicola comprising expressible foreign DNA. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: A map of pVALl and a schematic diagram of the construction of pRDB3, pRDB5 and pNFD13-2. A partial map of the TcΕm r DOT element is indicated at the bottom of the figure, and the regions of this element which were cloned into the vectors are indicated by brackets under the map. Abbreviations for restriction sites are: R, EcoRI and C, Clal. Only relevant restriction sites are shown: Ap r = ampicillin resistance; Tc r = tetracycline resistance; Em r = erythromycin resistance. Figure 2A: Total DNA from the B. uniformis 1108 donor containing pRDB5 (lane 1) , and four P. ruminicola B x 4 Tc r transconjugants (lanes 2-5) was digested with EcoRI. The Southern blot was probed with pFD160 which cross- hybridizes with pRDB5 but not with P. ruminicola Bj4 DNA. Figure 2B: Total DNA from the P. ruminicola B,4R recipient (lane 2), the B. uniformis 1108 donor (lane 3) and one of the P. ruminicola B x 4 transconjugants (lane 4) was digested with EcoRI and Hindlll. The Southern blot was probed with XBU4422: :pEG920. This probe hybridizes not only with pBR328 sequences on pRBD5 but also with the Tc r Em r 12256 element in the donor. The two bands corresponding to pRDB5 are indicated in lane 3 by arrows. Lane 1 contains DNA size standards. The largest four standards are 23.1 kb, 9.4 kb, 6.7 kb and 4.4 kb, respectivel . Figure 3: Results of pulse field electro- phoresis to verify the identity of P. ruminicola B,4 Tc r transconjugants. NotI digests of DNA from the donor, B. uniformis 1108 carrying pRDB5, are shown in lanes 1 and 5. NotI digested DNA from P. ruminicola GA33 (lane 2) , B j 4R (lane 3) and one of the B,4 pRDB5 transconjugants (lane 4) are also shown. The NotI pattern of P. ruminicola B,4 is identical to that of B,4R (data not shown) . The migration distances of some of the yeast chromosome size standards are shown in megabases (Mb) at the side of the gel. Figure 4 is a partial restriction map of the 2.7 kbp SstI clone of the Tc r gene from B. thetaiotaomicron DOT. Important deletion derivatives are indicated by the labeled brackets. The orientation and extent of the large open reading frame encoding TetQ are indicated below by the arrow. Figures 5A and 5B show products of the 2.7 kbp SstI clone in E. coli. Figure 5A is an autoradiogram of a polyacrylamide SDS gel of in vitro transcription- translation products. Lane 1 contains products from the vector control, pFD160. Shown also are products from Tc s deletion derivatives pNFD13-2ΔRV (lane 2) and pNFD13-2Δ5 (lane 3) , reduced Tc r deletion derivative pNFD13-2Δ4 (lane 4) , Tc r deletion derivatives pNFD13-2Δ3 (lane 5) and pNFD13-2Δl (lane 6), and intact pNFD13-2 (lane 7). The arrows in the right margin mark the two bands that were consistently unique to the SstI clone in maxicells. Figure 5B shows an autoradiogram of soluble and membrane fractions from maxicells containing pNFD13-2: Lane 1, soluble fraction; Lane 2, membrane fraction. Figure 6 shows the deduced amino acid sequence of TetQ aligned with representatives of TetO (Campylobacter ieiuni. and TetM (Streptococcus faecalis.. The consensus of the sequenced ribosomal protection Tc r genes is displayed below these sequences. Upper case denotes conservation among the ribosome protection Tc r proteins. The four barred regions are regions of conservation in GTP-binding proteins [Halliday, J. Nucleotide Prot. Phosphoryl. Res.. 9:435-448 (1984)]. Positions marked (*) were found to be involved directly in GTP binding and are invariant in all GTP-binding proteins [Jurnak, Science. 230:32-36 (1985)]. Figure 7 shows the upstream sequence of tetO. The endpoints of the pNFD13-2 deletions shown in Figure 4 are indicated by numbers above the sequence. Only the last three characters of the deletion designations are given. The first letter in parenthesis at each deletion denotes Tc r expression in E. coli (R = resistant; I = intermediate; S = sensitive) . The letter following the slash denotes Tc r expression in Bacteroides. The E. coli consensus -35 and -10 sequences are indicated by lines above the fretO sequence. Below the tetO upstream sequence is shown the upstream consensus of the tetM sequences from Staphylococcus aureus. Streptococcus faecalis. and Ureaplasma urealvticum and the tetO sequences from Campylobacter coli, Campylobacter ejuni, and Streptococcus mutans. Upper case letters denote bases that are conserved in all tetM and tetO sequences. Lower case letters denote bases that are not conserved in all cases, but are the consensus for that position. If data were not available for all six upstream sequences at a position, a lower case letter was used at that position. DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS The method of the present invention involves conjugal transfer of shuttle vectors to Prevotella ruminicola. As explained in the Background section, Prevotella ruminicola are strains of bacteria previously classified as Bacteroides ruminicola. The criteria for determining whether a bacterium should be classified as B. ruminicola have been loose in the past. Examples of very authentic B. ruminicola (now P. ruminicola) strains having characteristics quite different than the colonic Bacteroides are B t 4, GA33, 23 and 118B. The degree of homology of 16S ribosomal RNA will probably be used as the standard to classify Bacteroides and Prevotella in the near future. 'Based on this standard, it is expected that B x 4, GA33, 23, 118B and bacteria whose 16S ribosomal RNA is at least 70% homologous with that of these strains will be classified as P. ruminicola. The first step of the method of the invention is to transform an E. coli with a shuttle vector. Methods of transforming E. coli are well known in the art. Any strain of E. coli may be used, and numerous strains of E. coli are publicly available from such public depositories as the American Type Culture Collection (ATCC) . The E. coli must have, or be engineered to have, a mobilization element which functions to transfer the shuttle vector from E. coli to a recipient colonic Bacteroides species. These elements may be introduced into E. coli using methods known in the art. Preferably, the mobilization element is an IncP plasmid, and most preferably the IncP plasmid R751. IncP plasmids such as R751 may be introduced into the E. coli by conjugation methods known in the art. Alternatively, E. coli strains, such as S17- 1, are available which have the IncP plasmid inserted in their chromosomes. Next, the E. coli is contacted with a species of colonic Bacteroides under conditions sufficient so that the shuttle vector is transferred from the E. coli to the colonic Bacteroides species. Methods of mating E. coli and colonic Bacteroides are known and include those described in Shoemaker, et al., J. Bacteriol.. 166:959-965 (1986) and Thomson and Flint, FEMS Microbiol. Letters. 61: 101-104 (1989). Any species of colonic Bacteroides may be used, and many species are available from public depositories, including the ATCC and the Virginia Polytechnic Institute (VPI) Anaerobe Collection (Blacksburg, VA) . The colonic Bacteroides species must contain, or be engineered to contain, a mobilization element which functions to transfer the shuttle vector from the colonic Bacteroides to P. ruminicola. These elements may be introduced into the colonic Bacteroides using methods known in the art such as conjugation. The mobilization element is preferably the conjugal element Tc r Em r 12256. The TcΕm r 12256 element comprises approximately 120 kb of additional DNA not found in other Bacteroides conjugal elements. Although this segment of DNA has not been fully characterized, it is believed that it may enhance or increase efficiency of transfer. Most preferably, the colonic Bacteroides is Bacteroides uniformis containing the TcΕm r 12256 element. The colonic Bacteroides species is then contacted with a strain of P. ruminicola under conditions sufficient so that the shuttle vector is transferred from the colonic Bacteroides species to the P. ruminicola. Many suitable species of P. ruminicola are available from public depositories, including the ATCC and the VPI Anaerobe Collection. A preferred P. ruminicola is B,4. Since P. ruminicola is extremely sensitive to oxygen, conjugation must take place under anaerobic conditions. Further, the use of a modifed E (ME) medium has been found critical to obtaining transconjugants. The composition of ME medium is given in Example 1 below. The present invention also comprises shuttle vectors suitable for transferring heterologous DNA into P. ruminicola. A shuttle vector is a vector which contains one or more replicons which allow it to replicate in more than one type of organism. In particular, the shuttle vectors of the present invention must be able to replicate in E. coli and colonic Bacteroides. They may also be able to replicate in P. ruminicola. or the shuttle vectors, or fragments thereof, may integrate into the P. ruminicola chromosome. Suitable E. coli replicons are well-known and include the pUC and pBR series of plasmids. Replicons suitable for use in colonic Bacteroides include pB8-51, pBFTMlO, and pBI143 [Salyers, et al., CRC Critical Reviews in Microbiology. 14:49-71 (1987); Odelson, et al., Plasmid, 17:87-109 (1987); Smith, J. Bacteriol.. 164:294- 301 (1985)]. It has been found that the pB8-51 replicon also functions in P. ruminicola. Other P. ruminicola replicons can be identified using the teachings herein and, e.g.. the TetQ gene of the invention which is known to be expressed in P. ruminicola. The shuttle vectors of the invention must also be capable of being transferred from E. coli to a colonic Bacteroides species. Accordingly, they must contain a mobilization region which permits this transfer. The mobilization region must be one which is acted on by the mobilization element present in the E. coli to effect the transfer. Suitable mobilization regions are known. They include those on pBFTMlO (pDPl, pCG30) , pB8-51 (pEG920, pVALl) , and pBI143 (pFD160) which are mobilized by IncP plasmids [Salyers, et al., CRC Critical Reviews in Microbiology f 14:49-71 (1987); Odelson, et al., Plasmid. 17:87-109 (1987); Shoemaker, et al., J. Bacteriol.. 166:959-965 (1986)]. The shuttle vectors must also be capable of being transferred from the colonic Bacteroides species to P. ruminicola. and they must contain a mobilization region which permits this transfer. The mobilization region must be one which is acted on by the mobilization element present in the colonic Bacteroides to effect the transfer. Suitable mobilization regions include the mobilization region of pB8-51 which is mobilized by Tc r Em r 12256. Other mobilization regions can be identified using the teachings herein. The shuttle vector also comprises heterologous DNA sought to be transferred to P. ruminicola. "Heterologous DNA" is defined herein to mean DNA from a source other than the P. ruminicola strain which is to receive the heterologous DNA. The heterologous DNA may include DNA encoding enzymes involved in the fermentation of carbohydrates in the rumen, enzymes involved in the degradation of polysaccharides (such as xylanase or polysaccharases) , other enzymes involved in rumen • metabolism, and enzymes or groups of enzymes that synthesize substances that are beneficial to growth of cattle such as antibiotics. By transferring heterologous DNA to P. ruminicola. new and useful traits may be imparted to the recipient P. ruminicola. These traits can include those which will lead to more economical beef production. The heterologous DNA is operatively linked to a promoter functional in P. ruminicola. A preferred promoter is a promoter of a TetQ gene (see discussion of TetQ genes below) . Another preferred promoter is from the TcΕm r DOT element. A particularly preferred promoter comprises the sequence: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA, 180 [seq id no 1] or active variants thereof. This promoter is the promoter region of the Tc r gene of the TcΕm r element of B. thetaiotaomicron DOT and may be isolated from that gene or may be prepared by chemical synthesis. This promoter region is also strongly believed to be sufficient to initiate transcription in P. ruminicola. "Active variants" are promoters which have deletions, additions and/or substitutions of nucleotides as compared to the above sequence, but which are still able to initiate transcription in P. ruminicola. The shuttle vector will also include one or more selection markers. Selection markers must be used to distinguish transformed E. coli from untransformed E. coli and to distinguish transconjugant colonic Bacteroides and P. ruminicola from non-transconjugants. It is also necessary to include selection markers that distinguish donor from recipient in mating mixtures. Many suitable selection markers are known and include antibiotic resistance, amino acid or other nutrient requirements, pH, and combinations of these. Preferred selection markers for P. ruminicola are TetQ tetracycline resistance genes. Especially preferred is the TetQ tetracycline resistance gene isolated from the Tc r Em r -D0T element whose sequence is given below. The various components of the shuttle vector may be isolated or synthesized and then assembled using techniques that are well known in the art. Indeed, one the most important aspects of the present invention is that it allows for the engineering of DNA that is to be introduced into P. ruminicola. A preferred shuttle vector is pRDB5. The chimeric pRDB5 construct contains sequences from the plasmid pBR328, a cryptic colonic Bacteroides plasmid, pB8-51, and a colonic Bacteroides Tc r gene isolated from the TcΕm r -DOT conjugal element. The restriction map of pRDB5 is shown in Figure 1. Plasmid pRDB5 replicates in E. coli. colonic Bacteroides and P. ruminicola. Although it is not known whether pRDB5 replicates in, or transfers to, all colonic Bacteroides and P. ruminicola. this plasmid has a broad host range, and it is likely it can be used in many colonic Bacteroides and P. ruminicola. In a preferred embodiment of the method of the present invention, E. coli were transformed with pRDB5. Then pRDB5 was mobilized from E. coli into B. uniformis by the IncP plasmid R751 which was present.in the E. coli. Next, pRDB5 was conjugally transferred from B. uniformis to P. ruminicola B,4 by the conjugal element TcΕm r 12256 present in the B. uniformis. A combination of in vitro selections was utilized to identify P. ruminicola B,4 transconjugants. First, the P. ruminicola B,4 recipient used was a rifampicin resistant mutant (rif) produced by growing P. ruminicola B,4 on increasing levels of rifampicin to produce a spontaneous mutant. The r.if P. ruminicola B,4 transconjugants could then be selected against donor B. uniformis. a species that is rifampicin sensitive. B. uniformis 1100 was chosen as a donor because it is a thymidine auxotroph, and the lack of thymidine in the selection medium could be used to select against that donor after atings with P. ruminicola