# Patent

## Title
Insertion sequence-free bacteria

## Abstract

A bacteria lacking genomic and non-genomic IS elements is provided. The bacteria may be more stable and useful for the production of amino acids, polypeptides, nucleic acids and other products.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part of U.S. application Ser. No. 11/275,094, filed Dec. 9, 2005, which in turn claims the benefit of U.S. Provisional Application No. 60/634,611, filed Dec. 9, 2004, and is a continuation-in-part of U.S. application Ser. No. 10/896,739 filed Jul. 22, 2004 (abandoned), which is a continuation of International Application No. PCT/US03/01800, filed Jan. 22, 2003, which claims the benefit of U.S. Provisional Application No. 60/409,089, filed Sep. 6, 2002 and which is a continuation-in-part of U.S. Application Ser. No. 10/057,582, filed Jan. 23, 2002, now U.S. Pat. No. 6,989,265.


FIELD OF THE INVENTION
The present invention relates to strains of microorganisms and processes involving these microorganisms. More specifically, the present invention relates to modified strains of microorganisms lacking all Insertion Sequence elements and the use thereof.
BACKGROUND OF THE INVENTION
Bacteria have been used to produce a wide range of commercial products. For example, many Streptomyces strains and Bacillus strains have been used to produce antibiotics; Pseudomonas denitrificans and many Propionibacterium strains have been used to produce vitamin B12; some other bacteria have been used to produce vitamins such as Riboflavin; Brevibacterium flavum and Corynebacterium glutamicum have been used to produce lysine and glutamic acid, respectively, as food additives; other bacteria have been used to produce other amino acids used as food additives; Alcaligenes eutrophas has been used to produce biodegradable microbial plastics; and many Acetobacter and Gluconobacter strains have been used to produce vinegar. More recently, bacteria, such as Escherichia coli (E. coli), have been genetically engineered and used as host cells for the production of biological reagents, such as proteins and nucleic acids, in laboratory as well as industrial settings. The pharmaceutical industry supports several examples of successful products, which are human proteins manufactured in E. coli cultures cultivated in a fermenter.
E. coli K-12 is the most commonly used host for cloning and other molecular biology techniques and is the platform of choice for production of metabolites such as amino acids and many proteins of therapeutic or commercial interest. Recently it has been used or proposed for production of therapeutic DNA for use in gene therapy, DNA vaccines, and RNA interference applications. The complete genomes of two closely related E. coli K-12 strains, MG1655 and W3110, have been sequenced and are available from the National Center for Biotechnology Information microbial genomes database (NCBI database) (www.ncbi.nih.gov/genomes/lproks.cgi) as accession numbers U00096 and AP009048 respectively. Eighty-seven percent of E. coli K-12 genes have been assigned functions with some degree of confidence, making it one of the best understood organisms.
Desirable properties for a platform microorganism include efficiency of production, purity of product and stability of the genome during experimental manipulation, in production, and in storage. The chromosome of E. coli is littered with mobile genetic elements that mediate horizontal gene transfer, including insertion sequences (IS), transposases, defective phages, integrases, and site-specific recombinases. These elements can translocate, duplicate, and be maintained in the genome like an infectious agent, and are known to hop into plasmids as well. IS elements may cause inversions, duplications, and deletions mediated by homologous recombination. This can happen even when the transposase function has become inactive. Similar rearrangements also result from rRNA and Rhs repeats, but the instability is magnified when active transposases are involved.
Genome alterations due to IS translocation occur surprisingly frequently, and many commonly used laboratory and industrial strains have unrecognized genome alterations from this cause. For example, many of the differences between the two sequenced E. coli K-12 strains, which have been separated for about five decades from a common laboratory ancestor, are due to IS hops. The sequence databases provide ample evidence that IS hopping into plasmids is also common on the time scale of laboratory manipulations. Approximately one in every thousand eukaryotic sequences in the public databases is inadvertently contaminated with bacterial IS elements that apparently hopped into the cloned eukaryotic DNA during the brief period of propagation in E. coli prior to sequencing.
IS elements can also be inadvertently introduced into strains by laboratory manipulations. A case in point involves the E. coli K-12 derivatives DH10B and DH5α, which carry an IS10 not present in the ancestral K-12 genome. Despite a report that residual IS10 elements do not exhibit transpositional mutagenesis in recA strains, such as DH10B and DH5α, the prominence of IS10 contamination of the eukaryotic databases shows that this continues to be an issue. Thus, IS elements may lead to unpredictable consequences with important production hosts and pose a considerable impediment to the efficiency and accuracy of amino acid, protein, and nucleic acid production in E. coli. 
SUMMARY OF THE INVENTION
A non-naturally occurring bacterium is provided lacking genomic and non-genomic insertion sequences. The bacterium may be an E. coli. The genome of the bacterium may be less than 4.41 Mb, 4.27 Mb, 4.00 Mb, 3.71 Mb, 2.78 Mb or 1.86 Mb. The bacterium may be derived from strain E. coli K-12. The bacterium may also be derived from E. coli DH10B or E. coli DH5α. The bacterium may be competent to be transformed.
The bacterium may comprise an additional nucleic acid, which may lack insertion sequences. The additional nucleic acid may be a vector, which may be a plasmid. The additional nucleic acid may comprise another nucleic acid encoding a polypeptide. The polypeptide encoding nucleic acid may be operatively linked to an expression control sequence.
A method of propagating a nucleic acid is also provided. The nucleic acid may be toxic. A bacterium lacking genomic and non-genomic insertion sequences and an additional nucleic acid may be incubated under conditions allowing transformation of the bacterium with the nucleic acid which then may be grown under conditions allowing replication of the nucleic acid. Transformation may occur by electroporation. The nucleic acid may be amplified by propagating the bacterium, wherein the nucleic acid is amplified.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of the construction of an E. coli multiple deletion strain (MDS) lacking all IS elements. Concentric rings depict features mapped to the genome of the parental E. coli K-12 strain MG1655, numbered on the outer ring. Moving outward from the center, rings 1-5 (grey) show regions of K-12 that are absent in other sequenced E. coli genomes. Ring 6 shows the regions targeted for deletion. Ring 7 shows native IS and Rhs elements. Ring 8 shows experimental confirmation of the deletions in MDS43. The outer ring shows positions for the origin and terminus of replication and genes for rRNAs, tRNAs and other small stable RNAs.
FIG. 2 shows the PCR detection of IS elements in various DNA preparations. Each panel is loaded in the same order: 1 kb+ marker, IS1, IS2, IS3, IS5, IS10, IS186, 1 kb+ marker, for (1) Positive Control, (2) Negative Control, (3) pBR322 from Invitrogen (in DH10B), and (4) pBR322 produced in MDS42.
FIGS. 3a and 3b show the growth rate of strains MDS41, MDS42 and MDS43 in MOPS minimal medium at 37° C. (top) and a comparison of the growth rates and CAT expression of MG1655 and MDS42 in MOPS minimal medium at 37° C. (bottom).
FIG. 4 compares the frequency of mutation by IS hopping to the genome in MDS41 and MG1655 as measured by the acquired ability to utilize salicin as a carbon source.
FIG. 5 shows the restriction pattern of pCTXVP60 propagated in various strains: (M) molecular weight marker, 1 kbp ladder; (1) MDS41, no insertion; (2) MDS42, no insertion; (3) DH10B, IS10 insertion; (4) DH10B, IS10 insertion/deletion; (5) C600, IS5 insertion; (6) C600 IS1 insertion; and (7) C600, IS1 insertion. Relative positions of the IS insertions in the CTXVP60 reading frame are diagrammed below the gel.
FIG. 6 compares the frequency of mutation by IS hopping to the genome in MG1655 transformed with either pCTXVP60 or pCTX as measured by the acquired ability to utilize salicin as a carbon source.
FIG. 7 compares the rate of IS hopping in MG1655 carrying an expression plasmid for CAT in the presence and absence of IPTG induction, as measured by the appearance of D-cycloserine mutants. D-cycloserine mutants result almost exclusively from loss-of-function mutations in cycA.
FIG. 8 shows the restriction pattern of pT-ITR propagated in MDS42 and MG1655.