B,4. B. uniformis is also known to grow in medium containing vitamin K, whereas P. ruminicola B,4 has no vitamin K requirement. Thus, vitamin K was also omitted from the selection medium. Finally, pH was used in the selection method because P. ruminicola B^ grows well at pH 6.2, whereas B. uniformis does not grow well at pH values lower than 6.8. The combination of selection for antibiotic resistance, lack of thymidine and vitamin K, and low pH provided a relatively clean background for selecting P. ruminicola B j 4 transconjugants. The transconjugants were distinguished from non-transconjugant P. ruminicola Bj4 because they were tetracycline resistant due to the expression of the foreign Tc r gene on pRDB5. The P. ruminicola B x 4 transconjugants were also tested for other traits that characterize P. ruminicola B x 4 and differenti¬ ate that strain from the donor B. uniformis. The results of the tests demonstrated that true P. ruminicola transconjugants containing pRDB5 were produced by the method of the invention. The present invention also comprises transcon- jugant P. ruminicola prepared by the method of the invention and containing the shuttle vectors of the invention. A particularly preferred transconjugant is P. ruminicola B x 4 containing pRDB5. The invention further comprises a tetracycline resistance gene of the TetQ class, or fragments thereof that confer tetracycline resistance. The TetQ class is a new class of tetracycline resistance genes which confers tetracycline resistance by coding for proteins which protect ribosomes from the inhibitory binding of tetracyline. The invention also comprises the proteins encoded by the TetQ genes (hereinafter "TetQ class of proteins") , or active fragments thereof. "Active fragments" of these proteins are fragments which are still capable of conferring tetracycline resistance. The DNA sequence of one TetQ gene (isolated from the Bacteroides conjugal element TcΕm r -DOT) has been determined and is presented below in Example 2, along with the amino acid sequence of the protein encoded by the gene. The invention also comprises other DNA sequences which encode this same protein. Hybridization studies using a portion of the sequenced gene indicates that TetQ genes are widespread in colonic Bacteroides. Given the stringency used in these experiments, it is estimated that the Tc r genes found in other Bacteroides Tc r strains share at least 80% identity with the sequenced gene. Also, the Tc r gene on the P. ruminicola plasmid pRRI4 appears to be a TetQ gene. TetQ genes may be isolated from Bacteroides and Prevotella Tc r strains using known techniques. Alternatively, genes, or gene fragments, may be prepared using chemical synthesis. Finally, the invention provides an engineered P. ruminicola containing expressible foreign DNA. "Foreign DNA" is used herein to mean DNA from a source other than P. ruminicola. Thus, "foreign DNA" is more narrow than "heterologous DNA," and heterologous DNA includes foreign DNA. "Engineered" is used to mean P. ruminicola not found in nature. EXAMPLES The restriction enzymes used in the following examples were obtained from Bethesda Research Laboratory, Gaithersburg, MD. They were used according to the manufacturer 's instructions. EXAMPLE 1 A. Construction Of Shuttle Vectors Four shuttle vectors were constructed. They were pRDB5, pVALl, pRDB3, and pNFD13-2, shown in Figure 1. The vector pVALl carries the erythromycin resistance (Em r ) gene from the colonic Bacteroides transposon Tn4351 linked to portions of pBR328 (an E. coli replicon) and the cryptic Bacteroides plasmid pB8-51 (a colonic Bacteroides replicon) . It was prepared as described in Valentine, et al., J. Bacteriol.. 170:1319- 1324 (1988) . Briefly, pBR328 (available from Boehringer Mannheim) was digested with EcoRI. The EcoRI fragment of Tn4351 [preparation from pBF4 described in Shoemaker, et al., J. Bacteriol.. 162:626-632 (1985)] was ligated to the EcoRI-digested pBR328 to produce pTBl. Plasmid pB8-51 was isolated from Bacteroides eggerthi by standard plasmid isolation techniques [See Maniatis, et al.. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, New York 1982)]. It was then partially digested with Tagl. Next, Clal digests of pTBl were mixed with the TagI digests of pB8-51 and ligated with T4 DNA ligase to produce pVALl. Vector pRDB3 was prepared by cloning a 7 kbp Hindi fragment from a cosmid clone of the TcΕm r -D0T element into pVALl. The cosmid clone was prepared as described in Shoemaker, et al., J. Bacteriol.. 171:1294- 1302 (1989) . Then the cosmid clone was digested with Hindi, and the resulting 7 kbp fragment containing the Tc r gene was ligated to PvuII-digested pVALl to produce pRDB3. . Next, pRDB3 was digested with Clal and religated to produce pRDB5. The result of this manipulation was to remove the Tn435l Em r gene. The vector pNFD13-2 comprises pFD160 having a 2.7 kbp fragment containing the Tc r gene from TcΕm r -D0T cloned into the SstI site. Plasmid pFD160 was prepared as described in Smith, J. Bacteriol.. 164:294-301 (1985). It consists of Haell-cleaved pBI143 (a colonic Bacteroides replicon) ligated to Ndel-digested pUC19 (an E. coli replicon) . The 2.7 kbp fragment containing the Tc r gene was prepared as follows. TnlOOO insertions into pRDB3 were used to create convenient restriction sites. Transposon mutagenesis was performed by transforming an E. coli strain carrying the F plasmid on which TnlOOO resides with pRDB3. TnlOOO causes cointegrates to form between pRDB3 and the F plasmid. During conjugation, F::pRDB3 cointegrates are transferred to a recipient. In the recipient, the cointegrates resolve, leaving the F plasmid and pRDB3 with a TnlOOO insertion. Restriction digests of the resulting pRDB3: :TnlOOO isolates were screened by standard techniques (Maniatis, et al., supra) . and the smallest clone that would express Tc r in colonic Bacteroides was identified. This clone was the 2.7 kbp fragment containing the Tc r gene and was excised with SstI. B. Transformation Of E. coli E. coli donor strains were constructed by introducing pRDB5, pVALl, or pNFD13-2 into E. coli DH5C.MCR [obtained from Bethesda Research Laboratory] or S17-1 [obtained from R. Simon, Universitat Bielefeld, Postfach 86-40, D-4800 Bielefeld 1, FRG; described in Simon, et al.. Bio/Technology. 1:784-791 (1983)]. The plasmids were introduced into the E. coli strains by transformation techniques previously described [See Maniatis, et al., supra] . The IncP mobilizing plasmid R751 [See Meyer, et al., J. Bacteriol.. 143:1362-1373 (1980)] was introduced into E. coli DH5αMCR by conjugation as described in Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989); Thomson, et al., FEMS Microbiol. Letters. 61:101-104 (1989) . E. coli S17-1 had a copy of the IncP plasmid RP4 already inserted in its chromosome. Both R751 and RP4 mobilize pRDB5, pVALl, and pNFD13-2 from E. coli to B. uniformis at frequencies of 10 "4 per recipient. C. Preparation Of Colonic Bacteroides Donors B. uniformis donor strains containing TcΕm r element 12256 and pRDB5 (Tc r ) or pNFD13-2 (Tc r ) were constructed by first introducing the plasmid pRDB5 or pNFD13-2 into B. uniformis 1100 [obtained from the VPI Anaerobe Laboratory, Blacksburg, Virginia] , as described previously [Shoemaker, et al., J. Bacteriol.. 166:959-965 (1986); Thomson, et al., FEMS Microbiol. Letters- 61:101- 104 (1989)], and selecting for tetracycline resistance. Transconjugants carrying the Tc r plasmid were used as recipients in a mating with B. uniformis 1008 (TcΕm r ) [obtained from the VPI Anaerobe Laboratory] to transfer the Tc r Em r element 12256, with selection for Tc r and Em r . The resulting strains were designated B. uniformis 1108 (pRDB5) and B. uniformis 1108 (pNFD13-2) . Similarly, to construct B. uniformis carrying the TcΕm r element 12256 and pVALl (Em r ) , pVALl was first transferred from E. coli to B. uniformis 1100 by conjugation, with selection for Em r . Then, the Tc r Em r 12256 element was introduced by conjugation from B. uniformis 1008 to B. uniformis 1100 (pVALl) , with selection for Tc r and Em r . The final strain was designated B. uniformis 1108 (pVALl) . D. Mating With P. ruminicola Next, the recipient, P. ruminicola B,4 (obtained from Marvin Bryant, Dept. of Animal Sciences, University of Illinois, Urbana, IL) , was mated with E. coli or B. uniformis. έ. coli donor strains were grown in Luria broth (LB) to an O.D. (650 nm) of 0.15-0.20. B. uniformis 1108 strains were grown in TYG-Thy-K broth in 80% nitrogen-20% carbon dioxide to an O.D. (650 nm) of 0.15- 0.20. Optical densities were measured in 18 mm diameter culture tubes in a Spectronic 20 spectrophotometer (Milton Roy Co., Rochester, New York). TYG-Thy-K broth is trypticase-yeast extract-glucose broth [composition given in Holdeman, et al., Anaerobe Laboratory Manual (4th ed. , Virginia Polytechnic Institute, Blacksburg, Va. 1977)] containing 100 μg/ml thymidine and 1 μg/ml vitamin K 3 , with a final pH of 7.0-7.3. P. ruminicola B,4 was grown in MM10 broth at 80% nitrogen-20% carbon dioxide to an O.D. (650 nm) of 0.25- 0.30. MM10 is similar to M10 medium previously described rAnaerobe Laboratory Manual, supra] , except the concentration of trypticase and yeast extract was increased ten-fold and amylopectin was present as the carbohydrate source. Also, titanium citrate (0.15 M) was added drop-wise until the resazurin became colorless (approximately 0.2-0.3 ml per liter of medium) prior to the addition of cysteine. The pH of this medium was 6.5- 6.6. This medium, as were all media used for culturing P. ruminicola. was made in glass tubes sealed with a rubber stopper. The E. coli or B. uniformis donor (30 ml) was centrifuged in a Sorvall GLC28 bench top centrifuge (SP/X rotor; Dupont Instruments, Wilmington, Delaware) at 3,000 rpm for 15 minutes at room temperature to pellet the bacteria. The bacteria were then washed in 5 ml potassium phosphate buffer (0.1 M, pH 7.0) and resuspended in 1 ml of TYG-Thy-K medium. Manipulations of E. coli or B. uniformis were performed under aerobic conditions. ! ■ >. ruminicola B,4 (10 ml) was centrifuged in sealed culture tubes at 3,000 rpm for 15 minutes at room temperature as described above, and the supernatant fluid was removed with a sterile syringe. The resuspended donor (E. coli or B. uniformis) (1 ml) and 5 ml of anaerobic 0.1 M potassium phosphate buffer (pH 7.0) were injected into the tube. Anaerobic phosphate buffer was prepared by boiling phosphate buffer and cooling under a stream of oxygen-free carbon dioxide. After vortexing the tubes to dislodge the pelleted recipient, the bacterial mixture was centrifuged again in the sealed tubes, and the wash solution was withdrawn with a syringe. TYG-Thy-K medium (1.5 ml) and MM10 medium (1.5 ml) were injected into the tube, and the tube was vortexed to resuspend the bacteria. The resuspended mixture was injected into a sealed anaerobic tube containing a slant of modified E agar medium ("ME"), pH 6.8, for the mating. ME is the same as Sweet E medium previously described (Anaerobe Laboratory Manual, supra) , except it contains glucose, as the only carbohydrate, and 100 ug/ml thymidine. Agar was added to a final concentration of 2%. The tubes were then centrifuged as described above to pellet the bacteria on the slants. The tubes were inverted gently, and the supernatant fluid removed with a syringe. The tubes were then incubated upside down at 37°C for 15-18 hours. After incubation, 1 ml of MM10 (pH 6.6), containing no thymidine or vitamin K was added to the slant tubes, and the tubes were vortexed. Next, resuspended bacteria were removed with a sterile syringe which had been gassed out with nitrogen-carbon dioxide. To select for transconjugants, 0.1-0.2 ml of resuspended cells, or 0.1-0.2 ml of a 1:10 dilution, were inoculated into a roll tube containing MMIO-Rif-Tc or MMIO-Rif-Em selection medium. MMIO-Rif (pH 6.2) medium consisted of MM10 containing 2% agar and 40 ug/ml rifampicin. For selection of transconjugants, either tetracycline (final concentration of 5 ug/ml) or erythromycin (final concentration of 5 ug/ml) was added to the MMIO-Rif medium to produce MMIO-Rif-Tc and MMIO-Rif- Em, respectively. To enumerate the total number of B. uniformis donors, 0.1 ml of a 10 "6 dilution of the resuspended cells was plated on TYG-Thy-K agar plates and incubated in a GasPak jar. To enumerate the E. coli donors, 0.1 ml of a 10" 6 dilution was plated on LB agar and incubated aerobically. To enumerate the P. ruminicola Bj4 recipients, 0.1 ml of a 10 "6 and a 10 "8 dilution were inoculated into an MMIO-Rif roll tube. All incubations were done at 37°C for 3-4 days. E. Results Of E. coli-P. ruminicola Matings When the transfer of plasmids pVALl, pNFD13-2, and pRDB5 from E. coli to P. ruminicola was attempted, no Tc r or Em r P. ruminicola transconjugants were detected. As a result, B. uniformis was used as an intermediate donor for P. ruminicola as described in the next section. F. Results Of B. uniformis- E. coli Matings B. uniformis 1108 (pRDB5) , B. uniformis 1108 (pVALl) , or B. uniformis 1108 (pNFD13-2) , prepared as described above, were mated with E. coli HB101 or EM24 to determine whether the B. uniformis recipients carrying a conjugal TcΕm r 12256 element and a plasmid were capable of mobilizing the plasmid at high frequency. The procedure for the B. uniformis-E. coli mating has been described previously [See Shoemaker, et al., J. Bacteriol.. 166:959- 965 (1986); Thomson, et al., FEMS Microbiol. Letters. 61:101-104 (1989)]. Mobilization of these plasmids from B. uniformis to E. coli occurred at frequencies of lO^-lO" 5 per recipient (see Table 1 below) . G. Results Of B. uniformis- P. ruminicola Matings To test for transfer of the plasmids from B. uniformis to P. ruminicola B,4, a selective medium allowing growth of P. ruminicola but not B. uniformis had to be developed. Being able to detect transfer frequencies as low as 10" 9 per recipient was the criterion. First, the antibiotic sensitivity of P. ruminicola B,4 was tested. Minimal inhibitory concentrations for various antibiotics were determined by inoculating MM10 containing different concentrations of antibiotic and incubating for 48 hours. Antibiotic concentrations tested were 5, 10, 20, 50, 100 and 200 ug/ml. In the case of tetracycline and erythromycin, resistance levels on MM10 agar medium were also determined. P. ruminicola B,4 was found susceptible to rifampicin (10 ug/ml) , tetracycline (2 ug/ml) , erythromycin (1 ug/ml) , gentamicin (20 ug/ml) , and a picillin (5 ug/ml) . It was resistant to chloramphenicol (10 ug/ml) , kanamycin (50 ug/ml) , trimethoprim (200 ug/ml), and nalidixic acid (100 ug/ml). P. ruminicola B,4 was susceptible to all of the antibiotics which inhibited growth of B. uniformis except chloramphenicol. Accordingly, chloramphenicol was first used to select for P. ruminicola B,4 and against the donor. Mixtures of B. uniformis and P. ruminicola B,4 were plated on MM10 agar containing 10 ug/ml chloramphenicol. Donor B. uniformis colonies were still able to grow enough to obscure true transconjugants. Therefore, another resistance for selecting P. ruminicola recipients was required. A spontaneous rifampicin resistant (Rif r ) mutant of P. ruminicola Bj4 was isolated by inoculating the bacteria into MM10 broth medium containing progressively higher concentrations of rifampicin. By growing P. ruminicola B14 on successively higher concentrations, a spontaneous mutant of P. ruminicola Bj4 was obtained which would grow in rifampicin concentrations as high as 60 ug/ml. The spontaneous Rif r mutant was determined to be a derivative of P. ruminicola B x 4 by comparing its NotI digest pattern with that of the original B x 4 strain. The restriction enzyme digest patterns were identical. This Rif strain was used in matings to provide a selection for the P. ruminicola. This method of producing the rifampicin mutant is a well known method of producing antibiotic resistant mutants of bacteria, and other suitable P. ruminicola rifampicin resistant mutants can be produced in this manner. However, using the Rif r derivative, P. ruminicola B j 4R, as a recipient and selecting for rifampicin resistance did not allow for the detection of transfer frequencies as low as 10" 9 per recipient because spontaneous Rif r mutants of B. uniformis 1108 occurred at a frequency of 10" 7 . Accordingly, a combination of selections had to be used. First, the P. ruminicola Bj4 rifampicin resistant mutant was used. B. uniformis 1100 was chosen as a donor because it is a thymidine auxotroph, and the lack of thymidine in the selection medium could be used to select against that donor after matings with P. ruminicola B,4. However, spontaneous reversion to wild type occurs at relatively high frequencies (10" 6 ) . B. uniformis is also known to grow in medium containing vitamin K, whereas P. ruminicola B,4 has no vitamin K requirement. Thus, vitamin K was also omitted from the selection medium. Finally, pH was used in the selection method because P. ruminicola B,4 grows well at pH 6.2, whereas B. uniformis does not grow well at pH values lower than 6.8. The combination of selection for antibiotic resistance, lack of thymidine and • vitamin K, and low pH provided a relatively clean background for selecting P. ruminicola B,4 transconjugants. Using this selection medium and using a donor to recipient ratio of 1.5-3.0:1.0, Tc r transconjugants were detected in a mating between B. uniformis 1108 (pRDB5) and P. ruminicola B,4 (Rif r ) at frequencies of 10" 6 -10" 7 per recipient (see Table 1) . No transconjugants were detected in matings in which the donor was B. uniformis 1108 (pNFD13-2) or B. uniformis 1108 (pVALl) . The ability of the transconjugants to grow in various media was tested to rule out the possibility that the apparent transconjugants were spontaneous Rif r or Rif r Thy + mutants of the B. uniformis donor. Growth on TYG, no growth on TYG-Thy, no growth in MM10 containing gentamicin, and growth in MM10 containing xylan instead of glucose was observed. These phenotypic characteristics indicated that the transconjugants were of P. ruminicola origin rather than B. uniformis. DNA analysis of P. ruminicola B x 4 trans¬ conjugants was performed. Plasmids were isolated from P. ruminicola B,4 transconjugants by the Ish-Horowitz modification of the Birnbom and Doly procedure as described in Maniatis, et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1982) . Southern blots were also performed as described in Maniatis, et al., supra. Total DNA was prepared by standard methods as described in Saito, et al., Biochem. Biophys. Acta. 72:619-629 (1963); Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989); Shoemaker, et al., J. Bacteriol.. 166:959-965 (1986). Plasmid preparations made from P. ruminicola B,4R had a background staining material that made it difficult to see plasmid DNA unambigiously. However, when a plasmid preparation was used to transform E. coli and pRDB5 was recovered in E. coli. the restriction profile of this plasmid was identical to that of the original pRDB5. Additionally, total DNA (plasmid plus chromosome) was isolated from apparent P. ruminicola transconjugants, digested with EcoRI and subjected to Southern blot analysis. EcoRI cuts once in pRDB5 to produce a 15 kb linear segment. The DNA digests were separated on a 1.0% agarose gel and blotted onto Optibind (Schleicher and Schuell) . The digests were then probed with 32 P-labelled pFD160. The pFD160 plasmid hybridizes with the pBR328 sequences in pRDB5, but not with the Tc r gene. All of the putative transconjugants contained a single band of the correct size which hybridized with the probe (see Figure 2A) . Total DNA from B. uniformis. P. ruminicola B x 4, and P. ruminicola transconjugants were also digested with Hindlll and EcoRI restriction enzymes, and the digests - 27 - blotted onto Optibind. The blot was hybridized with labelled XBU4422: :pEG920 [prepared as described in Shoemaker, et al., J. Bacteriol., 172:1694-1702 (1990)], a probe which detects pRDB5 and the Tc r Em r 12256 element. If the apparent transconjugants were Thy + Rif r mutants of B. uniformis 1108 (pRDB5) , the Southern blot would show a number of bands, including the two bands produced from a Hindlll-EcoRI digest of pRDB5. As can be seen from Figure 2B, a mixture of bands due to pRDB5 and the TcΕm r 12256 element was seen in the B. uniformis donor, whereas only the bands associated with pRDB5 were seen in the transconjugant. These results indicated that the transconjugants were not revertants of the B. uniformis donor. The NotI restriction enzyme digest patterns of DNA from B. uniformis. P. ruminicola B,4R, and a P. ruminicola B,4 transconjugant were compared on pulsed field gels to determine whether a Tc r contaminant having properties similar to P. ruminicola had been isolated instead of true transconjugants. The NotI digest pattern of P. ruminicola B,4 differs not only from that of B. uniformis 1108, but also differs from that of other P. ruminicola strains (data not shown) . As shown in Figure 3, the NotI restriction patterns of the P. ruminicola B j 4R recipient and the Tc r transconjugant were identical to each other and to that of P. ruminicola B,4. The combined data show that true P. ruminicola transconjugants were obtained. No transfer of pNFD13-2 to P. ruminicola B,4 was detected. Since pNFD13-2 has the same Tc r gene as pRDB5 but derives its replication region from a different plasmid, the lack of transconjugants was most likely due to failure of the pNFD13-2 replication origin (pBI143) to work in P. ruminicola B,4. However, there is a 4 kbp region upstream of the Tc r gene which is present in pRDB5 but not in pNFD13-2. This region seems to have no effect on expression of the Tc r gene in B. uniformis. but it might affect expression in P. ruminicola Bj4. Since pNFD13-2 was mobilized from B. uniformis to E. coli at frequencies comparable to mobilization frequencies seen with pRDB5, it is possible that pNFD13-2 is getting into P. ruminicola Bj4 but is lost because it cannot replicate. If so, pNFD13-2 could serve as a suicide vector for introducing DNA into the chromosome of P. ruminicola B,4. Genetic manipulation of P. ruminicola would be easier if E. coli were the donor. Failure to demonstrate transfer of pRDB5 from E. coli to P. ruminicola could be due to the failure of IncP plasmids to mediate formation of mating pairs between E. coli and P. ruminicola. However, since IncP plasmids mediate mating between E. coli and the colonic Bacteroides strains, this seems unlikely. A more likely possibility is that the transfer frequency is lowered by the anaerobic mating conditions. Aerobic matings with the oxygen-sensitive P. ruminicola are not feasible. Nonetheless, it may be possible to find conditions that raise the frequency of mating and allow P. ruminicola to survive. Finally, restriction enzymes in P_j.. ruminicola may prevent survival of pRDB5 introduced from E. coli. Table 1: Transfer frequencies of various shuttle vectors from B. uniformis to either B. ruminicola B,4 or E. coli. Frequency of transfer from B. uniformis to Donor strain E. coli B. ruminicola B. uniformis 1108 (pRDB5) 3 x lO"* 10 -7_ 10 - β B. uniformis 1108 (pNFD13-2) 1 x 10" 4 < 10 -9 B. uniformis 1108 (pVALl) 1 x 10 J < IC 9 B. uniformis 1104 (pRBD3) 1 X 10 " < lo- 9 B. uniformis 1108 (pRDB5-2) b 2 x 10 -4 10 -7_ 10 -6 'Frequencies are given as transconjugants per recipient. Numbers represent the mean of at least three separate experiments. b pRDB5-2 is pRDB5 isolated from a B. ruminicola B,4 transconjugant in the B. uniformis 1108 background. EXAMPLE 2 The Bacteroides Tc r gene, originally derived from the Bacteroides conjugal element Tc r Em r -D0T [Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989)], was subcloned on a 2.7 kbp fragment , and the 2.7 kbp fragment was sequenced. The complete sequence of the fragment is shown below in Chart A. Computer analysis of the DNA sequence, translation into amino acid sequence, and comparisons to amino acid sequences of other tetracycline resistance peptides were performed. The amino acid sequence of the gene product is presented below in Chart B. A promoter region functional in Bacteroides species was identified. Its sequence is shown below in Chart C. This promoter region is also strongly believed to be sufficient to initiate transcription in P. ruminicola. The gene coded for a protein of the ribosome protection type of tetracycline resistance. However, the amino acid sequence coded for by the cloned gene was found to be only about 40% identical to sequences coded for by the TetM and TetO genes, two known classes of ribosome protection type tetracycline resistant genes. Accordingly, it was concluded that the Bacteroides Tc r was clearly in a separate DNA-DNA hybridization class from TetM and TetO and constituted its own DNA hybridization class. This new class of tetracycline resistance genes is designated TetQ. The experiments and analyses performed, and the Bacteroides Tc r gene and its gene product, will now be described in detail. A. Materials and Methods 1. Strains and growth conditions Strains used in this study are listed in Table 2. E. coli DH5α was obtained from Bethesda Research Laboratory. B. thetaiotaomicron strains BT 4001, BT4002, BT4004, BT4007 and BT4008 and B. uniformis BU10001 are described in Shoemaker and Salyers, J. Bacteriol.. 170:1651-1657 (1988). B. thetaiotaomicron strains 5482 and 2808, B. uniformis strains C7-17, 3537, Tl-1, B. distasonas strains 4243, C30-45, 6308, and B. caccae strains 3452A and 8608 are described in Johnson, J. Svst. Bacteriol.. 28:245-256 (1978). B. fragilis AK87 was obtained from A. Kuritza, Yale University Medical School, New Haven, CT. E. coli LCD44 was obtained from Dr. John Cronon, Jr., University of Illinois, Urbana, Illinois. Bacteroides strains were grown either in prereduced Trypticase (BBL Microbiology Systems)-yeast extract-glucose (TYG) [Holdeman, et al. , Anaerobe Laboratory Manual, supra1 under an 80% N 2 /20% C0 2 atmosphere or on TYG agar plates in a GasPak jar. E. coli strains were grown in Luria broth (LB) or on LB agar plates unless otherwise indicated. 2. Plasmids The preparation of pNFD13-2 is described above in Example 1. As discussed there, it contains a 2.7 kbp insert containing the Tc r gene of the TcΕm r -DOT element. Plasmid pNFD13-6 is identical to pNFD13-2, but with the 2.7 kbp insert in the opposite orientation. 3. DNA isolation and analysis Plasmids were isolated from E. coli by the Ish- Horowitz modification of the Birnboim and Doly method [Maniatis, et al., supra] . Chromosomal DNA from Bacteroides was isolated by the method of Saito and Miura, Biochim. Biophvs. Acta, 72:619-629 (1963). Restriction digestion and ligation with T4 DNA ligase followed standard procedures (Maniatis, et al., supra) . Electrophoretic resolution of restriction digests was done in 0.8-1.0% agarose slab gels in IX or 4X GGB (IX: 0.04M Tris, 0.02M sodium acetate, 0.002M EDTA). Gels were stained with ethidium bromide (l μg/ml) and photographed. Plasmids were introduced into E. coli employing the transformation procedure of Lederberg and Cohen, J. Bacteriol.. 119:1072-1074 (1974). 4. Southern hybridization For Southern blot hybridization analysis, DNA was digested with restriction enzymes and electrophoresed on a 1% agarose gel. The DNA was transferred to Millipore HAHY nitrocellulose paper by capillary blotting (Maniatis, et al., supra) . Nick translation was used to label DNA probes with [α- 32 P]-dCTP [Rigby, et al., J. Mol. Biol.. 113:237-251 (1977)]. Probes were hybridized to DNA on the nitrocellulose paper for 24 hours at 42°C in a hybridization solution containing 50% formamide (Maniatis, et al., supra) . Following hybridization, blots were washed twice for 30 minutes each with 2X SSC (0.3M NaCl and 0.03M sodium citrate) containing 0.2% sodium dodecyl sulfate (SDS) , then twice with 0.2% SDS in 0.5X SSC at 60°C. Blots were then analyzed using autoradiography. 5. Minimum Inhibitory Concentration (MIC) of tetracycline To test for expression of the 2.7 kbp clone of the Tc r gene and its various deletion derivatives in E. coli and B. thetaiotaomicron. MIC values were determined. When E. coli was the host, ampicillin (100 μg/ml) or tetracycline (3 μg/ml) was added to inoculum cultures to maintain plasmids in plasmid-bearing strains. In most experiments, MIC values were determined using the tube dilution method. Cells (0.1 ml) from overnight inoculum cultures were introduced into LB broth medium containing serially incremented concentrations of tetracycline. Increments of 5 μg/ml were used. Tubes were incubated at 37°C and scored visually for growth at 12 and 24 hours. In some experiments, the level of resistance was determined by patching cultures onto LB agar plates containing different concentrations of antibiotic and scoring growth after 24 hours. To test for expression in Bacteroides, vectors containing various subclones of the 2.7 kbp clone of the Tc r gene were mobilized into B. thetaiotaomicron as described previously [Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989); Shoemaker, et al., J. Bacteriol.. 162:626-632 (1985)], with selection for Tc r . Transfer frequencies were several logs above background. Thus, failure to obtain a Tc r transconjugant was a reasonable indication that the deletion clone failed to express Tc r in Bacteroides. In Bacteroides. MIC determinations were done in TYG broth medium with serially incremented concentrations of tetracycline. 6. Maxicells The maxicell procedure was executed as described by Sanσar, et al., J. Bacteriol.. 137:692-693 (1979), with E. coli LCD44 as host. Samples were solubilized by incubation in SDS or lithium dodecyl sulfate solubilizing solution at 37°C to avoid possible aggregation. Proteins from maxicells were separated by electrophoresis on 11% highly cross-linked SDS polyacrylamide gels as described by Hashimoto, et al.. Anal. Biochem.. 