DETAILED DESCRIPTION
The use of E. coli as a host organism for the production of biologically useful molecules has been plagued by genomic instability caused by mobile genetic elements such as IS elements. For example, IS elements can hop from host genomic nucleic acids into cloning vectors such as plasmids and thus are detrimental to the stability and efficiency of cloning. The role of extrachromosomal IS elements, such as IS mini-circles and other replicative and non-replicative IS derivatives is unappreciated and the presence of these in the host bacteria pose the same problem. A bacteria is provided lacking all genomic and non-genomic IS elements. The increased genetic stability of the bacteria is useful for such purposes as maintaining the integrity of cloned nucleic acids. The bacteria provides a more stable genetic environment for the production of nucleic acids, polypeptides, amino acids and other useful products.
1. Definitions
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
“Base pair” used herein may refer to the hydrogen bonded nucleotides of, for example, adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double-stranded DNA molecule. In RNA, uracil (U) is substituted for thymine. Base pair may also be used as a unit of measure for DNA length.
“Clone” used in reference to an insert sequence and a vector may mean ligation of the insert sequence into the vector or its introduction by recombination either homologous, site specific or illegitimate as the case may be. When used in reference to an insert sequence, a vector, and a host cell, the term may mean to make copies of a given insert sequence. The term may also refer to a host cell carrying a cloned insert sequence, or to the cloned insert sequence itself.
“Complement,” “complementary” or “complementarity” used herein may mean Watson-Crick or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. For example, the sequence 5′-A-G-T-3′ is complementary to the sequence 3′-T-C-A-5′. Complementarity may be “partial”, in which only some of the nucleotides are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands may have effects on the efficiency and strength of hybridization between nucleic acid strands.
“Encoding” or “coding” used herein when referring to a nucleic acid may mean a sequence of nucleotides, which upon transcription into RNA and subsequent translation into protein, would lead to the synthesis of a given protein, peptide, or amino acid sequence. Such transcription and translation may actually occur in vitro or in vivo, or may be strictly theoretical based on the standard genetic code.
“Expression control sequence” used herein may mean a promoter or array of transcription factor binding sites that direct transcription of a nucleic acid operatively linked thereto.
“Nucleic acid” used herein may mean any nucleic acid containing molecule including, but not limited to, DNA or RNA. The term encompasses sequences that include any base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5 carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracils, 5-methoxyaminomethyl-2-thiouracil, γ-D-maninosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
“Operably linked” used herein may refer to an expression control sequence and downstream polynucleotide, such that productive transcription of the polynucleotide is initiated at the expression control sequence.
“Overexpressing” used herein may mean that the total cellular activity of protein encoded by a gene is increased. The total cellular activity of a protein may be due to increased cellular amounts of a protein, or increased half-life of the protein. Total cellular amounts of a protein may be increased by methods including, but not limited to, amplification of the gene coding said protein, operatively linking a strong promoter to the gene coding said protein or by increasing the strength of the genes' native promoter by, for example, mutating the promoter.
“Plasmid” used herein may mean extrachromosomal genetic elements composed of DNA or RNA that are not part of a chromosome but can propagate themselves autonomously in cells. A plasmid may refer to not only those native plasmids isolated from cells, but also any modified or chimeric versions (e.g., having deletions, additions or substitutions or assembled from functional parts of different plasmids) so long as they retain the ability to propagate themselves autonomously in cells.
“Phage” used herein may mean extrachromosomal bacteriophage capable of propagating in cells, such as bacteriophage P1, and also includes lysogenic bacteriophage such as Lambda that can integrate into, and propagate within, the host chromosome. A phage may refer to not only naturally occurring bacteriophage, but also any modified or chimeric versions (e.g. having deletions, additions or substitutions or assembled from functional parts of different phage) so long as they retain the ability to propagate in cells either autonomously or with helper function provided, for example, by helper phage.
“Protein” used herein may mean a peptide, polypeptide and protein, whether native or recombinant, as well as fragments, derivatives, homologs, variants and fusions thereof.
“Region of comparison” used herein when referring to a genome may be 1×107, 1.5×107, 2×107, 2.5×107, 3.5×107, 4×107 or more nucleotides or base pairs, and when referring to a nucleic acid sequence may be 50, 100, 250, 500, 103, 5×103, 104, 5×104, 105, 5×105, 106 or more nucleotides or more base pairs.
“Substantially complementary” used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of comparison or that the two sequences hybridize under stringent hybridization conditions.
“Substantially identical” used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical or substantially complementary over a region of comparison. A reference sequence and a test sequence may be aligned, manually or by a computer algorithm (e.g., GAP, BESTFIT, FASTA and TFAST), and the percentage identity calculated by dividing “the total number of identical residues” by “the total number of residues in the reference sequence” and then multiplying by 100.
“Vector” as used herein may mean a carrier DNA molecule into which a nucleic acid sequence can be inserted for introduction into a new host cell where it may be replicated, and in some cases expressed. Vectors can be derived from plasmids, bacteriophages, plants, animals viruses, etc. The vector may be propagated in the host cell as an extrachromomal element or, alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
2. Bacteria Lacking Insertion Sequences
A non-naturally occurring bacteria is provided lacking genomic and non-genomic IS elements. IS elements and their associated transposases are often found in bacteria and are associated with instabilities that can interfere with standard industrial or laboratory practices and might entail costly and burdensome quality control procedures. IS elements may be contained not only in genomic host nucleic acids, but also in non-genomic host nucleic acids.
An IS element may be linear or circular. For example, the IS element may be circularized to form an IS-mini circle. Creation of IS mini-circles may be the first step in the transposition process of an IS element. For example, creation of an IS-mini-circle is the first step in the transposition process of IS elements belonging to the IS2 family. IS elements are common in E. coli and all of them may be deleted.
IS elements are currently grouped into families based on conserved motifs. IS families include, without limitation, IS, IS3, IS4, IS5, IS6, IS2J, IS30, IS66, IS91, IS110, IS200/605, IS256, IS481, IS630, IS982, IS1380, ISAs1, ISL3, Tn3, and variants thereof. A variant may contain any of the conserved regions that define any of the IS families. Representative conserved regions include, but are not limited to the DDE motif, conserved in most IS element families, and the N-terminal helix-turn-helix motif, conserved in members of the IS3 family. The ISFinder database (www-is.biotoul.fr) contains the sequences of various members of the IS families. Each member of each IS family may be deleted.
a. Parent
The parent of the ISfree bacteria may be any bacterial strain that contains IS elements, as well as an intermediate strain from which the bacterium is derived. Representative examples of parent strains include, but are not limited to, E. coli strains such as K-12 or B, or a strain with a genome sequence substantially identical thereto. The E. coli K-12 strain may be a derivative strain including, but not limited to MG1655, DH10B, DH5α, Invα, Top10, Top10F, JM103, JM105, JM109, MC1061, MC4100, XL1-Blue, EC100 or EC300. The nucleotide sequence of the genome of the parental strain may be partially or completely known. The complete genomic sequence of several strains of E. coli and other commonly used laboratory microorganisms is known (see, e.g., Blattner et al., Science, 277:1453-74, 1997; GenBank Accession No. U0096; NCBI database, Accession No. AP009048, Perna et al., Nature, 409, 529-533, 2001; Hayashi et al., DNA Res., 8, 11-22, 2001; Welch et al., Proc. Natl. Acad. Sci., USA 99:17020-17024, 2002 and GenBank Accession No. AE014075, each of which is incorporated herein by reference). The genomic sequence of DH10B is partially known (www.hgsc.bcm.tmc.edu/projects/microbial/EcoliDH10B).
E. coli strains MG1655 and W3110 have been sequenced and each contains a variety of IS elements, including: IS1, a member of the IS1 family; IS2, IS3 and IS150, members of the IS3 family; IS4 and IS 186, members of the IS4 family; IS5, a member of the IS5 family; and IS30, a member of the IS30 family. Additionally, partial sequences of IS600 and IS911, members of the IS3 family, are found in each strain. Frequency of occurrences of IS elements are presented in Table 1. Because DH10B is an E. coli K12 derivative, it is expected to have a similar IS composition to MG1655 and W3110.







TABLE 1







Frequency of IS elements in select E. coli K-12 strains












IS element
MG1655
W3110
DH10B
















IS1
7
8
present



IS2
6
10
present



IS3
5
5
present



IS4
1
1
present



IS5
11
18
present



IS30
3
3
present



IS150
1
1
present



IS186
3
3
present



IS600
partial
partial
?



IS911
partial
partial
?



IS10
0
0
present










The nucleic acid sequence of E. coli MG 155 (annotated version m56), (NCBI accession no. U00096.1) is set forth in SEQ ID NO: 1 with a total size of 4,639,675 nucleotides or base pairs. The original release of the genomic sequence of E. coli MG1655 was annotated version m54, (NCBI accession no. U00096.1) (4,639,221 nucleotides or base pairs). Positions of the IS elements on a genome map of E. coli MG1655 (annotated version m54) are shown in FIG. 1 and Table 2 of U.S. Patent Publication No. 20030138937 and International Patent Publication No. WO 2003/070880, the contents of which are incorporated herein by reference.
b. Genomic Deletions
The bacteria may be made by deleting IS elements using any of the several methods known to those of skill in the art for deleting genomic or non-genomic nucleic acid. The nucleic acid sequences may be deleted from genomic or from non-genomic genetic material.
Representative methods for making deletions in the genome of a bacterium are described in U.S. Patent Publication No. 20030138937 and International Patent Publication No. WO 2003/070880, Posfai, G. et al., J. Bacteriol. 179: 4426-4428 (1997), Muyrers, J. P. P. et al., Nucl. Acids Res. 27:1555-1557 (1999), Datsenko K. A. et al., Proc. Natl. Acad. Sci. 97:6640-6649 (2000) and Posfai, G. et al., Nucl. Acids. Res. 27: 4409-4415 (1999), each of which is incorporated herein by reference. The deletion methods may be classified to those that are based on linear DNA and those that are based on suicide plasmids. The methods disclosed in Muyrers, J. P. P. et al., Nucl. Acids Res. 27:1555-1557 (1999) and Datsenko, K. A., Proc. Natl. Acad. Sci. 97:6640-6649 (2000) are linear DNA-based methods and the methods disclosed in n Posfai, G. et al., J. Bacteriol. 179: 4426-4428 (1997) and Posfai, G. et al., Nucl. Acids Res. 27: 4409-4415 (1999) are suicide plasmid-based methods.
In addition to IS elements, additional nucleic acids regions may be deleted from the bacteria. For example, in addition to IS elements, the bacteria may also be lacking one or more of the nucleic acid regions set forth on Table 2, or sequences substantially similar thereto, and those set out in Table 1 of U.S. Provisional Application No. 60/709,960, incorporated herein by reference. The bacteria may be strain MDS39, MDS41, MDS42, MDS43, or a strain with a genome substantially identical thereto. FIG. 1 is a map of MDS41, MDS42, and MDS43, all of which lack IS elements. The bacteria may also be MDS42recA or a strain with a genome substantially identical thereto. The bacteria may have a genome that is less than 4.41 Mb, 4.27 Mb, 4.00 Mb, 3.71 Mb, 2.78 Mb, or 1.86 Mb. Elimination of unnecessary genes may improve metabolic efficiency and perhaps simplify the purification of desirable products.
c. Competent Bacteria
The bacteria may be competent for transformation by a foreign molecule, such as a nucleic acid. The bacteria may be made competent by methods well known in the art. Representative methods of making the bacteria competent may be found in U.S. Pat. No. 4,981,797 and U.S. Patent Publication No. 20050032225, which are hereby incorporated by reference.
Removal of IS elements may lead to increased electroporation efficiency. For example, electroporation efficiency of strains MDS41, MDS42, and MDS43, from which all genomic IS elements are deleted, is improved by 2 orders of magnitude over their MG1655 parent and is comparable to DH10B, normally considered to be the best E. coli for electroporation.
d. Bacteria Comprising a First Nucleic Acid
The bacteria may comprise an additional nucleic acid, which may lack IS elements. The additional nucleic acid may be a vector, which may, inter alia be a plasmid, cosmid, BAC, modified YAC, phagemid or phage. The vector may be a cloning vector or an expression vector.
The additional nucleic acid may comprise another nucleic acid encoding a polypeptide. The polypeptide may be a therapeutic product including, but not limited to, a vaccine component, a diagnostic product, or a research reagent. Further, the polypeptide may be a protein, including but not limited to, insulin, an interleukin, a cytokine, a growth hormone, a growth factor, erythropoietin, a colony stimulating factor, interferon, an antibody and an antibody fragment. Expression of the polypeptide may be under the control of an inducible promoter or a promoter that is constitutively expressed in the bacteria. For example, lac-based promoter/repressor, inducible by the non-metabolisable galactose derivative, IPTG, may be used.
A first nucleic acid lacking IS elements may be useful for cloning. For example, overexpressing even a well tolerated protein-of-interest may lead to elevated IS transposition rates. Such transposition may result in the insertion of an IS element into the nucleic acid encoding the protein-of-interest.
3. Methods
a. Cloning
The bacteria may be used to clone a nucleic acid. Briefly, the competent bacteria may be incubated with a nucleic acid under conditions allowing transformation of the bacteria by the nucleic acid. Conditions allowing transformation are well known in the art and may include, but are not limited to, electroporation, calcium or manganese chloride precipitation, lipofection, microinjection and natural transformation.
By providing a bacteria lacking genomic and non-genomic IS elements, cloning artifacts caused by transposable IS elements may be eliminated. Toxic nucleic acids may therefore be cloned in the bacteria. A “toxic” nucleic acid may be a nucleic acid which, when propagated in a host strain, results in an elevated rate of IS element transposition. Toxic nucleic acids are difficult to clone in bacterial hosts containing IS elements. For example, a nucleic acid encoding the open reading frame of VP60 of rabbit haemorrhagic disease virus fused to the B subunit of cholera toxin, previously incapable of being cloned due to the high rate of IS element transposition, has been successfully cloned in IS-free bacteria. In another example, pT-ITR, a plasmid possessing a stem-loop structure that prevents propagation in bacterial hosts containing IS elements, has been successfully propagated in IS-free bacteria.
Because IS element transposition may result in detectable insertion mutations, an elevated rate of IS element transposition of a toxic nucleic acid may be determined by comparison to the mutation rate of a host strain propagating a control nucleic acid. The insertion mutation rate of a host strain propagating the nucleic acid may be measured by the appearance of mutant cells that gain the ability to utilize salicin as a carbon source. Metabolism of salicin in E. coli K-12 requires activation of the bgl operon, which occurs primarily by integration of an IS element into the promoter region, as described in Hall, Mol. Biol. Evol., 15:1-5, 1998, which is incorporated herein by reference. The toxic nucleic acid may encode a polypeptide, in which case the rate of IS element transposition may be compared to that resulting from the propagation, in the same host strain, of a control nucleic acid of similar size encoding a different polypeptide. The toxic nucleic acid may also be a vector, in which case the rate of IS element transposition may be compared to that resulting from the propagation, in the same host strain, of a different vector of similar size. Representative vectors include, but are not limited to pBR322, pUC18, pGEM, and pBluescript.
b. Expression
The bacteria may also be used for the production of polypeptides. Briefly, a bacteria comprising an additional nucleic acid which comprises a nucleic acid encoding a polypeptide, as described above, may be incubated under conditions allowing expression of the polypeptide product.
Overexpression of even a well tolerated protein-of-interest may lead to elevated IS transposition rates. Such transposition may result in the insertion of an IS element into the nucleic acid encoding the protein-of-interest. A bacteria comprising a nucleic acid encoding a polypeptide and lacking genomic and non-genomic IS elements may provide an increased production of protein.
Recombinant proteins may be expressed in the periplasm or cytoplasm. The expression of proteins in the periplasm is routinely used for industrial use and has been reviewed in Hanahan, J. Mol. Biol., 166:557-80, 1983; Hockney, Trends Biotechnol., 12:456-632, 1994; and Hannig et al., Trends Biotechnol., 16:54-60, 1998, each of which is incorporated herein by reference. Recombinant proteins may be produced in the periplasm by expressing fusion proteins in which they are attached to a signal peptide that causes secretion into the periplasmic space. There the signal peptide may be cleaved off by specific signal peptidases. The protein transported into the periplasmic space may be biologically active.
The recombinant protein may also be co-expressed with chaperones/disulfide-bond forming enzymes, which may provide proper folding of the recombinant protein. Nucleic acid sequences of such proteins useful for periplasmic expression of recombinant protein include, but are not limited to, those described in U.S. Pat. Nos. 5,747,662; 5,578,464; 6,335,178; and 6,022,952; Thomas et al., Mol-Micro, (2001) 39 (1) 47-53; Weiner et al., Cell, (1998) 93, 93-101; and Current Protocols in Molecular Biology (1994) 16.6.1-16.6.14 (Copyrighted 2000 by John Wiley et al. and Sons), each of which is incorporated herein by reference.
c. Amplification
The reduced genome strain may also be used to amplify a nucleic acid. Briefly, a bacteria comprising a first nucleic acid lacking IS elements, may be incubated under conditions allowing the propagation of the first nucleic acid lacking IS elements.
The present invention has multiple aspects, illustrated by the following non-limiting examples.
EXAMPLE 1
Production of MDS39
Reduced genome strain MDS39 was produced as described in International Patent Publication No. WO 2003/070880, which is incorporated herein by reference. Briefly, a series of reduced genome strains (MDS01-MDS39) were produced by constructing a series of cumulative deletions of nucleic acid sequences from the parental strain E. coli MG1655 (annotated version m56)(SEQ ID NO: 1).
EXAMPLE 2
Production of MDS40-MDS43
Hybridization to genome scanning chips (NimbleGen Systems, Madison, Wis.) containing the K-12 sequence and all sequences in the IS database revealed that MDS39, the first strain designed to lack all IS elements, unexpectedly contained additional copies of IS elements that had hopped to new locations during its production. These IS elements were deleted to produce MDS40. The fhuACDB (the tonA locus) was deleted from MDS40 to produce MDS41. Strains lacking the tonA locus are resistant to infection by bacteriophage T1, a common laboratory scourge. The endA gene was deleted from MDS41 to produce MDS42. Loss of the endA-encoded endonuclease facilitates plasmid preparation. MDS43 was produced by deleting an additional 45 kb covering the lac operon from parental strain MDS42. The resulting MDS strains were again characterized by DNA chip hybridization. As shown in FIG. 1, for MDS43 (and for MDS41 and 42; data not shown) there is no evidence for any contaminating IS elements. Rings depict features mapped to the genome of the parental E. coli K-12 strain MG1655, numbered on the outer ring. Moving outward from the center, rings 1-5 (grey) show regions of K-12 that are absent in other sequenced E. coli genomes: RS218, CFT073, S. flexneri 2457T, EDL933 and DH10B. Ring 6 shows the regions targeted for deletion: MDS12 (red), MDS41 (yellow), MDS42 (blue), and MDS43 (purple); half-height black bars indicate 4 rogue IS elements detected and cleanly removed during strain construction. Ring 7: native IS (green) and Rhs elements (light blue). Ring 8: experimental confirmation of the deletions in MDS43 by a NimbleGen tiling chip; probes corresponding to the intended deletions are colored green, other probes are colored red. On the outer ring the positions of the origin and terminus of replication and genes for rRNAs (blue), tRNAs (turquoise) and other small stable RNAs (black) are indicated. The genome characteristics of new MDS strains MDS41-43 are summarized in the Table 3:







TABLE 3







Genome statistics of MG1655 and related multiple deletion strains













MG1655
MDS12
MDS41
MDS42
MDS43















total no.
4444
4029
3743
3742
3704


genes







genome size
4639675
4263492
3977067
3976359
3931408


(bp)







replichore
30517
141360
139331
138623
183574


imbalance







(bp)







total no.
0
415
701
702
740


genes







deleted







total bp
0
376183
662608
663316
708267


DNA







deleted







% genome
0
8.11%
14.28%
14.30%
15.27%


deleted







total no. ISs
0
22
42
42
42


deleted









EXAMPLE 3
Detection of IS Contamination of Plasmid Preparations
A commercial preparation of pBR322 plasmid DNA, grown on DH10B according to the manufacturer, was compared to MDS grown plasmid. PCR amplification was done with inward and outward primers specific for IS1, IS2, IS3, IS5, IS10 and IS186 (FIG. 2, lanes 1-6 respectively; M is 1 kb+size standard). Outward primers (FIG. 2, panels e-h) detect circular structures, whereas inward primers (FIG. 2, panels a-d) detect both linear and circular IS forms. Positive controls were constructed by cloning each IS type (minus about 20 base pairs from its ends to prevent mobilization) into pBR322 (FIG. 2, panels b and f). FIG. 2, panels a and e are negative controls containing no DNA. Both sets of primers detected IS elements in the DH10B-grown prep (FIG. 2, panels c and g). The circular forms include some with sizes expected for a simple insertion of the IS into the plasmid, while others are consistent with a circle the size of the element itself. Cloning and sequencing further characterized products of the outward primer reactions. IS1, IS2, IS5 and IS10 gave examples of simple insertions at various positions in the plasmid. To detect mini-circle forms of IS elements, outward primed PCR reactions were directly sequenced with one of the primers. IS2 gave sequence results consistent with the presence of mini-circles. Plasmids grown in MDS42, on the other hand, contained no contaminating IS elements (FIG. 2, panels d and h). This indicates that removal of IS elements from host nucleic acids eliminates the hopping of IS elements from host nucleic acids to plasmid nucleic acids during cloning.
EXAMPLE 4
Growth Rate of and Recombinant Protein Production in Strains Lacking IS Elements
Strains MDS41, MDS42 and MDS43 were characterized for growth in standard microbiological media. FIG. 3(a) shows that each of the MDS strains can be grown to high cell densities in fed-batch fermentations on minimal medium. MDS41 was cultured in a minimal medium. Three growth phases were used to reach a dry cell weight (DCW) of 44 g/l. The first phase was a simple batch process. The second phase was a fed-batch process where the growth rate was controlled to 0.15 h−1. The third growth phase (also a fed-batch process) had a significantly lower controlled growth rate to prevent exceeding the oxygen transfer rate of the fermenter. For the targeted cell density, a controlled growth rate of 0.03 h−1 was used. FIG. 3(b) shows that the growth rate of MDS42 was essentially unchanged relative to the parental strain MG1655 in MOPS minimal medium at 37° C. Doubling time was obtained by measuring OD600 of cultures grown in baffled flasks at 37° C. The log linear portion of the growth curve was used to calculate the average doubling times and standard deviation from six replicates on the plate. The doubling time of MG1655 was 61.3 minutes compared to a doubling time of 69.07 minutes for MDS42. FIG. 3(b) shows that expression of recombinant proteins was similar for the MDS and MG1655 strains based on expression of the model protein chloramphenicol acetyl transferase (CAT).
EXAMPLE 5
Transformation Efficiency of Strains Lacking IS Elements
Strain MDS42 was compared to MG1655 and DH10B for transformation efficiency. Cells were grown under standard growth conditions to an optical density of 2.0 at 600 nm. Electrocompetent cells were prepared according to the method of Dower et al., Nucleic Acids Res (1988) 16, 6127-6145, incorporated herein by reference, and stored as frozen aliquots in 15% glycerol at a final optical density of 200 at 600 nm. Electroporation in an Eppendorf model 2510 instrument was at 18 kv/cm using 0.1 pg pUC 19 or 50 pg pCC145 DNA added to 0.1 ml of competent cells. The median of five electroporations is presented, each with a different batch of competent cells. For commercial competent cells of DH10B the five determinations were from different tubes of the same batch. With 20 kV/cm as recommended by their manufacturer, the commercial cells gave slightly higher values of 82.3×108 for pUC19 and 6.1×106 for pCC145 DNA. A t-test (p=0.002) indicates the transformation efficiency of MDS42 is significantly improved over MG1655 for electroporation with both large and small plasmid DNAs. As shown in Table 4, MDS42 has a substantially higher transformation efficiency compared to MG1655. Commercial competent cells are indicated by *:







TABLE 4







Electroporation Efficiency (transformants/μg DNA)













pCC145





(pCC1BAC TM-145;



Strain
pUC19 (2.686 kb)
Epicentre) (153 kb)