112:192-199 (1983). Following electrophoresis, gels were stained with Fast Stain (Zoion Research Inc. , Allston, MA) , dried onto filter paper under vacuum, and autoradiographed. Molecular weight markers from BRL, Gaithersburg, MD, were used for size estimation. Maxicell fractionation was performed using an adaptation of the method of Tai and Kaplan, J. Bacteriol.. 164:83-88 (1985). 7. In vitro transcription and translation Proteins encoded by plasmid templates were compared using an E. coli-derived in vitro transcription- translation system [DeVries and Zubay, Proc. Nat. Acad. Sci. USA. 57:1010-1012 (1967)] in kit form (Amersham, Arlington Heights, IL) . Radiolabeled proteins were resolved on 11% highly cross-linked SDS polyacrylamide and detected by autoradiography as described above. 8. DNA seguencing and analysis The region sequenced in this study was the 2.7 kbp SstI fragment from pNFD13-2 and pNFD13-6. Progressive unidirectional deletions were introduced into the insert DNA using an adaptation of the exonuclease III procedure of Henikoff, Gene. 28:351 (1984), provided in kit form (Erase-a-Base System by Promega, Madison, WI) . (See Figure 4) Both strands were sequenced by the dideoxy chain termination reaction with the T7 DNA polymerase variant and reagents provided in the Sequenase 2.0 kit (U.S. Biochemicals, Cleveland, OH). Computer analysis of DNA sequence, translation into amino acid sequence and comparisons to amino acid sequences of other tetracycline resistance peptides were performed using Genetics Computer Group (GCG) software (Devereux, et al., Nucl. Acids Res.. 12:387-395 (1985)) on a MicroVAX computer system. The sequences of tetracycline resistance and elongation factor genes used in this study were obtained from GenBank and are listed with accession numbers in Table 3. B. Results 1. Expression of the Bacteroides tetracycline resistance gene in E. coli The Tc r gene from B. thetaiotaomicron DOT was localized to a 2.7 kbp SstI fragment in the constructs pNFD13-2 and pNFD13-6, which contained the insert in opposite orientations. Though these plasmids were originally constructed to test for expression in Bacteroides r we examined them for expression in E. coli because it was possible that the lac promoter adjacent to the cloned SstI fragment would drive Tc r expression in E. coli. Because E. coli carrying low copy number cosmid clones of the Tc r gene did not grow on LB plates containing 5 or 10 μg/ml tetracycline, Shoemaker, et al. had reported that the Bacteroides Tc r gene did not function in E. coli [Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989)]. However, it was found that the 2.7 kbp SstI insert in the higher copy number pUC19-based vector, pFD160R, allowed E. coli to grow on LB agar plates containing 5 μg/ml tetra¬ cycline. Following pregrowth in LB containing 100 μg/ml ampicillin, E. coli DH5α carrying the SstI clone had a tetracycline MIC value of 40 μg/ml for pNFD13-2 and a value of 25 μg/ml for pNFD13-6. However, when the inoculum culture was grown in LB containing sub-inhibitory tetracycline (1 μg/ml) , differences in MIC between the - 35 - clones diminished; the MIC values for pNFD13-2 and pNFD13-6 were 50 μg/ml and 40 μg/ml, respectively. The fact that the MIC values of both orientations were com¬ parable indicated that the promoter being recognized was on the cloned fragment, and therefore was not the lac promoter. Moreover, addition of IPTG to the growth medium had no effect upon MIC levels. Interestingly, MIC values obtained on LB agar plates for E. coli bearing pNFD13-2 and pNFD13-6 were significantly lower than the values obtained in broth medium (plate MIC of 10 μg/ml for pNFD13-2) . 2. Localization of the Bacteroides Tc r gene Initially, two deletions in the 2.7 kbp SstI segment were created by digesting pNFD13-2 with EcoRV and religating to form pNFD13-2ΔRV and by digesting pNFD13-6 with EcoRI and religating to form pNFD13-6ΔRI. The MIC of DH5α bearing pNFD13-2ΔRV or pNFD13-6ΔRI was the same as that for DH5α without plasmid (2 μg/ml) . Loss of resistance in both deletions indicated that the Tc r gene spanned the internal 0.9 kbp EcoRI-EcoRV region of the SstI clone. Further localization of the gene was undertaken using exonuclease III to create progressive unidirectional deletions in the 2.7 kbp SstI insert from the pFD160 polylinker. (See Figure 4) Deletion pNFD13- 2Δ3, which extended from the right to within 100 bp of the EcoRI site, did not affect resistance in E. coli. Deletion pNFD13-2Δ4, which extended to within 50 bp of the EcoRI site, decreased the MIC without completely eliminating resistance. Deletions into or through the EcoRI site abolished Tc r in E. coli. Deletion pNFD13-6Δl, which extended 200 bp into the other end of the SstI fragment, also abolished Tc r . Thus, it appeared that the genetic information essential for Tc r expression in E. coli spanned a 2.1 kbp region in the SstI insert DNA. A larger region was required for Tc r expression in Bacteroides than in E. coli. Deletion construct pNFD13-2Δ3, which conferred full resistance on E. coli. did not confer resistance on Bacteroides. The largest of the exonuclease III deletions from the right which retained full Tc r activity in Bacteroides was pNFD13-2Δ2. Thus, it appeared that an additional region of approxima¬ tely 200 bp was required for expression in Bacteroides. 3. Size and cellular location of the Tc r gene product In maxicell experiments, two major proteins were associated with the cloned 2.7 kbp SstI fragment. These were estimated to have molecular weights of 76 and 25.5 kDa (data not shown) . The two proteins were also seen when pNFD13-2 and its deletion derivatives were used as templates in an in vitro transcription-translation system. (See Figure 5A) Appearance of the 76 kDa band coincided with Tc r expression in E. coli. That is, the 76 kDa band was present in deletions that still conferred resistance on E. coli (pNFD13-2Δl, pNFD13-2Δ3) , was consistently fainter in the deletion which conferred reduced resistance (pNFD13-2Δ4) , and was missing in the Tc s deletions (pNFD13- 2Δ5, pNFD13-2Δ6) . By contrast, the 25.5 kDa band was produced from the Tc s deletions pNFD13-2Δ5 and pNFD13-2Δ6. The Tc s deletion in pNFD13-2ΔRV resulted in the loss of both of the major proteins associated with the SstI insert. Some additional proteins that were unique to the SstI clone were seen with the in vitro transcription- translation system, but these were also present in the Tc s deletions pNFD13-2Δ5 and pNFD13-2Δ6. Moreover, these proteins were not seen in the maxicell extracts. Cellular localization of the 76 kDa band by fractionation of maxicell extracts indicated that this protein partitioned predominately with the soluble fraction. (See Figure 5B) However, a portion of the - 37 - protein partitioned with the membrane fraction. The 25.5 kDa band clearly partitioned with the membrane fraction. 4. Relatedness to other Bacteroides tetracycline resistances Tetracycline resistance has been found to be widespread among strains of colonic Bacteroides. Previous hybridization studies of the Tc r conjugal elements resident in different Tc r colonic Bacteroides isolates have revealed extensive DNA hybridization [Shoemaker, et al., J. Bacteriol.. 171:1294-1302 (1989)]. To determine if the Tc r genes in other clinical strains were similar to the Tc r gene from B. thetaiotaomicron DOT, Southern hybridization was performed using the internal 0.9 kbp EcoRI-EcoRV segment of the Tc r gene to probe chromosomal DNA preparations digested with EcoRV and EcoRI. The Tc r strains analyzed were clinical isolates of B. fragilis. B. thetaiotaomicron r B. uniformis. Bacteroides caccae f and Bacteroides distasonas from the continental U.S., Hawaii and Japan. The 0.9 kbp probe hybridized with a 0.9 kbp band in all but one of the Tc r isolates probed. The only exception to this was B. distasonas C30-45, in which the probe hybridized to a fragment much larger than 0.9 kbp. This could be due to the modification or loss of one of the two restriction sites in C30-45. In another B. distasonas isolate, 6308, the probe hybridized strongly to a 0.9 kbp band. B. fragilis V479 exhibited weak hybridization relative to the other strains, but the cross-hybridizing band was the same 0.9 kbp size as the probe. The probe did not hybridize to DNA from Tc s type strain controls. These results indicated that the gene cloned in the 2.7 kbp SstI fragment is widespread among clinical isolates of colonic Bacteroides species. Given the stringency used in these experiments, it is estimated that the Tc r genes found in other Tc r strains of Bacteroides share at least 80% identity with the Tc r gene from B. thetaiotaomicron DOT. 5. DNA Sequence The DNA sequence of the 2.7 kbp SstI fragment was obtained. The sequence of the entire fragment is presented in Chart A below. Only one open reading frame within the SstI clone was sufficiently large to encode a protein of the estimated 76 kDa. (See Figure 4) All other open reading frames in the fragment were less than 400 bp. The start codon of the large open reading frame was 22 bp to the right of the EcoRI site in Figure 1. The open reading frame spanned the 0.9 kbp EcoRI-EcoRV region, which was determined to be internal to the Tc r gene. The location and extent of the open reading frame were also consistent with the exonuclease III deletion results. No additional open reading frames were found that might encode the 25.5 kDa protein seen in maxicells and in vitro transcription- translation. Presumably this protein was produced by a fusion between insert and vector DNA. The TetQ open reading frame codes for a protein of 642 amino acids (deduced molecular weight, 72,100 Da). The amino acid sequence of the protein is given below in Chart B. The tetQ gene had 40.1 mol% G+C, compared to 42 mol% G+C of the chromosome of B. thetaiotaomicron. the species from which the Tc r gene was cloned [Johnson, J. Svst. Bacteriol.. 28:245-256 (1979)]. 6. Relatedness to previously sequenced Tc r proteins The length of the deduced Bacteroides Tc r protein was similar to the lengths of proteins encoded by tetM and tetO [Martin, et al., Nucl. Acids Res. f 14:7047-7058 (1986); Nesin, et al., Antimicrob. Agents Chemother.. in press; Sanchez-Pescador, et al., Nucl. Acids Res. , 16:1216-1217 (1988); LeBlanc, et al., J. Bacteriol.. 170:3618-3626 (1988); Manavathu, et al.. Gene. 62:17-26 (1988) ] , which range from 638 amino acids to 640 amino acids. Comparisons of the Bacteroides Tc r amino acid sequence to those of TetM and TetO revealed extensive regions of similarity. (See Figure 6) However, the amino acid sequence of the Bacteroides Tc r protein was less closely related to the amino acid sequences of TetM and TetO (40.1 - 40.3% identity) than these sequences are to each other (75.6 - 76.9% identity; Table 4). In these comparisons, clusters of identity extended over the length of the alignment, but were concentrated in the amino- terminal region. The amino acid sequence of the Bacteroides Tc r protein had no significant similarity to those of sequenced Tc r genes belonging either to the efflux or to the tetracycline detoxification classes of resistance. The results of these comparisons indicated that the Bacteroides Tc r gene was likely to be a member of the ribosome protection class of Tc r , but was clearly in a different hybridization class from TetM and TetO. Accord¬ ingly, we have designated this new class TetQ. A hydrophobicity plot generated from the deduced amino acid sequence of TetQ was very similar to those generated for TetM and TetO. Since TetM and TetO are thought to be soluble proteins that function in the cytoplasm [Burdett, J. Bacteriol.. 165:564-569 (1986); Manavathu, et al., Antimicrob. Agents Chemother.. 34:71-77 (1990)], this suggests that TetQ is also a soluble protein. However, TetQ contained a relatively hydrophobic internal region (residues 205 - 247) that was ^not extant in TetM or TetO. This could explain why a portion of the Bacteroides Tc r protein fractionated with the membrane in maxicell separations. 7. Upstream region of tetQ The DNA sequence of the upstream region of tetQ is shown in Figure 7. An E. coli-like promoter sequence was found immediately upstream of the start of the open reading frame. The deletions in pNFD13-2Δl through PNFD13-2Δ3, which did not affect the tetracycline MIC in E. coli. left this promoter sequence intact. pNFD13-2Δ4, in which the -35 region of this promoter was deleted, reduced the MIC in E. coli. pNFD13-2Δ5, in which both the -35 and the -10 region of this promoter were deleted, abolished resistance in E. coli. Thus, the E. coli-like -10 and -35 regions probably constitute the promoter that is driving transcription in E. coli. Interestingly, this region was not sufficient for expression in Bacteroides. as evidenced by the observation that pNFD13-2Δ3 did not confer resistance on Bacteroides. The largest deletion that was still active in Bacteroides (pNFD13-2Δ2) contained the E. coli promoter plus about an additional 150 bp. The sequence of the smallest promoter region identified as functional in Bacteroides species is presented in Chart C below. The upstream regions of tetM and tetO genes showed remarkable sequence similarity. This region contained the putative Gram-positive ribosome binding site [Martin, et al. , Nucl. Acids Res.. 14:7047-7058 (1986)]. A comparison of the upstream region of tetM/O to that of tetQ disclosed no detectable similarity. (See Figure 6) The tetQ upstream region also lacked a distinguishable ribosome binding site. 8. Relatedness to Tc r of pRRI4 Plasmid pNFD13-2 labeled with P 32 was used as a probe to hybridize to pRRI4 digested with EcoRI , PvuII- HincII-EcoRV and Neil. Plasmid pRRI4 in P. ruminicola 223/M2/7 was obtained from Dr. Harry J. Flint, Rowett Research Institute, Bucksburn, Aberdeen, U.K. It was extracted from P. ruminicola 223/M2/7 by standard techniques [Maniatis, et al., supra1. A cross-hybridizing region was identified. To ascertain if this cross- hybridizing region contained the Tc r gene, a 5 kbp HincII- Pvuii segment which covers this region was cloned into pFD160 and mobilized from E. coli into B. uniformis. The resulting transconjugants were Tc r . Other hybridization experiments also indicated that the Tc r gene on pRRI4 was at least 80% homologous to the Tc r genes on pNFD13-2 and other Bacteroides Tc r Em r elements. Recently, sequencing of the Tc r gene on pRRI4 has been completed. Its sequence has been found to be 97% identical to that of the Tc r gene on pNFD13-2. Accordingly, it is in the TetQ class. C. Discussion By size and amino acid sequence similarity, the Bacteroides TetQ appeared to be a ribosome protection type of tetracycline resistance. However, TetQ clearly did not belong in either class TetM or class TetO because the amino acid identity with those classes is only 40.3 - 40.9%. All Tc r Bacteroides strains that we screened had DNA which hybridized to an internal fragment of the cloned Tc r gene under conditions of high stringency. Thus, TetQ is probably the predominant Tc r among the colonic Bacteroides. In fact, recent evidence indicates that Tc r determinants from colonic and oral Bacteroides have high similarity [Guiney and Bouic, J. Bacteriol.. 