MG1655
 0.7 × 108
0



DH10B
 35.0 × 108
 1.9 × 106



DH10B*
 35.4 × 108
 6.5 × 106



MDS42
130.0 × 108
10.0 × 106










EXAMPLE 6
DNA Stability in Strains Lacking IS elements
FIG. 4(a) shows that the measured frequency of mutation by IS hopping dropped to zero in strains lacking IS elements. Briefly, populations of MDS41 and parental MG1655 were monitored for appearance of mutant cells that gained the ability to utilize salicin as a carbon source. Metabolism of salicin in E. coli K-12 requires activation of the bgl operon, which occurs primarily by integration of an IS element into the promoter region. Cell populations were grown to saturation in glucose/minimal medium, then spread on minimal plates containing salicin as the sole carbon source. New colonies (adaptive mutants) were marked and counted each day for 9 days. The data shown in FIG. 4(a) are the mean from three independent experiments. Total plated cell numbers were calculated by plating appropriate dilutions from 2-5 parallel cultures onto rich medium. The mean colony numbers were normalized to 2.5×109 cells. A two-factor ANOVA analysis (α≦0.05) with post-hoc (t-tests) was used to determine if the adaptation rates (the mutant number/day) were significant with respect to strain or time. Both the time and strain/plasmid were determined to be significant (p≦0.001). The interaction of time and strain/plasmid was determined not to be significant. Relative to MG1655, MDS41 displayed a >92% decrease in the rate of activation of the bgl operon. PCR analysis of the adaptive mutants indicates the decrease is due to the complete absence of IS-generated mutations in MDS41. FIG. 4(b) shows the spectrum of mutations in the bglR region of MG1655 and MDS41 cells adapted to salicin/minimal medium on day 9.
EXAMPLE 7
Stability of BAC libraries in Strains Lacking IS elements
Many large-scale sequencing projects of medical and commercial importance rely on large insert libraries commonly referred to as Bacterial Artificial Chromosomes (BACs). These recombinant constructs are composed of very large contiguous sequences of the subject DNA, on the order of 100 kb or larger, in combination with a selectable marker and a stable, low-copy number origin of replication to allow the molecule to replicate in a bacterial host. These molecules represent large targets for IS element insertion and are frequently subject to IS-mediated rearrangements, including deletions and inversions, which are difficult to detect by electronic bioinformatics methods. Minimizing bacterial DNA contamination (via IS element movement) represents an enormous improvement in the utility of BAC strategies for sequencing genomes. IS free strains of E. coli allow a precise measurement of the extent to which IS elements contaminate BAC DNA libraries.
A human BAC library created and maintained in E. coli DH10B was used to test the extent of IS element incorporation in BAC DNA. Forty-five random clones were picked from the 32,000 clone tiled human library collection and grown overnight in 1 ml LB cultures supplemented with chloramphenicol. BAC DNA was prepared using an Autogen 9600 robot. Each purified BAC DNA was subsequently transformed into the IS free MDS42recA host and approximately 384 colonies of each transformant were transferred to several duplicate Nylon membranes for hybridization screening with transposon specific probes.
In addition to the 45 independent BAC clones, three types of controls were also hybridized to membranes. These include a positive control (a) consisting of 384 individual E. coli DH10B colonies that did not contain any plasmid or BAC DNA. Two additional controls involved transformation of BAC DNA directly into the IS free MDS42recA strain, so that these BACs have never been grown in a bacterial host containing IS elements. The first of these controls (b) consisted of an approximately 150 kb BAC clone isolated from MDS42recA and subsequently re-transformed into the same bacterial host, 384 colonies of which were then transferred to Nylon membranes for hybridization analysis. A final control (c) involved the same BAC DNA, but rather than directly transforming this DNA into the IS-free host, the BAC DNA was mixed with an extract of DH10B produced by the Autogen robot containing only host chromosomal DNA fragments. This mixture was then transformed into the IS-free host and 384 of the resulting transformants were arrayed on a Nylon membrane for hybridization analysis. These three controls test the ability of the analysis to differentiate IS elements present in host chromosomal DNA versus BAC or plasmid DNA (control a), the lack of IS elements in BAC DNA that has never been propagated in an IS containing host (control b), and whether IS elements can be transferred in vitro, or by co-transformation with linear chromosomal DNA that contains IS elements (control c). Altogether 48 BAC combinations were tested, including 45 samples and 3 controls by arraying approximately 384 transformants of each BAC onto Nylon membranes for a total of 18,046 hybridization targets.
Probes for each of six IS element classes known to be present in E. coli DH 10B were designed to test the Nylon membranes for the presence of IS elements within the BAC DNA. These probes were produced by synthesizing two overlapping complimentary oligonucleotides which, when annealed leave single stranded overhangs which can be subsequently filled using the Klenow fragment of DNA polymerase in the presence of radio-labeled nucleotides to produce high specific activity double stranded probes specific to the most highly conserved region of each of the six IS classes. The oligonucleotide and corresponding probe sequences are shown in Table 5.







TABLE 5







Probe designs for IS1, IS2, IS3, IS5,


IS10 and IS186 family detection










IS
Oligo
Primer sequence
Probe sequence





IS1
IS1-Ova
5′CTTATGAGCCTGCTGTCACCCTTT
5′CTTATGAGCCTGCTGTCACC-



IS1-OVb
5′TCCATATCACCACGTCAAAGGGTG
CTTTGACGTGGTGATATGGA


 


IS2
IS2-OVa
5′GTGGCTGACGGATAATGGTTCATG
5′GTGGCTGACGGATAATGGTT-



IS2-OVb
5′TTCATTAGCCCGGTAGCATGAACC
CATGCTACCGGGCTAATGAA


IS3
IS3-OVa
5′CAGACGTCTTCTGAACGTGAACTG
5′CAGACGTCTTCTGAACGTGA-



IS3-OVb
5′TCTCGGTAGACATCTCCAGTTCAC
ACTGGAGATGTCTACCGAGA


 


IS5
IS5-OVa
5′GAGCAGATTCTGCCATGGCAAAAC
5′GAGCAGATTCTGCCATGGCA-



IS5-OVb
5′CGATGACTTCCACCATGTTTTGCC
AAACATGGTGGAAGTCATCG


 


IS10
IS10-OVa
5′ATTGCGAGCTTCAGTCGCACTACA
5′ATTGCGAGCTTCAGTCGCAC-



IS10-OVb
5′AGTAACAGAACGACCGTGTAGTGC
TACACGGTCGTTCTGTTACT


 


IS186
IS186-OVa
5′TTGCGTGCAAAGGAACCTGAACTC
5′TTGCGTGCAAAGGAACCTGA-



IS186-OVb
5′ATATCCACGCTTTCGCGAGTTCAG
ACTCGCGAAAGCGTGGATAT









Hybridizations, washes and probe stripping procedures were carried out under standard conditions (Current Protocols in Molecular Biology (1994) sections 2.9-2.10). Each membrane was probed with a mixture of all six labeled probes and by each probe individually. Results are summarized in Table 6.







TABLE 6







Frequency of IS contamination in individual BAC library clones
















Number of







Clone
Name
subclones
IS1
IS2
IS5
IS10
IS186

















1
C12_RP_4_V2(73)A1
369
1
14
1
11
0


2
C22_RP_1_V2(107)A1
369
1
10
2
5
0


3
CX_RP_3_V2(111)K1
377
2
5
0
13
10


4
C12_RP_2_V2(71)A1
384
1
1
0
5
20


5
C10_RP_3_V2(62)A1
384
1
11
4
11
0


6
C2_RP_7_V2(15)A1
378
2
0
0
2
0


7
C1_RP_5_V2(5)A1
378
0
0
0
2
0


8
C22_RP_1_V2(107)K1
378
1
7
4
10
0


9
C5_RP_3_V2(32)A1
384
3
10
1
1
0


10
C5_RP_7_V2(36)A1
384
0
5
0
0
0


11
C6_RP_5_V2(42)A1
377
17
2
0
1
0


12
C5_RP_7_V2(36)I1
380
0
0
0
1
0


13
C12_RP_4_V2(73)C1
368
0
2
0
3
0


14
C5_RP_3_V2(32)I1
368
0
6
0
15
0


15
CX_RP_3_V2(111)C1
367
0
6
1
11
23


16
C4_RP_3_V2(26)I1
304
1
4
0
186
1


17
C10_RP_3_V2(62)C1
379
0
0
0
0
0


18
C2_RP_7_V2(15)C1
377
0
2
0
2
0


19
C1_RP_5_V2(5)C1
380
2
10
2
5
33


20
C4_RP_3_V2(26)C1
380
2
52
0
14
0


21
C22_RP_1_V2(107)A1
375
0
12
0
1
6


22
C12_RP_4_V2(73)I1
379
4
15
2
4
0


23
Control a (1A5 in DH10B)
384
0
0
0
0
0


24
C9_RP_3_V2(57)C1
371
1
35
0
2
0


25
C12_RP_4_V2(73)E1
383
10
31
0
14
3


26
C22_RP1_1_V2(107)E1
379
1
10
1
27
0


27
CX_RP_3_V2(111)E1
382
0
11
1
33
0


28
C12_RP_2_V2(71)E1
384
1
6
0
6
0


29
C10_RP_3_V2(62)E1
381
4
9
1
7
0


30
C2_RP_7_V2(15)E1
383
0
11
0
5
0


31
C1_RP_5_V2(5)E1
384
1
3
1
1
0


32
C4_RP_3_V2(26)E1
382
2
14
1
8
0


33
C5_RP_3_V2(32)E1
382
6
7
0
4
0


34
C5_RP_7_V2(36)E1
351
4
4
6
14
0


35
C6_RP_5_V2(42)E1
380
2
12
0
1
3


36
C9_RP_3_V2(57)E1
381
6
15
5
16
5


37
C12_RP_4_V2(73)G1
382
0
7
0
1
0


38
Control b (1A5 in MDS42)
382
0
0
0
0
0


39
CX_RP_3_V2(111)G1
384
5
8
2
14
0


40
C12_RP_2_V2(71)G1
384
3
6
1
9
0


41
C10_RP_3_V2(62)G1
384
2
13
6
6
10


42
C2_RP_7_V2(15)G1
382
1
29
0
4
17


43
C1_RP_5_V2(5)G1
376
2
11
0
6
0


44
C4_RP_3_V2(26)G1
379
2
379
3
29
4


45
C5_RP_3_V2(32)G1
345
4
5
0
2
2


46
C5_RP_7_V2(36)G1
374
1
20
4
0
1


47
C6_RP_5_V2(42)G1
364
4
3
0
5
1


48
Control c (1A5 in MDS42 + DH10B DNA)
384
0
0
0
0
0









The lack of hybridization signal in any of the control hybridizations (clones 23, 38 and 48 in the table above) indicates that (a) DH 10B chromosomal DNA containing IS elements is not present in detectable quantities on the membranes, (b) no IS elements are present in DNA propagated solely in the IS-free strain MDS42recA and (c) no IS elements are transferred from DH10B chromosomal DNA to the BAC DNA in the course of the transformation procedure. The presence of IS elements in all but one (clone 17, BAC C10_RP—3_V2(62)C1) of the BACs isolated from DH10B, indicates that the hybridization strategy is an effective method for detecting IS elements on these membranes and that IS contamination is common in BACs propagated on strains containing IS elements. Together these data demonstrate that BAC libraries maintained in IS free bacterial hosts remain free of IS elements and therefore represent a superior technology for producing and maintaining BAC libraries.
In addition, IS free bacterial hosts can also be used to identify and isolate IS-free BACs from existing libraries by the transformation, arraying and probing strategy outlined in this example. For example, each of the 45 DH10B derived clones listed in Table 6 contains IS contaminated progeny as well as IS free progeny and the use of an IS free host, coupled with the hybridization screening strategy described here not only identifies, but also isolates, IS free clones from clones contaminated with IS elements. The resulting IS-free library would obviously be superior to the contaminated variant produced from IS containing bacteria.
EXAMPLE 8
Cloning of a Difficult to Clone Sequence in Strains Lacking IS Elements
Attempts to clone the open reading frame encoding the VP60 of rabbit haemorrhagic disease virus fused to the B subunit of cholera toxin (“CTXVP60 fusion construct”) using standard strains of bacteria have been unsuccessful. By providing a strain lacking IS elements, the CTXVP60 fusion construct was capable of being cloned. The surprising efficiency with which the CTXVP60 fusion construct was cloned in the strain lacking IS elements indicates that the presence of IS elements in the host chromosomal or extrachromosomal nucleic acids may be the primary obstacle to cloning such toxic genes. MDS42 was used to prepare pCTXVP60, carrying the CTXVP60 open reading frame. The plasmid DNA was then propagated in various hosts, isolated, then digested with NcoI and EcoRI. FIG. 5 shows a representative restriction pattern. The fragments were then purified and sequenced. MDS41 (lane 1) and MDS42 (lane 2) did not contain any insertion sequences. Plasmid DNA from DH10B contained an IS1 insertion (lane 3) and also an IS10 insertion/deletion (lane 4). Plasmid DNA from strain C600 contained an IS5 insertion (lane 5), and an IS1 insertion (lanes 6 and 7). This shows that strains lacking IS elements may be used to clone sequences that would be difficult to clone (e.g., toxic genes) in other strains.
EXAMPLE 9
Detection of IS Contamination of the Genome of a Host Carrying a Toxic Gene
Bystander mutation tests were performed on MG1655 transformed with pCTXVP60. Briefly, MG1655 cells were electroporated with either pCTX, carrying the CTX open reading frame (MG1655(pCTX)), or the toxic construct pCTXVP60 (MG1655(pCTXVP60)) and the cultures were grown to saturation, followed by spreading on salicin/minimal plates. New colonies (adaptive mutants) were marked and counted daily. The data shown in FIG. 6 were derived from five replicate experiments. Total plated cell numbers were calculated by plating appropriate dilutions from 2-5 parallel cultures onto rich medium. The mean colony numbers were normalized to 2.5×109 cells. A two-factor ANOVA analysis (α≦0.05) with post-hoc (t-tests) was used to determine if the adaptation rates (the mutant number/day) were significant with respect to strain or time. Both the time and strain/plasmid were determined to be significant (p≦0.001). The interaction of time and strain/plasmid was determined not to be significant. Counting the mutant colonies appearing on the plates revealed a>4-fold increase in the mutation rate due to pCTXVP60 (FIG. 6). Most of the bgl mutants of MG1655(pCTXVP60) also carry insertions in the plasmid.
EXAMPLE 10
Effect of Protein Overexpression on the Mutational Spectrum
Cultures of MG1655 carrying an expression plasmid for the well tolerated CAT enzyme were compared with and without IPTG induction. Using fluctuation tests, a 2.5-fold increase in IS transposition rates into cycA in cultures derived from IPTG-treated cells was found. Insertions involving IS1, IS2, IS5 and IS150 were observed, while point mutation rates remained virtually unchanged. (FIG. 7). This indicates that the over-expression of recombinant proteins may induce IS transposition. The strains lacking IS elements may therefore be a more stable host for protein production.
EXAMPLE 11
Cloning of a Difficult to Clone Plasmid that Does Not Encode a Protein Product in a Strain Lacking IS Elements
The plasmid pT-ITR, which contains a pair of G-C rich hairpins known as “hammerheads,” which are thermodynamically very stable, was propagated for 4 serial passages with a 106 dilution on each passage, in strains MDS42 and MG1655. FIG. 8 illustrates that 9T-ITR can be propagated intact for at least 4 serial passages in MDS42. However, 9T-ITR in the undeleted parent strain MG1655 is subject to deletion and rearrangement within the first passage.












TABLE 2





Strain
Type
Coordinates
Name
B
Function







MDS01
CDS
complement(262552 . . . 262893)

b0245
orf, hypothetical protein


MDS01
CDS
complement(262914 . . . 263231)
yafW
b0246
orf, hypothetical protein


MDS01
CDS
complement(263480 . . . 263956)
ykfG
b0247
putative DNA repair protein


MDS01
CDS
complement(263972 . . . 264430)
yafX
b0248
orf, hypothetical protein


MDS01
CDS
complement(264528 . . . 264767)
ykfF
b0249
orf, hypothetical protein


MDS01
CDS
complement(264844 . . . 265311)
ykfB
b0250
orf, hypothetical protein


MDS01
CDS
complement(265334 . . . 266191)
yafY
b0251
hypothetical transcriptional regulator


MDS01
CDS
complement(266408 . . . 267244)
yafZ
b0252
orf, hypothetical protein


MDS01
CDS
complement(267321 . . . 268187)
ykfA
b0253
putative GTP-binding protein


MDS01
CDS
complement(268513 . . . 269406)
perR
b0254
peroxide resistance protein


MDS01
CDS
269466 . . . 269870
yi91a
b0255



MDS01
CDS
269827 . . . 270978
tra8_1
b0256
IS30 transposase


MDS01
CDS
271054 . . . 271479

b0257
putative transposase


MDS01
CDS
272086 . . . 273216
ykfC
b0258
orf, hypothetical protein


MDS01
CDS
complement(273325 . . . 274341)
trs5_1
b0259
IS5 transposase


MDS01
CDS
274525 . . . 275952
ykfB
b0260
orf, hypothetical protein


MDS01
CDS
275939 . . . 276871
yafY
b0261
hypothetical transcriptional regulator


MDS01
CDS
complement(276980 . . . 278038)
afuC
b0262
putative ATP-binding component of a







transport system


MDS01
CDS
complement(278038 . . . 278400)

b0263



MDS01
CDS
complement(278402 . . . 278905)
insB_2
b0264
IS1 protein InsB


MDS01
CDS
complement(278824 . . . 279099)
insA_2
b0265
IS1 protein InsA


MDS01
CDS
complement(279609 . . . 279986)
yagB
b0266
orf, hypothetical protein


MDS01
CDS
complement(280053 . . . 281207)
yagA
b0267
orf, hypothetical protein


MDS01
CDS
281481 . . . 282410
yagE
b0268
putative lyase/synthase


MDS01
CDS
282425 . . . 284392
yagF
b0269
putative dehydratase


MDS01
CDS
284619 . . . 286001
yagG
b0270
putative permease


MDS01
CDS
286013 . . . 287623
yagH
b0271
putative beta-xylosidase (EC 3.2.1.37


MDS01
CDS
complement(287628 . . . 288386)
yagI
b0272
putative regulator


MDS01
CDS
complement(288525 . . . 289529)
argF
b0273
ornithine carbamoyltransferase 2, chain F


MDS01
CDS
complement(289873 . . . 290376)
insB_3
b0274
IS1 protein InsB


MDS01
CDS
complement(290295 . . . 290570)
insA_3
b0275
IS1 protein InsA


MDS01
CDS
290724 . . . 291455
yagJ
b0276
orf, hypothetical protein


MDS01
CDS
complement(291546 . . . 292172)
yagK
b0277
orf, hypothetical protein


MDS01
CDS
complement(292444 . . . 293142)
yagL
b0278
DNA-binding protein


MDS01
CDS
complement(293169 . . . 294023)
yagM
b0279
orf, hypothetical protein


MDS01
CDS
complement(294363 . . . 294803)
yagN
b0280
orf, hypothetical protein


MDS01
CDS
complement(294920 . . . 296320)
intF
b0281
putative phage integrase


MDS01
CDS
complement(296605 . . . 297015)
yagP
b0282
putative transcriptional regulator LYSR-type


MDS01
CDS
complement(296994 . . . 297950)
yagQ
b0283
orf, hypothetical protein


MDS01
CDS
complement(297960 . . . 300158)
yagR
b0284
orf, hypothetical protein


MDS01
CDS
complement(300155 . . . 301111)
yagS
b0285
orf, hypothetical protein


MDS01
CDS
complement(301108 . . . 301797)
yagT
b0286
putative xanthine dehydrogenase (EC







1.1.1.20


MDS01
CDS
302215 . . . 302829
yagU
b0287
orf, hypothetical protein


MDS01
CDS
complement(303077 . . . 303406)
ykgJ
b0288
putative ferredoxin


MDS01
CDS
complement(303719 . . . 304474)
yagV
b0289
orf, hypothetical protein


MDS01
CDS
complement(304398 . . . 306041)
yagW
b0290
putative receptor


MDS01
CDS
complement(306031 . . . 308556)
yagX
b0291
putative enzyme


MDS01
CDS
complement(308582 . . . 309250)
yagY
b0292
orf, hypothetical protein


MDS01
CDS
complement(309308 . . . 309895)
yagZ
b0293
orf, hypothetical protein


MDS01
CDS
complement(309970 . . . 310560)
ykgK
b0294
putative regulator


MDS01
CDS
311336 . . . 311563
ykgL
b0295
orf, hypothetical protein


MDS01
CDS
complement(311738 . . . 312001)
ykgM
b0296
putative ribosomal protein


MDS01
CDS
313581 . . . 314468
eaeH
b0297
attaching and effacing protein,







pathogenesis factor


MDS01
CDS
314506 . . . 314814

b0298



MDS01
CDS
314811 . . . 315677
tra5_5
b0299



MDS01
CDS
complement(315674 . . . 316393)
ykgA
b0300
putative ARAC-type regulatory protein


MDS01
CDS
complement(316950 . . . 317552)
ykgB
b0301
orf, hypothetical protein


MDS01
CDS
complement(317555 . . . 317806)
ykgI
b0303
orf, hypothetical protein


MDS01
CDS
complement(317900 . . . 319252)
ykgC
b0304
putative oxidoreductase


MDS01
CDS
319451 . . . 320305
ykgD
b0305
putative ARAC-type regulatory protein


MDS01
CDS
320832 . . . 321551
ykgE
b0306
putative dehydrogenase subunit


MDS01
CDS
321562 . . . 322989
ykgF
b0307
orf, hypothetical protein


MDS01
CDS
322829 . . . 323677
ykgG
b0308
putative transporter


MDS01
CDS
complement(323632 . . . 323844)

b0309
orf, hypothetical protein


MDS01
CDS
complement(323920 . . . 324588)
ykgH
b0310
orf, hypothetical protein


MDS02
CDS
complement(1398271 . . . 1399803)
ydaH
b1336



MDS02
CDS
complement(1399834 . . . 1401279)

b1337



MDS02
CDS
complement(1401279 . . . 1402604)
ydaJ
b1338



MDS02
CDS
1402765 . . . 1403673
ydaK
b1339
putative transcriptional regulator LYSR-type