172:495-497 (1990) ] . This suggests that TetQ may be ubiquitous in the genus Bacteroides. Also, at least one P. ruminicola Tc r gene is of the TetQ class. Previously sequenced ribosome protection Tc r genes were remarkable for their sequence similarity (Table 4) . For instance, the TetO found in Campylobacter ieiuni shared 98.1% intraclass amino acid identity with the TetO found in Streptococcus mutans. The C. ήeiuni TetO shared 75.1 - 76.8% interclass amino acid identity with the TetM's found in Staphylococcus. Streptococcus, and Ureaplasma. Genes that are similar enough to cross- hybridize with tetM and tetO on Southern blots have been found in Clostridium. Eikenella. Fusobacterium. Gardnerella. Hemophilus. Kingella. Mycoplasma, Neisseria. and Veillonella [Salyers, et al., Mol. Microbiol.. 4:151- 156 (1989)]. The amino-terminal regions of TetM and TetO have high amino acid similarity to the amino-terminal region of the elongation factors [Sanchez-Pescador, et al., Nucl. Acids Res. , 16, 1218 (1988); Manavathu et al., Antimicrob. Agents Chemother.. 34:71-77 (1990)]. This region is responsible for guanosine nucleotide binding by one elongation factor (EF-Tu) [Jacquet and Parmeggiani, The EMBO J. , 7:2861-2867 (1988); Jurnak, Science. 230:32-36 (1985) ], and is conserved in GTP-binding proteins [Halliday, J. Nucleotide Prot. Phosphoryl. Res., 9:435-448 (1984)]. Though TetQ is the most diverged ribosome protection Tc r , it maintains high amino acid conservation in this GDP/GTP-binding domain. (See Figure 6) This indicates that this functional domain may be involved directly in the ribosome protection resistance mechanism. Manavathu, et al., Antimicrob. Agents Chemother. , 34:71-77 (1990) , suggested that TetO may have the potential to bind GTP, but no such binding has yet been demonstrated. The upstream regions of tetM and tetO genes, which are virtually identical, contain a Gram-positive ribosome binding site. The mol% G+C of tetQ (40.1%) is similar to that of tetM and tetO, but is also similar to the mol% G+C of chromosomal DNA from colonic Bacteroides [39 - 46%; Johnson, J. Svst. Bacteriol.. 28:245-256 (1978) ] . By contrast, the upstream region of tetQ is completely different from that of tetM and tetO. - 43 - Table 2: Strains Used In Connection With Sequencing Of Tc r Strain or Relevant Plasmid Phenotype E. coli strains DH5α RecA Δ (argF-lacA)U169 ø80dlacΔM15 LCD44 RecA MetE Tc s derivative of RK5173. Bacteroides strains B. thetaoitaomicron 5482 BT4001 Tc s Em s ; Spontaneous Rif derivative of B. thetaiotaomicron 5482 'Resistance phenotype expressed in E. coli is indicated in parenthesis. Table 3: GenBank Access Codes For Seguences Source organism Staphylococcus aureus Streptococcus faecalis Ureaplasma urealyticum Campylobacter ieiuni Streptococcus mutans Escherichia coli Micrococcus luteus Spirulina platensis Thermus thermophilus EF-Tu Tthtufl X05977 EF-G X16278 X16278 Thermotoga maritima EF-Tu Tmoeftu M27479 Euglena gracilis chloroplast EF-TU Egrcpeftu X00044 Methanococcus vannielii EF-1 Mvatuf X05698 EF-2 Mvafus X12384 Saccharomyces cerevisiae EF-lα Yscefla X00779 Mucor racemonsus EF-lα Mratefla J02605 Dictyostelium discoideum EF-2 Ddief2 M26017 Drosophila melanogaster EF-2 X15805 X15805 Xenopus laevis EF-lα Xeleflal M25697 Mesocricetus sp. Mus musculus Rattus norvegicus Homo sapiens Table 4: Percent amino acid similarity and percent amino acid identity between deduced peptide sequences of ribosome protection tetracycline resistance genes percent similarity 1 2 3 4 percent identity Chart A GAGCTCTAAA TTTAAATATA AACAACGAAT TATCTCCTTA ACGTACGTTT 50 TCGTTCCATT GGCCCTCAAA CCCCGTTATA TACATTCATG TCCATTTATG 100 TAAAAAATCC TGCTGACCTT GTTTATGTCT TGTCAGTCAC CATTTGCAAA 150 ACCATATTTG ACCCTCAAAG AGGCTGAATT TGATAAGCAA CTTGCTACAT 200 ACTCATAATA AGGAGCTAAA TAGAACACGA ATGGGAAATA CTCAAATGCC 250 AAACTAAAGA AGATATTGGC CAAAATAAAC GCTATACCGA GAGAGAAACT 300 TGATTTTTCA ACTTCCTAAA ACAGTGTTGT TCAAACATTT CTACTTATTT 350 GTACTTACCA GTTGAACCTA CGTTTCCCTA ATAAAATGTC TATGGTAAAA 400 AGTTAAAAAA TCCTCCTACT TTTGTTAGAT ATATTTTTTT GTGTAATTTT 450 GTAATCGTTA TGCGGCAGTA ATAATATACA TATTAATACG AGTTATTAAT 500 CCTGTAGTTC TCATATGCTA CGAGGAGGTA TTAAAAGGTG CGTTTCGACA 550 ATGCATCTAT TGTAGTATAT TATTGCTTAA TCCAA ATG AAT ATT ATA 597 Met Asn lie lie AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA GGA AAA ACT TCC 639 Asn Leu Gly lie Leu Ala His lie Asp Ala Gly Lys Thr Ser 5 10 15 GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA GCA ACG GAA AAG 681 Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr Glu Lys 20 25 30 TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA ACG GAC TCT ATG 723 Cys Gly Cys Val Asp Asn Gly Asp Thr lie Thr Asp Ser Met 35 40 45 GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT CGG GCT TCT ACG 765 Asp lie Glu Lys Arg Arg Gly lie Thr Val Arg Ala Ser Thr 50 55 60 ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC AAT ATC ATT GAC 807 Thr Ser lie lie Trp Asn Gly Val Lys Cys Asn lie lie Asp 65 70 ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA GTG GAG CGG ACA 849 Thr Pro Gly His Met Asp Phe lie Ala Glu Val Glu Arg Thr 75 80 85 TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC TTA TCC GCA AAG 891 Phe Lys Met Leu Asp Gly Ala Val Leu lie Leu Ser Ala Lys 90 95 100 GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG TTC AAT ACT TTA 933 Glu Gly lie Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu 105 110 115 CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT ATC AAT AAG ATT 975 Gin Lys Leu Gin lie Pro Thr lie lie Phe lie Asn Lys lie 120 125 130 GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG TAT CTG GAT ATA 1017 Asp Arg Ala Gly Val Asn Leu Glu Arg Leu Tyr Leu Asp lie 135 140 AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT ATG CAA AAT GTT 1059 Lys Ala Asn Leu Ser Gin Asp Val Leu Phe Met Gin Asn Val 145 150 155 GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC CAA ACA TAT ATA 1101 Val Asp Gly Ser Val Tyr Pro Val Cys Ser Gin Thr Tyr lie 160 165 170 AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC CAT GAC GAC AAT 1143 Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn His Asp Asp Asn 175 180 185 ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA ATT TCA CCG GCT 1185 lie Leu Glu Arg Tyr Leu Ala Asp Ser Glu lie Ser Pro Ala 190 195 200 GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG GCA AAA GCC AAA 1227 Asp Tyr Trp Asn Thr lie lie Ala Leu Val Ala Lys Ala Lys 205 210 GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG TTC AAT ATC GGT 1269 Val Tyr Pro Val Leu His Gly Ser Ala Met Phe Asn lie Gly 215 . 220 225 ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT TTT ATA CTT CCT 1311 lie Asn Glu Leu Leu Asp Ala lie Thr Ser Phe lie Leu Pro 230 235 240 CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT TAT CTT TAT AAG 1353 Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr Lys 245 250 255 ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA AGT TTT CTA AAA 1395 lie Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC GTT GTA AGA ATC 1437 lie lie Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg lie 275 280 AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA AAT CTA AAA ACT 1479 Asn Asp Ser Glu Lys Phe lie Lys lie Lys Asn Leu Lys Thr 285 290 295 ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT GAA GTG GGC GCC 1521 lie Asn Gin Gly Arg Glu lie Asn Val Asp Glu Val Gly Ala 300 305 310 AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT GAT TTT CGA ATC 1563 Asn Asp lie Ala lie Val Glu Asp Met Asp Asp Phe Arg lie 315 320 325 GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG ATT CAA GGA TTA 1605 Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu lie Gin Gly Leu 330 335 340 TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC GTC CGG CCA GAC 1647 Ser His Gin His Pro Ala Leu Lys Ser Ser Val Arg Pro Asp 345 350 AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC GCT CTG AAT ACA 1689 Arg Pro Glu Glu Arg Ser Lys Val lie Ser Ala Leu Asn Thr 355 360 365 TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT TCC ATA AAC TCA 1731 Leu Trp lie Glu Asp Pro Ser Leu Ser Phe Ser lie Asn Ser 370 375 380 TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT GGT TTA ACC CAA 1773 Tyr Ser Asp Glu Leu Glu lie Ser Leu Tyr Gly Leu Thr Gin 385 390 395 AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA CGA TTT TCC GTA 1815 Lys Glu lie lie Gin Thr Leu Leu Glu Glu Arg Phe Ser Val 400 405 410 AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA TAC AAA GAA GGA 1857 Lys Val His Phe Asp Glu lie Lys Thr lie Tyr Lys Glu Arg 415 420 CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG ATC GAA GTG CCG 1899 Pro Val Lys Lys Val Asn Lys lie lie Gin lie Glu Val Pro 425 430 435 CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG ACT CTT GAT CCC 1941 Pro Asn Pro Tyr Trp Ala Thr lie Gly Leu Thr Leu Glu Pro 440 445 450 TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA AGT GAC ATC TCC 1983 Leu Pro Leu Gly Thr Gly Leu Gin lie Glu Ser Asp lie Ser 455 460 465 TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT GCC GTT TTT GAA 2025 Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA CAT GGA TGG GAA 2067 Gly lie Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu 485 490 GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA GCC GAG TAT TAT 2109 Val Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr 495 500 505 AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA CAG CTG ACC CCT 2151 Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro 510 515 520 TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA GGT GTG GAC ATT 2193 Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser Gly Val Asp lie 525 530 535 CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG ATA CCC CAA GCG 2235 Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin lie Pro Gin Ala 540 545 550 GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA AAA ATG ATG TCT 2277 Ala Ser Ser Lys Ala lie Thr Asp Leu Gin Lys Met Met Ser 555 560 GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG TGG TGT CAT ATT 2319 Glu lie Glu Asp lie Ser Cys Asn Asn Glu Trp Cys His lie 565 570 575 AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA GAC TAT GCA TCA 2361 Lys Gly Lys Val Pro Leu Asn Thr Ser Lys Asp Tyr Ala Ser 580 585 590 GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC ATT TTT ATG GTT 2403 Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly lie Phe Met Val 595 600 605 AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC GGT TAT TCT GAT 2445 Lys Pro Cys Gly Tyr Gin lie Thr Lys Gly Gly Tyr Ser Asp 610 615 620 AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT TTA TTC ATG TTC 2487 Asn lie Arg Met Asn Glu Lys Asp Lys Leu Leu Phe Met Phe 625 630 CAA AAA TCA ATG TCA TCA AAA TAATGGAGCG GTCAGGAAAT 2528 Gin Lys Ser Met Ser Ser Lys 635 640 TTCTATAAGG CAATACAGTT GGGATATATA CTTATCTCCA TTCTTATCGG 2578 ATGTATGGCA TATAATAGCC TCTATGAATG GCAGGAGATA GAAGCATTAG 2628 AACTTGGCAA TAAAAAAATA GACGAGCTC 2657 [seq id no 2] chart B Met Asn lie lie Asn Leu Gly lie Leu Ala His lie Asp Ala Gly 5 10 15 Lys Thr Ser Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr 20 25 30 Glu Lys Cys Gly Cys Val Asp Asn Gly Asp Thr lie Thr Asp Ser 35 40 45 Met Asp lie Glu Lys Arg Arg Gly lie Thr Val Arg Ala Ser Thr 50 55 60 Thr Ser lie lie Trp Asn Gly Val Lys Cys Asn lie lie Asp Thr 65 70 75 Pro Gly His Met Asp Phe lie Ala Glu Val Glu Arg Thr Phe Lys 80 85 90 Met Leu Asp Gly Ala Val Leu lie Leu Ser Ala Lys Glu Gly lie 95 100 105 Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu Gin Lys Leu Gin 110 115 120 lie Pro Thr lie lie Phe lie Asn Lys lie Asp Arg Ala Gly Val 125 130 135 Asn Leu Glu Arg Leu Tyr Leu Asp lie Lys Ala Asn Leu Ser Gin 140 145 150 Asp Val Leu Phe Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro 155 160 165 Val Cys Ser Gin Thr Tyr lie Lys Glu Glu Tyr Lys Glu Phe Val 170 175 180 Cys Asn His Asp Asp Asn lie Leu Glu Arg Tyr Leu Ala Asp Ser 185 190 195 Glu lie Ser Pro Ala Asp Tyr Trp Asn Thr lie lie Ala Leu Val 200 205 210 Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met Phe 215 220 225 Asn He Gly He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He 230 235 240 Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr 245 250 255 Lys He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He Asn 275 280 285 Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr He Asn 290 295 300 Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala Asn Asp He 305 310 315 Ala He Val Glu Asp Met Asp Asp Phe Arg He Gly Asn Tyr Leu 320 325 330 Gly Ala Glu Pro Cys Leu He Gin Gly Leu Ser His Gin His Pro 335 340 345 Ala Leu Lys Ser Ser Val Arg Pro Asp Arg Pro Glu Glu Arg Ser 350 355 360 Lys Val He Ser Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser 365 370 375 Leu Ser Phe Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser 380 385 390 Leu Tyr Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu 395 400 405 Glu Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin He 425 430 435 Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu 440 445 450 Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He 455 460 465 Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu Val 485 490 495 Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr Ser Pro 500 505 510 Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro Tyr Val Phe 515 520 525 Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He Leu Glu Pro Met 530 535 540 Leu Tyr Phe Glu Leu Gin He Pro Gin Ala Ala Ser Ser Lys Ala 545 550 555 He Thr Asp Leu Gin Lys Met Met Ser Glu He Glu Asp He Ser 560 565 570 Cys Asn Asn Glu Trp Cys His He Lys Gly Lys Val Pro Leu Asn 575 580 585 Thr Ser Lys Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly 590 595 600 Leu Gly He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys 605 610 615 Gly Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 [seq id no 3] Chart C AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA, 180 [seq id no 1] SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Salyers, Abigail A., Shoemaker, Nadja B. , Nikolich, Mikeljon P. (ii) TITLE OF INVENTION: Method and Materials For Introducing DNA Into Prevotella ruminicola (iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Willian Brinks Olds Hofer Gilson and Lione (B) STREET: P.O. Box 10395 (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: USA (F) ZIP: 60610 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMPUTER: IBM XT (C) OPERATING SYSTEM: MS-DOS 3.31 (D) SOFTWARE: WordPerfect 5.1 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 07/718,535 (B) FILING DATE: 05-JUN-1991 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Crook, Wannell M. (B) REGISTRATION NUMBER: 31,071 (C) REFERENCE/DOCKET NUMBER: 3617/22 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (312) 321-4200 (B) TELEFAX: (312) 321-4299 (2) INFORMATION FOR SEQUENCE ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:double stranded (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA 180 SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2657 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAGCTCTAAA TTTAAATATA AACAACGAAT TATCTCCTTA ACGTACGTTT 50 TCGTTCCATT GGCCCTCAAA CCCCGTTATA TACATTCATG TCCATTTATG 100 TAAAAAATCC TGCTGACCTT GTTTATGTCT TGTCAGTCAC CATTTGCAAA 150 ACCATATTTG ACCCTCAAAG AGGCTGAATT TGATAAGCAA CTTGCTACAT 200 ACTCATAATA AGGAGCTAAA TAGAACACGA ATGGGAAATA CTCAAATGCC 250 AAACTAAAGA AGATATTGGC CAAAATAAAC GCTATACCGA GAGAGAAACT 300 TGATTTTTCA ACTTCCTAAA ACAGTGTTGT TCAAACATTT CTACTTATTT 350 GTACTTACCA GTTGAACCTA CGTTTCCCTA ATAAAATGTC TATGGTAAAA 400 AGTTAAAAAA TCCTCCTACT TTTGTTAGAT ATATTTTTTT GTGTAATTTT 450 GTAATCGTTA TGCGGCAGTA ATAATATACA TATTAATACG AGTTATTAAT 500 CCTGTAGTTC TCATATGCTA CGAGGAGGTA TTAAAAGGTG CGTTTCGACA 550 ATGCATCTAT TGTAGTATAT TATTGCTTAA TCCAA ATG AAT ATT ATA 597 Met Asn He He AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA GGA AAA ACT TCC 639 Asn Leu Gly He Leu Ala His He Asp Ala Gly Lys Thr Ser 5 10 15 GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA GCA ACG GAA AAG 681 Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr Glu Lys 20 25 30 TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA ACG GAC TCT ATG 723 Cys Gly Cys Val Asp Asn Gly Asp Thr He Thr Asp Ser Met 35 40 45 GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT CGG GCT TCT ACG 765 Asp He Glu Lys Arg Arg Gly He Thr Val Arg Ala Ser Thr 50 55 60 SUBSTITUT SHEE1 ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC AAT ATC ATT GAC 807 Thr Ser He He Trp Asn Gly Val Lys Cys Asn He He Asp 65 70 ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA GTG GAG CGG ACA 849 Thr Pro Gly His Met Asp Phe He Ala Glu Val Glu Arg Thr 75 80 85 TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC TTA TCC GCA AAG 891 Phe Lys Met Leu Asp Gly Ala Val Leu He Leu Ser Ala Lys 90 95 100 GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG TTC AAT ACT TTA 933 Glu Gly He Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu 105 110 115 CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT ATC AAT AAG ATT 975 Gin Lys Leu Gin He Pro Thr He He Phe He Asn Lys He 120 125 130 GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG TAT CTG GAT ATA 1017 Asp Arg Ala Gly Val Asn Leu Glu Arg Leu Tyr Leu Asp He 135 140 AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT ATG CAA AAT GTT 1059 Lys Ala Asn Leu Ser Gin Asp Val Leu Phe Met Gin Asn Val 145 150 155 GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC CAA ACA TAT ATA 1101 Val Asp Gly Ser Val Tyr Pro Val Cys Ser Gin Thr Tyr He 160 165 170 AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC CAT GAC GAC AAT 1143 Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn His Asp Asp Asn 175 180 185 ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA ATT TCA CCG GCT 1185 He Leu Glu Arg Tyr Leu Ala Asp Ser Glu He Ser Pro Ala 190 195 200 GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG GCA AAA GCC AAA 1227 Asp Tyr Trp Asn Thr He He Ala Leu Val Ala Lys Ala Lys 205 210 GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG TTC AAT ATC GGT 1269 Val Tyr Pro Val Leu His Gly Ser Ala Met Phe Asn He Gly 215 220 225 ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT TTT ATA CTT CCT 1311 He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He Leu Pro 230 235 240 SUBSTITUTESHEET CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT TAT CTT TAT AAG 1353 Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr Lys 245 250 255 ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA AGT TTT CTA AAA 1395 He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC GTT GTA AGA ATC 1437 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He 275 280 AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA AAT CTA AAA ACT 1479 Asn Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr 285 290 295 ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT GAA GTG GGC GCC 1521 He Asn Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala 300 305 310 AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT GAT TTT CGA ATC 1563 Asn Asp He Ala He Val Glu Asp Met Asp Asp Phe Arg He 315 320 325 GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG ATT CAA GGA TTA 1605 Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu He Gin Gly Leu 330 335 340 TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC GTC CGG CCA GAC 1647 Ser His Gin His Pro Ala Leu Lys Ser Ser Val Arg Pro Asp 345 350 AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC GCT CTG AAT ACA 1689 Arg Pro Glu Glu Arg Ser Lys Val He Ser Ala Leu Asn Thr 355 360 365 TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT TCC ATA AAC TCA 1731 Leu Trp He Glu Asp Pro Ser Leu Ser Phe Ser He Asn Ser 370 375 380 TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT GGT TTA ACC CAA 1773 Tyr Ser Asp Glu Leu Glu He Ser Leu Tyr Gly Leu Thr Gin 385 390 395 AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA CGA TTT TCC GTA 1815 Lys Glu He He Gin Thr Leu Leu Glu Glu Arg Phe Ser Val 400 405 410 AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA TAC AAA GAA GGA 1857 Lys Val His Phe Asp Glu He Lys Thr He Tyr Lys Glu Arg 415 420 CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG ATC GAA GTG CCG 1899 Pro Val Lys Lys Val Asn Lys He He Gin He Glu Val Pro 425 430 435 CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG ACT CTT GAT CCC 1941 Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu Glu Pro 440 445 450 TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA AGT GAC ATC TCC 1983 Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He Ser 455 460 465 TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT GCC GTT TTT GAA 2025 Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA CAT GGA TGG GAA 2067 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu 485 490 GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA GCC GAG TAT TAT 2109 Val Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr 495 500 505 AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA CAG CTG ACC CCT 2151 Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro 510 515 520 TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA GGT GTG GAC ATT 2193 Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He 525 530 535 CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG ATA CCC CAA GCG 2235 Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin He Pro Gin Ala 540 545 550 GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA AAA ATG ATG TCT 2277 Ala Ser Ser Lys Ala He Thr Asp Leu Gin Lys Met Met Ser 555 560 GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG TGG TGT CAT ATT 2319 Glu He Glu Asp He Ser Cys Asn Asn Glu Trp Cys His He 565 570 575 AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA GAC TAT GCA TCA 2361 Lys Gly Lys Val Pro Leu Asn Thr Ser Lys Asp Tyr Ala Ser 580 585 590 GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC ATT TTT ATG GTT 2403 Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly He Phe Met Val 595 600 605 SUBSTITU E SK AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC GGT TAT TCT GAT 2445 Lys Pro Cys Gly Tyr Gin He Thr Lys Gly Gly Tyr Ser Asp 610 615 620 AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT TTA TTC ATG TTC 2487 Asn He Arg Met Asn Glu Lys Asp Lys Leu Leu Phe Met Phe 625 630 CAA AAA TCA ATG TCA TCA AAA TAATGGAGCG GTCAGGAAAT 2528 Gin Lys Ser Met Ser Ser Lys 635 640 TTCTATAAGG CAATACAGTT GGGATATATA CTTATCTCCA TTCTTATCGG 2578 ATGTATGGCA TATAATAGCC TCTATGAATG GCAGGAGATA GAAGCATTAG 2628 AACTTGGCAA TAAAAAAATA GACGAGCTC 2657 (2) INFORMATION FOR SEQUENCE ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 641 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: SUBSTITUTE SHEET He Pro Thr He He Phe He Asn Lys He Asp Arg Ala Gly Val 125 130 135 Asn Leu Glu Arg Leu Tyr Leu Asp He Lys Ala Asn Leu Ser Gin 140 145 150 Asp Val Leu Phe Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro 155 160 165 Val Cys Ser Gin Thr Tyr He Lys Glu Glu Tyr Lys Glu Phe Val 170 175 180 Cys Asn His Asp Asp Asn He Leu Glu Arg Tyr Leu Ala Asp Ser 185 190 195 Glu He Ser Pro Ala Asp Tyr Trp Asn Thr He He Ala Leu Val 200 205 210 Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met Phe 215 220 225 Asn He Gly He.Asn Glu Leu Leu Asp Ala He Thr Ser Phe He 230 235 240 Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr 245 250 255 Lys He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He Asn 275 280 285 Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr He Asn 290 295 300 Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala Asn Asp He 305 310 315 Ala He Val Glu Asp Met Asp Asp Phe Arg He Gly Asn Tyr Leu 320 325 330 Gly Ala Glu Pro Cys Leu He Gin Gly Leu Ser His Gin His Pro 335 340 345 Ala Leu Lys Ser Ser Val Arg Pro Asp Arg Pro Glu Glu Arg Ser 350 355 360 Lys Val He Ser Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser 365 370 375 Leu Ser Phe Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser 380 385 390 SUBS ITUTE Leu Tyr Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu 395 400 405 Glu Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin He 425 430 435 Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu 440 445 450 Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He 455 460 465 Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu Val 485 490 495 Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr Ser Pro 500 505 510 Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro Tyr Val Phe 515 520 525 Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He Leu Glu Pro Met 530 535 540 Leu Tyr Phe Glu Leu Gin He Pro Gin Ala Ala Ser Ser Lys Ala 545 550 555 He Thr Asp Leu Gin Lys Met Met Ser Glu He Glu Asp He Ser 560 565 570 Cys Asn Asn Glu Trp Cys His He Lys Gly Lys Val Pro Leu Asn 575 580 585 Thr Ser Lys Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly 590 595 600 Leu Gly He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys 605 610 615 Gly Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2106 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA GGAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA ATG AAT ATT ATA 192 Met Asn He He AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA GGA AAA ACT TCC 234 Asn Leu Gly He Leu Ala His He Asp Ala Gly Lys Thr Ser 5 10 15 GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA GCA ACG GAA AAG 276 Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr Glu Lys 20 25 30 TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA ACG GAC TCT ATG 318 Cys Gly Cys Val Asp Asn Gly Asp Thr He Thr Asp Ser Met 35 40 45 GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT CGG GCT TCT ACG 360 Asp He Glu Lys Arg Arg Gly He Thr Val Arg Ala Ser Thr 50 55 60 ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC AAT ATC ATT GAC 402 Thr Ser He He Trp Asn Gly Val Lys Cys Asn He He Asp 65 70 ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA GTG GAG CGG ACA 444 Thr Pro Gly His Met Asp Phe He Ala Glu Val Glu Arg Thr 75 80 85 TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC TTA TCC GCA AAG 486 Phe Lys Met Leu Asp Gly Ala Val Leu He Leu Ser Ala Lys 90 95 100 GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG TTC AAT ACT TTA 528 Glu Gly He Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu 105 110 115 SUBS JT ≤ CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT ATC AAT AAG ATT 570 Gin Lys Leu Gin He Pro Thr He He Phe He Asn Lys He 120 125 130 GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG TAT CTG GAT ATA 612 Asp Arg Ala Gly Val Asn Leu Glu Arg Leu Tyr Leu Asp He 135 140 AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT ATG CAA AAT GTT 654 Lys Ala Asn Leu Ser Gin Asp Val Leu Phe Met Gin Asn Val 145 150 155 GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC CAA ACA TAT ATA 696 Val Asp Gly Ser Val Tyr Pro Val Cys Ser Gin Thr Tyr He 160 165 170 AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC CAT GAC GAC AAT 738 Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn His Asp Asp Asn 175 180 185 ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA ATT TCA CCG GCT 780 He Leu Glu Arg Tyr Leu Ala Asp Ser Glu He Ser Pro Ala 190 195 200 GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG GCA AAA GCC AAA 822 Asp Tyr Trp Asn Thr He He Ala Leu Val Ala Lys Ala Lys 205 210 GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG TTC AAT ATC GGT 864 Val Tyr Pro Val Leu His Gly Ser Ala Met Phe Asn He Gly 215 220 225 ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT TTT ATA CTT CCT 906 He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He Leu Pro 230 235 240 CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT TAT CTT TAT AAG 948 Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr Lys 245 250 255 ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA AGT TTT CTA AAA 990 He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC GTT GTA AGA ATC 1032 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He 275 280 AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA AAT CTA AAA ACT 1074 Asn Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr 285 290 295 SUBSTITUTE SHEET ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT GAA GTG GGC GCC 1116 He Asn Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala 300 305 310 AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT GAT TTT CGA ATC 1158 Asn Asp He Ala He Val Glu Asp Met Asp Asp Phe Arg He 315 320 325 GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG ATT CAA GGA TTA 1200 Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu He Gin Gly Leu 330 335 340 TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC GTC CGG CCA GAC 1242 Ser His Gin His Pro Ala Leu Lys Ser Ser Val Arg Pro Asp 345 350 AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC GCT CTG AAT ACA 1284 Arg Pro Glu Glu Arg Ser Lys Val He Ser Ala Leu Asn Thr 355 360 365 TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT TCC ATA AAC TCA 1326 Leu Trp He Glu Asp Pro Ser Leu Ser Phe Ser He Asn Ser 370 375 380 TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT GGT TTA ACC CAA 1368 Tyr Ser Asp Glu Leu Glu He Ser Leu Tyr Gly Leu Thr Gin 385 390 395 AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA CGA TTT TCC GTA 1410 Lys Glu He He Gin Thr Leu Leu Glu Glu Arg Phe Ser Val 400 405 410 AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA TAC AAA GAA GGA 1452 Lys Val His Phe Asp Glu He Lys Thr He Tyr Lys Glu Arg 415 420 CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG ATC GAA GTG CCG 1494 Pro Val Lys Lys Val Asn Lys He He Gin He Glu Val Pro 425 430 435 CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG ACT CTT GAT CCC 1536 Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu Glu Pro 440 445 450 TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA AGT GAC ATC TCC 1578 Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He Ser 455 460 465 TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT GCC GTT TTT GAA 1620 Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA CAT GGA TGG GAA 1662 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu 485 490 GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA GCC GAG TAT TAT 1704 Val Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr 495 500 505 AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA CAG CTG ACC CCT 1746 Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro 510 515 520 TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA GGT GTG GAC ATT 1788 Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He 525 530 535 CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG ATA CCC CAA GCG 1830 Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin He Pro Gin Ala 540 545 550 GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA AAA ATG ATG TCT 1872 Ala Ser Ser Lys Ala He Thr Asp Leu Gin Lys Met Met Ser 555 560 GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG TGG TGT CAT ATT 1914 Glu He Glu Asp He Ser Cys Asn Asn Glu Trp Cys His He 565 570 575 AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA GAC TAT GCA TCA 1956 Lys Gly Lys Val Pro Leu Asn Thr Ser Lys Asp Tyr Ala Ser 580 585 590 GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC ATT TTT ATG GTT 1998 Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly He Phe Met Val 595 600 605 AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC GGT TAT TCT GAT 2040 Lys Pro Cys Gly Tyr Gin He Thr Lys Gly Gly Tyr Ser Asp 610 615 620 AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT TTA TTC ATG TTC 2082 Asn He Arg Met Asn Glu Lys Asp Lys Leu Leu Phe Met Phe 625 630 CAA AAA TCA ATG TCA TCA AAA TAA 2106 Gin Lys Ser Met Ser Ser Lys 635 640 (2) INFORMATION FOR SEQUENCE ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1926 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: circular ( i) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATG AAT ATT ATA AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA 42 Met Asn He He Asn Leu Gly He Leu Ala His He Asp Ala 5 10 GGA AAA ACT TCC GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA 84 Gly Lys Thr Ser Val Thr Glu Asn Leu Leu Phe Ala Ser Gly 15 20 25 GCA ACG GAA AAG TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA 126 Ala Thr Glu Lys Cys Gly Cys Val Asp Asn Gly Asp Thr He 30 35 40 ACG GAC TCT ATG GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT 168 Thr Asp Ser Met Asp He Glu Lys Arg Arg Gly He Thr Val 45 50 55 CGG GCT TCT ACG ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC 210 Arg Ala Ser Thr Thr Ser He He Trp Asn Gly Val Lys Cys 60 65 70 AAT ATC ATT GAC ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA 252 Asn He He Asp Thr Pro Gly His Met Asp Phe He Ala Glu 75 80 GTG GAG CGG ACA TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC 294 Val Glu Arg Thr Phe Lys Met Leu Asp Gly Ala Val Leu He 85 90 95 TTA TCC GCA AAG GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG 336 Leu Ser Ala Lys Glu Gly He Gin Ala Gin Thr Lys Leu Leu 100 105 110 TTC AAT ACT TTA CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT 378 Phe Asn Thr Leu Gin Lys Leu Gin He Pro Thr He He Phe 115 120 125 ATC AAT AAG ATT GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG 420 He Asn Lys He Asp Arg Ala Gly Val Asn Leu Glu Arg Leu 130 135 140 TAT CTG GAT ATA AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT 462 Tyr Leu Asp He Lys Ala Asn Leu Ser Gin Asp Val Leu Phe 145 150 SUBSTITUTE SHEET ATG CAA AAT GTT GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC 504 Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro Val Cys Ser 155 160 165 CAA ACA TAT ATA AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC 546 Gin Thr Tyr He Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn 170 175 180 CAT GAC GAC AAT ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA 588 His Asp Asp Asn He Leu Glu Arg Tyr Leu Ala Asp Ser Glu 185 190 195 ATT TCA CCG GCT GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG 630 He Ser Pro Ala Asp Tyr Trp Asn Thr He He Ala Leu Val 200 205 210 GCA AAA GCC AAA GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG 672 Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met 215 220 TTC AAT ATC GGT ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT 714 Phe Asn He Gly He Asn Glu Leu Leu Asp Ala He Thr Ser 225 230 235 TTT ATA CTT CCT CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT 756 Phe He Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser 240 245 250 TAT CTT TAT AAG ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA 798 Tyr Leu Tyr Lys He Glu His Asp Pro Lys Gly His Lys Arg 255 260 265 AGT TTT CTA AAA ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC 840 Ser Phe Leu Lys He He Asp Gly Ser Leu Arg Leu Arg Asp 270 275 280 GTT GTA AGA ATC AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA 882 Val Val Arg He Asn Asp Ser Glu Lys Phe He Lys He Lys 285 290 AAT CTA AAA ACT ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT 924 Asn Leu Lys Thr He Asn Gin Gly Arg Glu He Asn Val Asp 295 300 305 GAA GTG GGC GCC AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT 966 Glu Val Gly Ala Asn Asp He Ala He Val Glu Asp Met Asp 310 315 320 GAT TTT CGA ATC GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG 1008 Asp Phe Arg He Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu 325 330 335 ATT CAA GGA TTA TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC 1050 He Gin Gly Leu Ser His Gin His Pro Ala Leu Lys Ser Ser 340 345 350 GTC CGG CCA GAC AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC 1092 Val Arg Pro Asp Arg Pro Glu Glu Arg Ser Lys Val He Ser 355 360 GCT CTG AAT ACA TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT 1134 Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser Leu Ser Phe 365 370 375 TCC ATA AAC TCA TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT 1176 Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser Leu Tyr 380 385 390 GGT TTA ACC CAA AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA 1218 Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu Glu 395 400 405 CGA TTT TCC GTA AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA 1260 Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 TAC AAA GAA GGA CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG 1302 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin 425 430 ATC GAA GTG CCG CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG 1344 He Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu 435 440 445 ACT CTT GAT CCC TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA 1386 Thr Leu Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu 450 455 460 AGT GAC ATC TCC TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT 1428 Ser Asp He Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn 465 470 475 GCC GTT TTT GAA GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA 1470 Ala Val Phe Glu Gly He Arg Met Ser Cys Gin Ser Gly Leu 480 485 490 CAT GGA TGG GAA GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA 1512 His Gly Trp Glu Val Thr Asp Leu Lys Val Thr Phe Thr Gin 495 500 GCC GAG TAT TAT AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA 1554 Ala Glu Tyr Tyr Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg 505 510 515 CAG CTG ACC CCT TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA 1596 Gin Leu Thr Pro Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser 520 525 530 GGT GTG GAC ATT CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG 1638 Gly Val Asp He Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin 535 540 545 ATA CCC CAA GCG GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA 1680 He Pro Gin Ala Ala Ser Ser Lys Ala He Thr Asp Leu Gin 550 555 560 AAA ATG ATG TCT GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG 1722 Lys Met Met Ser Glu He Glu Asp He Ser Cys Asn Asn Glu 565 570 TGG TGT CAT ATT AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA 1764 Trp Cys His He Lys Gly Lys Val Pro Leu Asn Thr Ser Lys 575 580 585 GAC TAT GCA TCA GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC 1806 Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly 590 595 600 ATT TTT ATG GTT AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC 1848 He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys Gly 605 610 615 GGT TAT TCT GAT AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT 1890 Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 TTA TTC ATG TTC CAA AAA TCA ATG TCA TCA AAA TAA 1926 Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 SUBSTITUTE SHEET

Claims: WE CLAIM: 1. A method of introducing heterologous DNA into a Prevotella ruminicola. comprising: (a) transforming an Escherichia coli with a shuttle vector comprising: (i) a mobilization region which permits transfer of the shuttle vector from Escherichia coli to a colonic Bacteroides species; (ii) a mobilization region which permits transfer of the shuttle vector from the colonic Bacteroides species to the P. ruminicola; and (iii) the heterologous DNA operatively linked to a promoter functional in the P. ruminicola (b) contacting the E. coli with a colonic Bacteroides species under conditions sufficient so that the shuttle vector is transferred from the E. coli to the colonic Bacteroides species; and (c) contacting the colonic Bacteroides species with the P. ruminicola under conditions sufficient so that the shuttle vector is transferred from the colonic Bacteroides species to the P. ruminicola. 2. The method of Claim 1 wherein the colonic Bacteroides species contains the Tc r Em r 12256 element. 3. The method of Claim 1 wherein the colonic Bacteroides species is Bacteroides uniformis. 4. The method of Claim 1 wherein the shuttle vector is pRDB5. 5. The method of Claim 1 wherein the . ruminicola is P. ruminicola Bj4. - 71 - 6. The method of Claim l wherein the promoter comprises the following sequence: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA, 180 [seq id no 1] or active variants thereof. 7. The P. ruminicola produced by the method of any one of Claims 1-6. 8. A shuttle vector comprising: a mobilization region which permits transfer of the shuttle vector from Escherichia coli to a colonic Bacteroides species; a mobilization region which permits transfer of the shuttle vector from the colonic Bacteroides species to a Prevotella ruminicola: and heterologous DNA operatively linked to a promoter functional in P. ruminicola. 9. The shuttle vector of Claim 8 which is pRDB5. 10. The shuttle vector of Claim 8 wherein the promoter comprises the following sequence: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA, 180 [seq id no 1] or active variants thereof. 11. Prevotella ruminicola containing the shuttle vector of Claim 8, 9 or 10. 12. The P. ruminicola of Claim 11 which is P. ruminicola B t 4. 13. A tetracycline resistance gene of the TetQ class, or fragments thereof that confer tetracycline resistance. 14. The gene of Claim 13 which comprises the following sequence: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA GGAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA ATG AAT ATT ATA 192 Met Asn He lie AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA GGA AAA ACT TCC 234 Asn Leu Gly He Leu Ala His He Asp Ala Gly Lys Thr Ser 5 10 15 GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA GCA ACG GAA AAG 276 Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr Glu Lys 20 25 30 TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA ACG GAC TCT ATG 318 Cys Gly Cys Val Asp Asn Gly Asp Thr He Thr Asp Ser Met 35 40 45 GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT CGG GCT TCT ACG 360 Asp He Glu Lys Arg Arg Gly He Thr Val Arg Ala Ser Thr 50 55 60 ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC AAT ATC ATT GAC 402 Thr Ser He He Trp Asn Gly Val Lys Cys Asn He He Asp 65 70 ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA GTG GAG CGG ACA 444 Thr Pro Gly His Met Asp Phe He Ala Glu Val Glu Arg Thr 75 80 85 SUBSTITUTE SHEET TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC TTA TCC GCA AAG 486 Phe Lys Met Leu Asp Gly Ala Val Leu He Leu Ser Ala Lys 90 95 100 GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG TTC AAT ACT TTA 528 Glu Gly He Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu 105 110 115 CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT ATC AAT AAG ATT 570 Gin Lys Leu Gin He Pro Thr He He Phe He Asn Lys He 120 125 130 GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG TAT CTG GAT ATA 612 Asp Arg Ala Gly Val Asn Leu Glu Arg Leu Tyr Leu Asp He 135 140 AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT ATG CAA AAT GTT 654 Lys Ala Asn Leu Ser Gin Asp Val Leu Phe Met Gin Asn Val 145 150 155 GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC CAA ACA TAT ATA 696 Val Asp Gly Ser Val Tyr Pro Val Cys Ser Gin Thr Tyr He 160 165 170 AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC CAT GAC GAC AAT 738 Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn His Asp Asp Asn 175 180 185 ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA ATT TCA CCG GCT 780 He Leu Glu Arg Tyr Leu Ala Asp Ser Glu He Ser Pro Ala 190 195 200 GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG GCA AAA GCC AAA 822 Asp Tyr Trp Asn Thr He He Ala Leu Val Ala Lys Ala Lys 205 210 GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG TTC AAT ATC GGT 864 Val Tyr Pro Val Leu His Gly Ser Ala Met Phe Asn He Gly 215 220 225 ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT TTT ATA CTT CCT 906 He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He Leu Pro 230 235 240 CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT TAT CTT TAT AAG 948 Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr Lys 245 250 255 ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA AGT TTT CTA AAA 990 He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 SUBSTITUTE SHEET ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC GTT GTA AGA ATC 1032 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He 275 280 AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA AAT CTA AAA ACT 1074 Asn Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr 285 290 295 ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT GAA GTG GGC GCC 1116 He Asn Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala 300 305 310 AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT GAT TTT CGA ATC 1158 Asn Asp He Ala He Val Glu Asp Met Asp Asp Phe Arg He 315 320 325 GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG ATT CAA GGA TTA 1200 Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu He Gin Gly Leu 330 335 340 TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC GTC CGG CCA GAC 1242 Ser His Gin His Pro Ala Leu Lys Ser Ser Val Arg Pro Asp 345 350 AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC GCT CTG AAT ACA 1284 Arg Pro Glu Glu Arg Ser Lys Val He Ser Ala Leu Asn Thr 355 360 365 TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT TCC ATA AAC TCA 1326 Leu Trp He Glu Asp Pro Ser Leu Ser Phe Ser He Asn Ser 370 375 380 TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT GGT TTA ACC CAA 1368 Tyr Ser Asp Glu Leu Glu He Ser Leu Tyr Gly Leu Thr Gin 385 390 395 AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA CGA TTT TCC GTA 1410 Lys Glu He He Gin Thr Leu Leu Glu Glu Arg Phe Ser Val 400 405 410 AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA TAC AAA GAA GGA 1452 Lys Val His Phe Asp Glu He Lys Thr He Tyr Lys Glu Arg 415 420 CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG ATC GAA GTG CCG 1494 Pro Val Lys Lys Val Asn Lys He He Gin He Glu Val Pro 425 430 435 CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG ACT CTT GAT CCC 1536 Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu Glu Pro 440 445 450 TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA AGT GAC ATC TCC 1578 Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He Ser 455 460 465 TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT GCC GTT TTT GAA 1620 Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA CAT GGA TGG GAA 1662 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu 485 490 GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA GCC GAG TAT TAT 1704 Val Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr 495 500 505 AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA CAG CTG ACC CCT 1746 Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro 510 515 520 TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA GGT GTG GAC ATT 1788 Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He 525 530 535 CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG ATA CCC CAA GCG 1830 Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin He Pro Gin Ala 540 545 550 GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA AAA ATG ATG TCT 1872 Ala Ser Ser Lys Ala He Thr Asp Leu Gin Lys Met Met Ser 555 560 GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG TGG TGT CAT ATT 1914 Glu He Glu Asp He Ser Cys Asn Asn Glu Trp Cys His He 565 570 575 AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA GAC TAT GCA TCA 1956 Lys Gly Lys Val Pro Leu Asn Thr Ser Lys Asp Tyr Ala Ser 580 585 590 GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC ATT TTT ATG GTT 1998 Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly He Phe Met Val 595 600 605 AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC GGT TAT TCT GAT 2040 Lys Pro Cys Gly Tyr Gin He Thr Lys Gly Gly Tyr Ser Asp 610 615 620 AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT TTA TTC ATG TTC 2082 Asn He Arg Met Asn Glu Lys Asp Lys Leu Leu Phe Met Phe 625 630 CAA AAA TCA ATG TCA TCA AAA TAA 2106 Gin Lys Ser Met Ser Ser Lys 635 640 [seq id no 4] or fragments thereof that confer tetracycline resistance. 