MDS02
CDS
1404003 . . . 1404566
ydaL
b1340
orf, hypothetical protein


MDS02
CDS
complement(1404587 . . . 1405879)

b1341
orf, hypothetical protein


MDS02
CDS
1406074 . . . 1407057

b1342
orf, hypothetical protein


MDS02
CDS
1407535 . . . 1408908
dbpA
b1343
ATP-dependent RNA helicase


MDS02
CDS
complement(1409037 . . . 1409972)
ydaO
b1344
orf, hypothetical protein


MDS02
CDS
complement(1410024 . . . 1411259)

b1345



MDS02
CDS
complement(1411261 . . . 1411500)
ydaQ
b1346
putative lambdoid prophage Rac







excisionase


MDS02
CDS
complement(1411555 . . . 1411764)
ydaC
b1347
orf, hypothetical protein


MDS02
CDS
complement(1411757 . . . 1411951)
lar
b1348
restriction alleviation and modification







enhancement


MDS02
CDS
complement(1412008 . . . 1412817)
recT
b1349
recombinase, DNA renaturation


MDS02
CDS
complement(1412810 . . . 1415410)
recE
b1350
exonuclease VIII, ds DNA exonuclease 5′-->







3′ specific


MDS02
CDS
complement(1415512 . . . 1415787)
racC
b1351
RacC protein


MDS02
CDS
complement(1416032 . . . 1416265)
kil
b1352
Kil protein (killing function) of lambdoid







prophage Rac


MDS02
CDS
1416572 . . . 1417183
sieB
b1353
phage superinfection exclusion protein


MDS02
CDS
1417192 . . . 1417368

b1354
orf, hypothetical protein


MDS02
CDS
complement(1417346 . . . 1417525)

b1355
orf, hypothetical protein


MDS02
CDS
complement(1417789 . . . 1418265)
ydaR
b1356



MDS02
CDS
1418389 . . . 1418685
ydaS
b1357
orf, hypothetical protein


MDS02
CDS
1418708 . . . 1419130
ydaT
b1358
orf, hypothetical protein


MDS02
CDS
1419143 . . . 1420000
ydaU
b1359
orf, hypothetical protein


MDS02
CDS
1420007 . . . 1420753

b1360
putative DNA replication factor


MDS02
CDS
1420725 . . . 1421336
ydaW
b1361
orf, hypothetical protein


MDS02
CDS
1421363 . . . 1421668

b1362
putative Rac prophage endopeptidase


MDS02
CDS
1421806.1423263
trkG
b1363



MDS02
CDS
1423202 . . . 1423483

b1364
orf, hypothetical protein


MDS02
CDS
1423401 . . . 1423664

b1365
orf, hypothetical protein


MDS02
CDS
1423645 . . . 1424004
ydaY
b1366
orf, hypothetical protein


MDS02
CDS
1424079 . . . 1424312

b1367
orf, hypothetical protein


MDS02
CDS
1424478 . . . 1425506

b1368
putative alpha helix protein


MDS02
CDS
1425482 . . . 1425637

b1369
orf, hypothetical protein


MDS02
CDS
complement(1425770 . . . 1426750)
trs5_5
b1370



MDS02
CDS
1426547 . . . 1427008

b1371
orf, hypothetical protein


MDS02
CDS
1427067 . . . 1430435

b1372



MDS02
CDS
1430435 . . . 1431010

b1373
tail fiber assembly protein homolog from







lambdoid prophage Rac


MDS02
CDS
complement(1431108 . . . 1431698)

b1374



MDS02
CDS
complement(1432015 . . . 1432281)
ynaE
b1375
orf, hypothetical protein


MDS02
CDS
complement(1433209 . . . 1433715)

b1376
ynaF putative filament protein


MDS02
CDS
complement(1433784 . . . 1434917)

b1377



MDS02
CDS
complement(1435284 . . . 1438808)
ydbK
b1378
putative oxidoreductase, Fe—S subunit


MDS02
CDS
complement(1439345 . . . 1439767)
hslJ
b1379
heat shock protein HslJ


MDS02
CDS
complement(1439878 . . . 1440867)
ldhA
b1380
fermentative D-lactate dehydrogenase NAD-







dependent


MDS02
CDS
1441075 . . . 1443714
ydbH
b1381
orf, hypothetical protein


MDS02
CDS
1443711 . . . 1443896
ynbE
b1382
orf, hypothetical protein


MDS02
CDS
1443898 . . . 1444230
ydbL
b1383
orf, hypothetical protein


MDS02
CDS
complement(1444402 . . . 1445307)
feaR
b1384
regulatory protein for 2-phenylethylamine







catabolism


MDS02
CDS
1445540 . . . 1447042
feaB
b1385
phenylacetaldehyde dehydrogenase


MDS02
CDS
complement(1447100 . . . 1449373)
tynA
b1386
copper amine oxidase (tyramine oxidase)


MDS02
CDS
complement(1449621 . . . 1451666)
maoC
b1387



MDS02
CDS
1451951 . . . 1452880
ydbO
b1388



MDS02
CDS
1452892 . . . 1453179
ynbF
b1389



MDS02
CDS
1453188 . . . 1453934
ydbP
b1390



MDS02
CDS
1453943 . . . 1454446

b1391



MDS02
CDS
1454454 . . . 1455524

b1392



MDS02
CDS
1455521 . . . 1456288
ydbS
b1393



MDS02
CDS
1456288 . . . 1457076

b1394



MDS02
CDS
1457078 . . . 1458505
ydbU
b1395



MDS02
CDS
1458495 . . . 1458917

b1396



MDS02
CDS
1458917 . . . 1460122

b1397



MDS02
CDS
1460149 . . . 1461462

b1398



MDS02
CDS
1461563 . . . 1462513

b1399



MDS02
CDS
1462495 . . . 1463085

b1400



MDS02
CDS
1463416 . . . 1465974
ydbA_1
b1401



MDS02
CDS
complement(1465945 . . . 1466850)
yi22_2
b1402
IS2 hypothetical protein


MDS02
CDS
complement(1466808 . . . 1467218)
yi21_2
b1403
IS2 hypothetical protein


MDS02
CDS
1467382 . . . 1468533
tra8_2
b1404
IS30 transposase


MDS02
CDS
1468714 . . . 1472037
ydbA_2
b1405



MDS02
CDS
1472245 . . . 1473105
ydbC
b1406
putative dehydrogenase


MDS02
CDS
1473162 . . . 1475474
ydbD
b1407
orf, hypothetical protein


MDS02
CDS
1475639 . . . 1476250

b1408



MDS02
CDS
1476250 . . . 1477146

b1409
putative phosphatidate cytidiltransferase


MDS02
CDS
1477162 . . . 1478919

b1410
orf, hypothetical protein


MDS02
CDS
1478933 . . . 1480225
ynbD
b1411
putative enzymes


MDS02
misc_RNA
complement(1403676 . . . 1403833)
IS061
b4426



MDS02
misc_RNA
1435145 . . . 1435252
tke8
b4427



MDS03
CDS
complement(2555340 . . . 2556743)
eutB
b2441
ethanolamine ammonia-lyase, heavy chain


MDS03
CDS
2556793 . . . 2558088

b2442
putative prophage integrase


MDS03
CDS
2558279 . . . 2558920

b2443
orf, hypothetical protein


MDS03
CDS
2559390 . . . 2559635

b2444
orf, hypothetical protein


MDS03
CDS
2559632 . . . 2560015

b2445
orf, hypothetical protein


MDS03
CDS
2560133 . . . 2560549

b2446
orf, hypothetical protein


MDS03
CDS
2560546 . . . 2561139

b2447
orf, hypothetical protein


MDS03
CDS
2561599 . . . 2561991

b2448
orf, hypothetical protein


MDS03
CDS
2562002 . . . 2562394

b2449
orf, hypothetical protein


MDS03
CDS
2562515 . . . 2563354

b2450
orf, hypothetical protein


MDS04
CDS
2754181 . . . 2755422
intA
b2622
prophage CP4-57 integrase


MDS04
CDS
complement(2755666 . . . 2756622)
yfjH
b2623
putative histone


MDS04
CDS
2756666 . . . 2756878
alpA
b2624
prophage CP4-57 regulatory protein alpA


MDS04
CDS
2757007 . . . 2758416
yfjI
b2625
orf, hypothetical protein


MDS04
CDS
2758569 . . . 2759195
yfjJ
b2626
orf, hypothetical protein


MDS04
CDS
complement(2759373 . . . 2761562)
yfjK
b2627
orf, hypothetical protein


MDS04
CDS
complement(2761559 . . . 2763175)
yfjL
b2628
orf, hypothetical protein


MDS04
CDS
complement(2763535 . . . 2763798)
yfjM
b2629
orf, hypothetical protein


MDS04
CDS
2763940 . . . 2765013
yfjN
b2630
putative cell division protein


MDS04
CDS
2765057 . . . 2765377
yfjO
b2631
orf, hypothetical protein


MDS04
CDS
2765732 . . . 2766595
yfjP
b2632
putative GTP-binding protein


MDS04
CDS
2766687 . . . 2767508
yfjQ
b2633
orf, hypothetical protein


MDS04
CDS
2767725 . . . 2768426
yfjR
b2634
orf, hypothetical protein


MDS04
CDS
2768311 . . . 2768703

b2635
orf, hypothetical protein


MDS04
CDS
2768454 . . . 2769146

b2636
orf, hypothetical protein


MDS04
CDS
2769170 . . . 2769637
yfjT
b2637
orf, hypothetical protein


MDS04
CDS
complement(2769862 . . . 2770176)

b2638
orf, hypothetical protein


MDS04
CDS
complement(2770189 . . . 2770707)

b2639
putative pump protein


MDS04
CDS
complement(2770858 . . . 2771058)

b2640
orf, hypothetical protein


MDS04
CDS
complement(2770998 . . . 2771114)

b2641
orf, hypothetical protein


MDS04
CDS
2771340 . . . 2773043
yfjW
b2642
orf, hypothetical protein


MDS04
CDS
2773941 . . . 2774399
yfjX
b2643
orf, hypothetical protein


MDS04
CDS
2774408 . . . 2774890
yfjY
b2644
putative DNA repair protein


MDS04
CDS
2775137 . . . 2775454
yfjZ
b2645
orf, hypothetical protein


MDS04
CDS
2775475 . . . 2775804
ypjF
b2646
orf, hypothetical protein


MDS04
CDS
complement(2776168 . . . 2780877)
ypjA
b2647
putative ATP-binding component of a







transport system


MDS04
CDS
complement(2781087 . . . 2781230)

b2648
orf, hypothetical protein


MDS04
CDS
complement(2781660 . . . 2782451)

b2649
orf, hypothetical protein


MDS04
CDS
complement(2782551 . . . 2783033)

b2650
orf, hypothetical protein


MDS04
CDS
2783243 . . . 2783374

b2651
orf, hypothetical protein


MDS04
tRNA
complement(2783784 . . . 2783859)
ileY
b2652
tRNA-Ile


MDS04
CDS
complement(2783822 . . . 2783995)

b2653
orf, hypothetical protein


MDS04
CDS
2784419 . . . 2784751

b2654
orf, hypothetical protein


MDS04
CDS
2785628 . . . 2786260

b2657
putative enzyme


MDS04
CDS
2786399 . . . 2786671

b2658
orf, hypothetical protein


MDS04
CDS
2786902 . . . 2787984

b2659
orf, hypothetical protein


MDS04
CDS
2787938 . . . 2789272
ygaF
b2660
orf, hypothetical protein


MDS04
CDS
2784770 . . . 2785456
ygaR
b4462
orf, hypothetical protein


MDS05
CDS
complement(2064329 . . . 2065345)
trs5_6
b1994
IS5 transposase


MDS05
CDS
2066632 . . . 2067051

b1995
orf, hypothetical protein


MDS05
CDS
complement(2066976 . . . 2067881)
yi22_3
b1996
IS2 hypothetical protein


MDS05
CDS
complement(2067839 . . . 2068249)
yi21_3
b1997
IS2 hypothetical protein


MDS05
CDS
2068268 . . . 2068528

b1998
orf, hypothetical protein


MDS05
CDS
2068525 . . . 2069235
yeeP
b1999
putative histone


MDS05
CDS
2069563 . . . 2072682
flu
b2000
antigen 43, phase-variable bipartite outer







membrane fluffing protein


MDS05
CDS
2072797 . . . 2074335

b2001
orf, hypothetical protein


MDS05
CDS
2074332 . . . 2074778
yeeS
b2002
putative DNA repair protein, RADC family


MDS05
CDS
2074841 . . . 2075062
yeeT
b2003
orf, hypothetical protein


MDS05
CDS
2075136 . . . 2075504
yeeU
b2004
putative structural protein


MDS05
CDS
2075593 . . . 2075967
yeeV
b2005
orf, hypothetical protein


MDS05
CDS
2075964 . . . 2076158
yeeW
b2006
orf, hypothetical protein


MDS05
CDS
complement(2077056 . . . 2077451)
veeX
b2007
putative alpha helix protein


MDS05
CDS
complement(2077557 . . . 2078615)
yeeA
b2008
orf, hypothetical protein


MDS05
misc_RNA
2069339 . . . 2069542

b4435
IS102


MDS06
CDS
complement(3451530 . . . 3451949)

b3322
calcium-binding protein required for







initiation of chromosome replication


MDS06
CDS
complement(3451951 . . . 3453420)
yheD
b3323
putative export protein A for general







secretion pathway (GSP)


MDS06
CDS
3453600 . . . 3454415
yheE
b3324



MDS06
CDS
3454387 . . . 3456351
yheF
b3325



MDS06
CDS
3456361 . . . 3457842
yheG
b3326



MDS06
CDS
3457839 . . . 3459035
hofF
b3327



MDS06
CDS
3459045 . . . 3459482
hofG
b3328



MDS06
CDS
3459490 . . . 3459999
hofH
b3329



MDS06
CDS
3459957 . . . 3460373
yheH
b3330



MDS06
CDS
3460366 . . . 3460953
yheI
b3331



MDS06
CDS
3460946 . . . 3461929
yheJ
b3332



MDS06
CDS
3461941 . . . 3463107
yheK
b3333



MDS06
CDS
3463080 . . . 3463565
pshM
b3334



MDS06
CDS
3463565 . . . 3464242
hofD
b3335



MDS06
CDS
complement(3464271 . . . 3464747)
bfr
b3336
bacterioferrin, an iron storage homoprotein


MDS06
CDS
complement(3464819 . . . 3465013)
yheA
b3337



MDS06
CDS
complement(3465182 . . . 3467875)
yheB
b3338



MDS07
CDS
2464567 . . . 2465724
intC
b2349



MDS07
CDS
2465877 . . . 2466239

b2350



MDS07
CDS
2471542 . . . 2471988

b2350
orf, hypothetical protein


MDS07
CDS
2466236 . . . 2467156

b2351



MDS07
CDS
2467153 . . . 2468484

b2352
putative ligase


MDS07
CDS
2468783 . . . 2469127

b2353



MDS07
CDS
complement(2469099 . . . 2469539)

b2354
orf, hypothetical protein


MDS07
CDS
complement(2469566 . . . 2470084)
yfdL
b2355
putative RNA polymerase beta


MDS07
CDS
complement(2470134 . . . 2470442)
yfdM
b2356
orf, hypothetical protein


MDS07
CDS
complement(2470409 . . . 2470903)
yfdN
b2357
orf, hypothetical protein


MDS07
CDS
complement(2470900 . . . 2471268)
yfdO
b2358
orf, hypothetical protein


MDS07
CDS
2472054 . . . 2472878

b2360
orf, hypothetical protein


MDS07
CDS
2472979 . . . 2473542

b2361
orf, hypothetical protein


MDS07
CDS
2473533 . . . 2473895

b2362
orf, hypothetical protein


MDS07
CDS
2473895 . . . 2474200

b2363
orf, hypothetical protein


MDS08
CDS
1625541 . . . 1626287
ydfG
b1539
putative oxidoreductase


MDS08
CDS
1626376 . . . 1627062
ydfH
b1540
orf, hypothetical protein


MDS08
CDS
1627239 . . . 1627442

b1541
orf, hypothetical protein


MDS08
CDS
complement(1627477 . . . 1628937)
ydfI
b1542
putative oxidoreductase


MDS08
CDS
complement(1629026 . . . 1630309)

b1543
putative transport protein


MDS08
CDS
1631063 . . . 1631329
ydfK
b1544
orf, hypothetical protein


MDS08
CDS
1631646 . . . 1632236

b1545



MDS08
CDS
complement(1632334 . . . 1632909)
ydfM
b1546
tail fiber assembly protein homolog from







lambdoid prophage Qin


MDS08
CDS
complement(1632909 . . . 1633871)

b1547



MDS08
CDS
complement(1633822 . . . 1634391)
nohA
b1548
DNA packaging protein NU1 homolog from







lambdoid prophages


MDS08
CDS
1635056 . . . 1635481
ydfO
b1549
orf, hypothetical protein


MDS08
CDS
complement(1635633 . . . 1635809)

b1550



MDS08
CDS
complement(1635978 . . . 1636169)

b1551
orf, hypothetical protein


MDS08
CDS
complement(1636479 . . . 1636691)
cspI
b1552
cold shock-like protein


MDS08
CDS
complement(1637054 . . . 1637551)

b1553
orf, hypothetical protein


MDS08
CDS
complement(1637548 . . . 1638081)

b1554



MDS08
CDS
complement(1638078 . . . 1638389)

b1555
orf, hypothetical protein


MDS08
CDS
complement(1638394 . . . 1638684)

b1556



MDS08
CDS
complement(1639363 . . . 1639578)
cspB
b1557



MDS08
CDS
1639879 . . . 1640091
cspF
b1558



MDS08
CDS
complement(1640513 . . . 1641295)

b1559



MDS08
CDS
complement(1641279 . . . 1642367)

b1560
orf, hypothetical protein


MDS08
CDS
complement(1642675 . . . 1642926)
rem
b1561
orf, hypothetical protein


MDS08
CDS
complement(1643143 . . . 1643298)
hokD
b1562
polypeptide destructive to membrane







potential


MDS08
CDS
complement(1643370 . . . 1643657)
relE
b1563
orf, hypothetical protein


MDS08
CDS
complement(1643657 . . . 1643896)
relB
b1564
negative regulator of translation


MDS08
CDS
1643921 . . . 1644226

b1565
orf, hypothetical protein


MDS08
CDS
1644429 . . . 1644761
flxA
b1566
orf, hypothetical protein


MDS08
CDS
complement(1645198 . . . 1645347)

b1567
orf, hypothetical protein


MDS08
CDS
complement(1645370 . . . 1645660)

b1568
orf, hypothetical protein


MDS08
CDS
complement(1645644 . . . 1645874)
dicC
b1569
regulator of dicB


MDS08
CDS
1645958 . . . 1646365
dicA
b1570
regulator of dicB


MDS08
CDS
1646532 . . . 1646687
ydfA
b1571
orf, hypothetical protein


MDS08
CDS
1646647 . . . 1646817
ydfB
b1572
orf, hypothetical protein


MDS08
CDS
1646847 . . . 1647065
ydfC
b1573
orf, hypothetical protein


MDS08
misc_RNA
1647406 . . . 1647458
dicF
b1574
DicF antisense RNA, inhibits ftsZ







translation


MDS08
CDS
1647633 . . . 1647821
dicB
b1575
inhibition of cell division


MDS08
CDS
1647818 . . . 1648009
ydfD
b1576
orf, hypothetical protein


MDS08
CDS
1648102 . . . 1649022
ydfE
b1577
orf, hypothetical protein


MDS08
CDS
1648905 . . . 1649561

b1578
orf, hypothetical protein


MDS08
CDS
1649536 . . . 1650732

b1579



MDS09
CDS
complement(4493213 . . . 4494274)
yjgB
b4269
putative oxidoreductase


MDS09
tRNA
4494428 . . . 4494512
leuX
b4270
tRNA-Leu


MDS09
CDS
4494773 . . . 4495963
intB
b4271
prophage P4 integrase


MDS09
CDS
4496250 . . . 4496660
yi21_6
b4272
IS2 hypothetical protein


MDS09
CDS
4496618 . . . 4497523
yi22_6
b4273
IS2 hypothetical protein


MDS09
CDS
4497622 . . . 4497957
yjgW
b4274
orf, hypothetical protein


MDS09
CDS
complement(4498066 . . . 4498512)
yjgX
b4275
orf, hypothetical protein


MDS09
CDS
complement(4498455 . . . 4498904)
yjgY
b4276
orf, hypothetical protein


MDS09
CDS
4499283 . . . 4499612
yjgZ
b4277
orf, hypothetical protein


MDS09
CDS
complement(4500126 . . . 4501454)
yi41
b4278
IS4 hypothetical protein


MDS09
CDS
4502021 . . . 4503298
yjhB
b4279
putative transport protein


MDS09
CDS
4503295 . . . 4504428
yjhC
b4280
putative dehydrogenase


MDS09
CDS
complement(4504649 . . . 4505023)
yjhD
b4281
orf, hypothetical protein


MDS09
CDS
4504929 . . . 4505132
yjhE
b4282
orf, hypothetical protein


MDS09
CDS
4505184 . . . 4505486
yi91b
b4283



MDS09
CDS
complement(4505489 . . . 4506640)
tra8_3
b4284
IS30 transposase


MDS09
CDS
4506981 . . . 4507577

b4285
putative transposase


MDS09
CDS
4507743 . . . 4508156

b4286
orf, hypothetical protein


MDS09
CDS
complement(4508713 . . . 4509480)
fecE
b4287
ATP-binding component of citrate-







dependent


MDS09
CDS
complement(4509481 . . . 4510437)
fecD
b4288
citrate-dependent iron transport, membrane-







bound protein


MDS09
CDS
complement(4510434 . . . 4511432)
fecC
b4289
citrate-dependent iron(III) transport protein,







cytosolic


MDS09
CDS
complement(4511429 . . . 4512337)
fecB
b4290
citrate-dependent iron transport,







periplasmic protein


MDS09
CDS
complement(4512376 . . . 4514700)
fecA
b4291
outer membrane receptor; citrate-







dependent iron


MDS09
CDS
complement(4514787 . . . 4515740)
fecR
b4292
outer membrane receptor; citrate-







dependent iron transport, outer membrane







receptor


MDS09
CDS
complement(4515737 . . . 4516258)
fecI
b4293
probable RNA polymerase sigma factor