15. A promoter functional in Prevotella ruminicola which is the promoter of a TetQ gene. 16. The promoter of Claim 15 which comprises the sequence: AAAAATCCTC CTACTTTTGT TAGATATATT TTTTTGTGTA ATTTTGTAAT 50 CGTTATGCGG CAGTAATAAT ATACATATTA ATACGAGTTA TTAATCCTGT 100 AGTTCTCATA TGCTACGAGG AGGTATTAAA AGGTGCGTTT CGACAATGCA 150 TCTATTGTAG TATATTATTG CTTAATCCAA, 180 [seq id no 1] or active variants thereof. 17. A protein of the TetQ class that provides resistance to tetracycline by protecting ribosomes from tetracycline, or active fragments thereof. 18. The protein of Claim 17 which has the following amino acid sequence: Met Asn He He Asn Leu Gly He Leu Ala His He Asp Ala Gly 5 10 15 Lys Thr Ser Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr 20 25 30 Glu Lys Cys Gly Cys Val Asp Asn Gly Asp Thr He Thr Asp Ser 35 40 45 Met Asp He Glu Lys Arg Arg Gly He Thr Val Arg Ala Ser Thr 50 55 60 Thr Ser He He Trp Asn Gly Val Lys Cys Asn He He Asp Thr 65 70 75 Pro Gly His Met Asp Phe He Ala Glu Val Glu Arg Thr Phe Lys 80 85 90 Met Leu Asp Gly Ala Val Leu He Leu Ser Ala Lys Glu Gly He 95 100 105 Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu Gin Lys Leu Gin 110 115 120 He Pro Thr He He Phe He Asn Lys He Asp Arg Ala Gly Val 125 130 135 Asn Leu Glu Arg Leu Tyr Leu Asp He Lys Ala Asn Leu Ser Gin 140 145 150 Asp Val Leu Phe Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro 155 160 165 Val Cys Ser Gin Thr Tyr He Lys Glu Glu Tyr Lys Glu Phe Val 170 175 180 Cys Asn His Asp Asp Asn He Leu Glu Arg Tyr Leu Ala Asp Ser 185 190 195 Glu He Ser Pro Ala Asp Tyr Trp Asn Thr He He Ala Leu Val 200 205 210 Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met Phe 215 220 225 Asn He Gly He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He 230 235 240 Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr 245 250 255 Lys He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He Asn 275 280 285 Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr He Asn 290 295 300 Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala Asn Asp He 305 310 315 Ala He Val Glu Asp Met Asp Asp Phe Arg He Gly Asn Tyr Leu 320 325 330 Gly Ala Glu Pro Cys Leu He Gin Gly Leu Ser His Gin His Pro 335 340 345 Ala Leu Lys Ser Ser Val Arg Pro Asp Arg Pro Glu Glu Arg Ser 350 355 360 Lys Val He Ser Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser 365 370 375 Leu Ser Phe Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser 380 385 390 Leu Tyr Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu 395 400 405 Glu Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin He 425 430 435 Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu 440 445 450 Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He 455 460 465 Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu Val 485 490 495 Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr Ser Pro 500 505 510 Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro Tyr Val Phe 515 520 525 Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He Leu Glu Pro Met 530 535 540 Leu Tyr Phe Glu Leu Gin He Pro Gin Ala Ala Ser Ser Lys Ala 545 550 555 He Thr Asp Leu Gin Lys Met Met Ser Glu He Glu Asp He Ser 560 565 570 Cys Asn Asn Glu Trp Cys His He Lys Gly Lys Val Pro Leu Asn 575 580 585 Thr Ser Lys Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly 590 595 600 Leu Gly He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys 605 610 615 Gly Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 - 79 - Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 [seq id no 3] or active fragments thereof. 19. A DNA sequence encoding the following amino acid sequence: Met Asn He He Asn Leu Gly He Leu Ala His He Asp Ala Gly 5 10 15 Lys Thr Ser Val Thr Glu Asn Leu Leu Phe Ala Ser Gly Ala Thr 20 25 30 Glu Lys Cys Gly Cys Val Asp Asn Gly Asp Thr He Thr Asp Ser 35 40 45 Met Asp He Glu Lys Arg Arg Gly He Thr Val Arg Ala Ser Thr 50 55 60 Thr Ser He He Trp Asn Gly Val Lys Cys Asn He He Asp Thr 65 70 75 Pro Gly His Met Asp Phe He Ala Glu Val Glu Arg Thr Phe Lys 80 85 90 Met Leu Asp Gly Ala Val Leu He Leu Ser Ala Lys Glu Gly He 95 100 105 Gin Ala Gin Thr Lys Leu Leu Phe Asn Thr Leu Gin Lys Leu Gin 110 115 120 He Pro Thr He He Phe He Asn Lys He Asp Arg Ala Gly Val 125 130 135 Asn Leu Glu Arg Leu Tyr Leu Asp He Lys Ala Asn Leu Ser Gin 140 145 150 Asp Val Leu Phe Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro 155 160 165 Val Cys Ser Gin Thr Tyr He Lys Glu Glu Tyr Lys Glu Phe Val 170 175 180 Cys Asn His Asp Asp Asn He Leu Glu Arg Tyr Leu Ala Asp Ser 185 190 195 Glu He Ser Pro Ala Asp Tyr Trp Asn Thr He He Ala Leu Val 200 205 210 ISTITUTE SHEET Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met Phe 215 220 225 Asn He Gly He Asn Glu Leu Leu Asp Ala He Thr Ser Phe He 230 235 240 Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser Tyr Leu Tyr 245 250 255 Lys He Glu His Asp Pro Lys Gly His Lys Arg Ser Phe Leu Lys 260 265 270 He He Asp Gly Ser Leu Arg Leu Arg Asp Val Val Arg He Asn 275 280 285 Asp Ser Glu Lys Phe He Lys He Lys Asn Leu Lys Thr He Asn 290 295 300 Gin Gly Arg Glu He Asn Val Asp Glu Val Gly Ala Asn Asp He 305 310 315 Ala He Val Glu Asp Met Asp Asp Phe Arg He Gly Asn Tyr Leu 320 325 330 Gly Ala Glu Pro Cys Leu He Gin Gly Leu Ser His Gin His Pro 335 340 345 Ala Leu Lys Ser Ser Val Arg Pro Asp Arg Pro Glu Glu Arg Ser 350 355 360 Lys Val He Ser Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser 365 370 375 Leu Ser Phe Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser 380 385 390 Leu Tyr Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu 395 400 405 Glu Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin He 425 430 435 Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu Thr Leu 440 445 450 Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu Ser Asp He 455 460 465 Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn Ala Val Phe Glu 470 475 480 Gly He Arg Met Ser Cys Gin Ser Gly Leu His Gly Trp Glu Val 485 490 495 Thr Asp Leu Lys Val Thr Phe Thr Gin Ala Glu Tyr Tyr Ser Pro 500 505 510 Val Ser Tyr Pro Ala Asp Phe Arg Gin Leu Thr Pro Tyr Val Phe 515 520 525 Arg Leu Ala Leu Gin Gin Ser Gly Val Asp He Leu Glu Pro Met 530 535 540 Leu Tyr Phe Glu Leu Gin He Pro Gin Ala Ala Ser Ser Lys Ala 545 550 555 He Thr Asp Leu Gin Lys Met Met Ser Glu He Glu Asp He Ser 560 565 570 Cys Asn Asn Glu Trp Cys His He Lys Gly Lys Val Pro Leu Asn 575 580 585 Thr Ser Lys Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly 590 595 600 Leu Gly He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys 605 610 615 Gly Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 [seq id no 3] 20. The DNA sequence of Claim 19 which is: ATG AAT ATT ATA AAT TTA GGA ATT CTT GCT CAC ATT GAT GCA 42 Met Asn He He Asn Leu Gly He Leu Ala His He Asp Ala 5 10 GGA AAA ACT TCC GTA ACC GAG AAT CTG CTG TTT GCC AGT GGA 84 Gly Lys Thr Ser Val Thr Glu Asn Leu Leu Phe Ala Ser Gly 15 20 25 GCA ACG GAA AAG TGC GGC TGT GTG GAT AAT GGT GAC ACC ATA 126 Ala Thr Glu Lys Cys Gly Cys Val Asp Asn Gly Asp Thr He 30 35 40 ACG GAC TCT ATG GAT ATA GAG AAA CGT AGA GGA ATT ACT GTT 168 Thr Asp Ser Met Asp He Glu Lys Arg Arg Gly He Thr Val 45 50 55 CGG GCT TCT ACG ACA TCT ATT ATC TGG AAT GGT GTG AAA TGC 210 Arg Ala Ser Thr Thr Ser He He Trp Asn Gly Val Lys Cys 60 65 70 AAT ATC ATT GAC ACT CCG GGA CAC ATG GAT TTT ATT GCG GAA 252 Asn He He Asp Thr Pro Gly His Met Asp Phe He Ala Glu 75 80 GTG GAG CGG ACA TTC AAA ATG CTT GAT GGA GCA GTC CTC ATC 294 Val Glu Arg Thr Phe Lys Met Leu Asp Gly Ala Val Leu He 85 90 95 TTA TCC GCA AAG GAA GGC ATA CAA GCG CAG ACA AAG TTG CTG 336 Leu Ser Ala Lys Glu Gly He Gin Ala Gin Thr Lys Leu Leu 100 105 110 TTC AAT ACT TTA CAG AAG CTG CAA ATC CCG ACA ATT ATA TTT 378 Phe Asn Thr Leu Gin Lys Leu Gin He Pro Thr He He Phe 115 120 125 ATC AAT AAG ATT GAC CGA GCC GGT GTG AAT TTG GAG CGT TTG 420 He Asn Lys He Asp Arg Ala Gly Val Asn Leu Glu Arg Leu 130 135 140 TAT CTG GAT ATA AAA GCA AAT CTG TCT CAA GAT GTC CTG TTT 462 Tyr Leu Asp He Lys Ala Asn Leu Ser Gin Asp Val Leu Phe 145 150 ATG CAA AAT GTT GTC GAT GGA TCG GTT TAT CCG GTT TGC TCC 504 Met Gin Asn Val Val Asp Gly Ser Val Tyr Pro Val Cys Ser 155 160 165 CAA ACA TAT ATA AAG GAA GAA TAC AAA GAA TTT GTA TGC AAC 546 Gin Thr Tyr He Lys Glu Glu Tyr Lys Glu Phe Val Cys Asn 170 175 180 CAT GAC GAC AAT ATA TTA GAA CGA TAT TTG GCG GAT AGC GAA 588 His Asp Asp Asn He Leu Glu Arg Tyr Leu Ala Asp Ser Glu 185 190 195 ATT TCA CCG GCT GAT TAT TGG AAT ACG ATA ATC GCT CTT GTG 630 He Ser Pro Ala Asp Tyr Trp Asn Thr He He Ala Leu Val 200 205 210 GCA AAA GCC AAA GTC TAT CCG GTG CTA CAT GGA TCA GCA ATG 672 Ala Lys Ala Lys Val Tyr Pro Val Leu His Gly Ser Ala Met 215 220 TTC AAT ATC GGT ATC AAT GAG TTG TTG GAC GCC ATC ACT TCT 714 Phe Asn He Gly He Asn Glu Leu Leu Asp Ala He Thr Ser 225 230 235 SUBSTITUTE SHEET TTT ATA CTT CCT CCG GCA TCG GTC TCA AAC AGA CTT TCA TCT 756 Phe He Leu Pro Pro Ala Ser Val Ser Asn Arg Leu Ser Ser 240 245 250 TAT CTT TAT AAG ATA GAG CAT GAC CCC AAA GGA CAT AAA AGA 798 Tyr Leu Tyr Lys He Glu His Asp Pro Lys Gly His Lys Arg 255 260 265 AGT TTT CTA AAA ATA ATT GAC GGA AGT CTG AGA CTT CGA GAC 840 Ser Phe Leu Lys He He Asp Gly Ser Leu Arg Leu Arg Asp 270 275 280 GTT GTA AGA ATC AAC GAT TCG GAA AAA TTC ATC AAG ATT AAA 882 Val Val Arg He Asn Asp Ser Glu Lys Phe He Lys He Lys 285 290 AAT CTA AAA ACT ATC AAT CAG GGC AGA GAG ATA AAT GTT GAT 924 Asn Leu Lys Thr He Asn Gin Gly Arg Glu He Asn Val Asp 295 300 305 GAA GTG GGC GCC AAT GAT ATC GCG ATT GTA GAG GAT ATG GAT 966 Glu Val Gly Ala Asn Asp He Ala He Val Glu Asp Met Asp 310 315 320 GAT TTT CGA ATC GGA AAT TAT TTA GGT GCT GAA CCT TGT TTG 1008 Asp Phe Arg He Gly Asn Tyr Leu Gly Ala Glu Pro Cys Leu 325 330 335 ATT CAA GGA TTA TCG CAT CAG CAT CCC GCT CTC AAA TCC TCC 1050 He Gin Gly Leu Ser His Gin His Pro Ala Leu Lys Ser Ser 340 345 350 GTC CGG CCA GAC AGG CCC GAA GAG AGA AGC AAG GTG ATA TCC 1092 Val Arg Pro Asp Arg Pro Glu Glu Arg Ser Lys Val He Ser 355 360 e GCT CTG AAT ACA TTG TGG ATT GAA GAC CCG TCT TTG TCC TTT 1134 Ala Leu Asn Thr Leu Trp He Glu Asp Pro Ser Leu Ser Phe 365 370 375 TCC ATA AAC TCA TAT AGT GAT GAA TTG GAA ATC TCG TTA TAT 1176 Ser He Asn Ser Tyr Ser Asp Glu Leu Glu He Ser Leu Tyr 380 385 390 GGT TTA ACC CAA AAG GAA ATC ATA CAG ACA TTG CTG GAA GAA 1218 Gly Leu Thr Gin Lys Glu He He Gin Thr Leu Leu Glu Glu 395 400 405 CGA TTT TCC GTA AAG GTC CAT TTT GAT GAG ATC AAG ACT ATA 1260 Arg Phe Ser Val Lys Val His Phe Asp Glu He Lys Thr He 410 415 420 TAC AAA GAA GGA CCT GTA AAA AAG GTC AAT AAG ATT TAA CAG 1302 Tyr Lys Glu Arg Pro Val Lys Lys Val Asn Lys He He Gin 425 430 ATC GAA GTG CCG CCC AAC CCT TAT TGG GCC ACA ATA GGG CTG 1344 He Glu Val Pro Pro Asn Pro Tyr Trp Ala Thr He Gly Leu 435 440 445 ACT CTT GAT CCC TTA CCG TTA GGG ACA GGG TTG CAA ATC GAA 1386 Thr Leu Glu Pro Leu Pro Leu Gly Thr Gly Leu Gin He Glu 450 455 460 AGT GAC ATC TCC TAT GGT TAT CTG AAC CAT TCT TTT CAA AAT 1428 Ser Asp He Ser Tyr Gly Tyr Leu Asn His Ser Phe Gin Asn 465 470 475 GCC GTT TTT GAA GGG ATT CGT ATG TCT TGC CAA TCC GGG TTA 1470 Ala Val Phe Glu Gly He Arg Met Ser Cys Gin Ser Gly Leu 480 485 490 CAT GGA TGG GAA GTG ACT GAT CTG AAA GTA ACT TTT ACT CAA 1512 His Gly Trp Glu Val Thr Asp Leu Lys Val Thr Phe Thr Gin 495 500 GCC GAG TAT TAT AGC CCG GTA AGT ACA CCT GCT GAT TTC AGA 1554 Ala Glu Tyr Tyr Ser Pro Val Ser Tyr Pro Ala Asp Phe Arg 505 510 515 CAG CTG ACC CCT TAT GTC TTC AGG CTG GCC TTG CAA CAG TCA 1596 Gin Leu Thr Pro Tyr Val Phe Arg Leu Ala Leu Gin Gin Ser 520 525 530 GGT GTG GAC ATT CTC GAA CCG ATG CTC TAT TTT GAG TTG CAG 1638 Gly Val Asp He Leu Glu Pro Met Leu Tyr Phe Glu Leu Gin 535 540 545 ATA CCC CAA GCG GCA AGT TCC AAA GCT ATT ACA GAT TTG CAA 1680 He Pro Gin Ala Ala Ser Ser Lys Ala He Thr Asp Leu Gin 550 555 560 AAA ATG ATG TCT GAG ATT GAA GAC ATC AGT TGC AAT AAT GAG 1722 Lys Met Met Ser Glu He Glu Asp He Ser Cys Asn Asn Glu 565 570 TGG TGT CAT ATT AAA GGG AAA GTT CCA TTA AAT ACA AGT AAA 1764 Trp Cys His He Lys Gly Lys Val Pro Leu Asn Thr Ser Lys 575 580 585 GAC TAT GCA TCA GAA GTA AGT TCA TAC ACT AAG GGC TTA GGC 1806 Asp Tyr Ala Ser Glu Val Ser Ser Tyr Thr Lys Gly Leu Gly 590 595 600 ATT TTT ATG GTT AAG CCA TGC GGG TAT CAA ATA ACA AAA GGC 1848 He Phe Met Val Lys Pro Cys Gly Tyr Gin He Thr Lys Gly 605 610 615 GGT TAT TCT GAT AAT ATC CGC ATG AAC GAA AAA GAT AAA CTT 1890 Gly Tyr Ser Asp Asn He Arg Met Asn Glu Lys Asp Lys Leu 620 625 630 TTA TTC ATG TTC CAA AAA TCA ATG TCA TCA AAA TAA 1926 Leu Phe Met Phe Gin Lys Ser Met Ser Ser Lys 635 640 [seq id no 5] 21. An engineered Prevotella ruminicola comprising expressible foreign DNA. 22. The P. ruminicola of Claim 21 wherein the foreign DN is under control of the promoter of Claim 15 or 16.

Inventors: NIKOLICH MIKELJON P, SALYERS ABIGAIL A, SHOEMAKER NADJA B, NIKOLICH, MIKELJON, P., SALYERS, ABIGAIL, A., SHOEMAKER, NADJA, B.
Applicants: BIOTECHNOLOGY RES & DEV, UNIV ILLINOIS, BIOTECHNOLOGY RESEARCH AND DEVELOPMENT CORPORATION, BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS
Application year: 1992
Main classification: C07H21
IPC codes: C07K14, C12N1, C12N15