MDS09
CDS
4516550 . . . 4516825
insA_7
b4294
IS1 protein InsA


MDS09
CDS
complement(4517361 . . . 4518161)
yjhU
b4295
orf, hypothetical protein


MDS09
CDS
complement(4518694 . . . 4520043)
yihF
b4296
putative transport system permease


MDS09
CDS
complement(4520150 . . . 4522117)
yjhG
b4297
putative dehydratase


MDS09
CDS
complement(4522128 . . . 4523087)
yjhH
b4298
putative lyase/synthase


MDS09
CDS
complement(4523038 . . . 4523826)
yjhI
b4299
putative regulator


MDS09
CDS
complement(4524129 . . . 4524911)
sgcR
b4300
putative DEOR-type transcriptional







regulator


MDS09
CDS
complement(4524928 . . . 4525560)
sgcE
b4301
putative epimerase


MDS09
CDS
complement(4525572 . . . 4526003)
sgcA
b4302
putative PTS system enzyme II A







component


MDS09
CDS
complement(4526134 . . . 4526940)
sgcQ
b4303
putative nucleoside triphosphatase


MDS09
CDS
complement(4526953 . . . 4528266)
sgcC
b4304
putative PTS system enzyme IIC







component


MDS09
CDS
complement(4528553 . . . 4529704)
sgcX
b4305
putative lyase/synthase


MDS09
CDS
complement(4530460 . . . 4531206)
yjhP
b4306
putative methyltransferase


MDS09
CDS
complement(4531262 . . . 4531807)
yjhQ
b4307
orf, hypothetical protein


MDS09
CDS
4533038 . . . 4534054
yjhR
b4308
putative frameshift suppressor′′


MDS09
CDS
complement(4534637 . . . 4535617)
yjhS
b4309
orf, hypothetical protein


MDS09
CDS
complement(4535682 . . . 4536896)
yjhT
b4310
orf, hypothetical protein


MDS09
CDS
complement(4536808 . . . 4537533)
yjhA
b4311
orf, hypothetical protein


MDS09
CDS
4538980 . . . 4539582
fimB
b4312
recombinase involved in phase variation;







regulator for fimA′′


MDS09
CDS
4540060 . . . 4540656
fimE
b4313
recombinase involved in phase variation;







regulator for fimA′′


MDS09
CDS
4541138 . . . 4541686
fimA
b4314
major type 1 subunit fimbrin (pilin)


MDS09
CDS
4541643 . . . 4542290
fimI
b4315
fimbrial protein


MDS09
CDS
4542327 . . . 4543052
fimC
b4316
periplasmic chaperone, required for type 1







fimbriae


MDS09
CDS
4543119 . . . 4545755
fimD
b4317
outer membrane protein; export and







assembly of type 1 fimbriae, interrupted


MDS09
CDS
4545765 . . . 4546295
fimF
b4318
fimbrial morphology


MDS09
CDS
4546308 . . . 4546811
fimG
b4319
fimbrial morphology


MDS09
CDS
4546831 . . . 4547733
fimH
b4320
minor fimbrial subunit, D-mannose specific







adhesin


MDS10
CDS
complement(3108612 . . . 3109148)
yghD
b2968
putative secretion pathway protein


MDS10
CDS
complement(3109150 . . . 3110010)
yghE
b2969
putative general secretion pathway for







protein export (GSP)


MDS10
CDS
complement(3110076 . . . 3110942)

b2970
putative general secretion pathway for







protein export (GSP)


MDS10
CDS
complement(3111089 . . . 3111499)

b2971
orf, hypothetical protein


MDS10
CDS
complement(3111565 . . . 3112497)

b2972



MDS10
CDS
complement(3117619 . . . 3119301)
yghK
b2975
putative permease


MDS10
CDS
complement(3119656 . . . 3121827)
glcB
b2976
malate synthase G


MDS10
CDS
complement(3121849 . . . 3122253)
glcG
b2977
orf, hypothetical protein


MDS10
CDS
complement(3124544 . . . 3126043)
glcD
b2979
glycolate oxidase subunit D


MDS10
CDS
3126294 . . . 3127058
glcC
b2980
transcriptional activator for glc operon


MDS10
CDS
complement(3127065 . . . 3128237)

b2981
orf, hypothetical protein


MDS10
CDS
3128200 . . . 3129216
trs5_9
b2982
IS5 transposase


MDS10
CDS
complement(3129363 . . . 3130430)
yghQ
b2983
orf, hypothetical protein


MDS10
CDS
complement(3130476 . . . 3131234)
yghR
b2984
orf, hypothetical protein


MDS10
CDS
complement(3131266 . . . 3131979)
yghS
b2985
orf, hypothetical protein


MDS10
CDS
3132153 . . . 3132845
yghT
b2986
orf, hypothetical protein


MDS10
CDS
complement(3132894 . . . 3134393)
pitB
b2987
low-affinity phosphate transport


MDS10
CDS
complement(3112572 . . . 3117134)
yghJ
b4466
putative lipoprotein


MDS10
CDS
complement(3122258 . . . 3123481)
glcF
b4467
glycolate oxidase iron-sulfur subunit


MDS10
CDS
complement(3123492 . . . 3124544)
glcE
b4468
glycolate oxidase iron-sulfur subunit


MDS11
CDS
complement(1196090 . . . 1196755)
ymfD
b1137
orf, hypothetical protein


MDS11
CDS
complement(1196756 . . . 1197460)
ymfE
b1138
orf, hypothetical protein


MDS11
CDS
1197918 . . . 1198811
lit
b1139
phage T4 late gene expression; at locus of







e14 element


MDS11
CDS
complement(1198902 . . . 1200029)
intE
b1140
prophage e14 integrase


MDS11
CDS
complement(1200010 . . . 1200255)

b1141



MDS11
CDS
complement(1200292 . . . 1200603)
ymfH
b1142



MDS11
CDS
1200675 . . . 1201061
ymfI
b1143



MDS11
CDS
complement(1200999 . . . 1201283)
ymfJ
b1144



MDS11
CDS
complement(1201482 . . . 1202156)

b1145



MDS11
CDS
1201944 . . . 1202447

b1146



MDS11
CDS
1202479 . . . 1203048
ymfL
b1147



MDS11
CDS
1203045 . . . 1203383
ymfM
b1148



MDS11
CDS
1203393 . . . 1204760
ymfN
b1149



MDS11
CDS
1204772 . . . 1204954
ymfR
b1150



MDS11
CDS
1204954 . . . 1205427
ymfO
b1151



MDS11
CDS
1205354 . . . 1206145

b1152



MDS11
CDS
1206136 . . . 1206720

b1153



MDS11
CDS
1206724 . . . 1207353
ycfK
b1154



MDS11
CDS
1207355 . . . 1207768

b1155



MDS11
CDS
complement(1207740 . . . 1208342)
ycfA
b1156



MDS11
CDS
complement(1208342 . . . 1208881)

b1157



MDS11
CDS
1208908 . . . 1209462
pin
b1158
inversion of adjacent DNA; at locus of e14







element


MDS11
CDS
1209569 . . . 1210402
mcrA
b1159
restriction of DNA at 5-methylcytosine







residues; at locus of e14 element


MDS11
CDS
complement(1210903 . . . 1211226)
ycgW
b1160
orf, hypothetical protein


MDS11
CDS
complement(1211926 . . . 1212330)
ycgX
b1161
orf, hypothetical protein


MDS11
CDS
complement(1212551 . . . 1213282)
ycgE
b1162
putative transcriptional regulator


MDS11
CDS
complement(1213487 . . . 1214698)

b1163
orf, hypothetical protein


MDS11
CDS
1215012 . . . 1215248
ycgZ
b1164
orf, hypothetical protein


MDS11
CDS
1215291 . . . 1215563
ymgA
b1165
orf, hypothetical protein


MDS11
CDS
1215592 . . . 1215858
ymgB
b1166
orf, hypothetical protein


MDS11
CDS
1215971 . . . 1216219
ymgC
b1167
orf, hypothetical protein


MDS11
CDS
1216509 . . . 1218074

b1168
putative proteases


MDS11
CDS
1218824 . . . 1220344

b1169
putative ATP-binding component of a







transport system


MDS11
CDS
1220429 . . . 1221445

b1170



MDS11
CDS
complement(1221528 . . . 1221863)

b1171
orf, hypothetical protein


MDS11
CDS
complement(1221867 . . . 1222151)

b1172
orf, hypothetical protein


MDS12
CDS
complement(564038 . . . 565201)
intD
b0537
prophage DLP12 integrase


MDS12
CDS
565195 . . . 565755

b0538
putative sensory transduction regulator


MDS12
CDS
complement(565321 . . . 565584)

b0539



MDS12
CDS
566056 . . . 566364

b0540



MDS12
CDS
566361 . . . 567227
tra5_2
b0541



MDS12
CDS
567333 . . . 567470

b0542
orf, hypothetical protein


MDS12
CDS
567538 . . . 567870
emrE
b0543
methylviologen resistance


MDS12
CDS
568125 . . . 569651
ybcK
b0544
orf, hypothetical protein


MDS12
CDS
570116 . . . 570667
ybcL
b0545
orf, hypothetical protein


MDS12
CDS
570677 . . . 571474
ybcM
b0546
putative ARAC-type regulatory protein


MDS12
CDS
571689 . . . 572144
ybcN
b0547
orf, hypothetical protein


MDS12
CDS
572144 . . . 572314
ninE
b0548
similar to phage 82 and lambda proteins


MDS12
CDS
572307 . . . 572597
ybcO
b0549
orf, hypothetical protein


MDS12
CDS
572594 . . . 572956
rus
b0550
endodeoxyribonuclease RUS (Holliday







junction resolvase)


MDS12
CDS
573179 . . . 573562
ybcQ
b0551
orf, hypothetical protein


MDS12
CDS
complement(573960 . . . 574976)
trs5_2
b0552
IS5 transposase


MDS12
CDS
complement(574981 . . . 576108)
nmpC
b0553
outer membrane porin protein; locus of qsr







prophage


MDS12
CDS
576621 . . . 576836
ybcR
b0554
orf, hypothetical protein


MDS12
CDS
576836 . . . 577333
ybcS
b0555
bacteriophage lambda lysozyme homolog


MDS12
CDS
577330 . . . 577791
ybcT
b0556
bacteriophage lambda endopeptidase







homolog


MDS12
CDS
complement(577823 . . . 578116)
ybcU
b0557
bacteriophage lambda Bor protein homolog


MDS12
CDS
complement(578407 . . . 578859)
ybcV
b0558
putative an envelop protein


MDS12
CDS
579103 . . . 579309
ybcW
b0559
orf, hypothetical protein


MDS12
CDS
580057 . . . 580602
nohB
b0560
bacteriophage DNA packaging protein


MDS12
CDS
580577 . . . 581320
ybcX
b0561
orf, hypothetical protein


MDS12
CDS
complement(581375 . . . 581959)
ybcY
b0562
orf, hypothetical protein


MDS12
CDS
582098 . . . 582283
ylcE
b0563
orf, hypothetical protein


MDS12
CDS
582904 . . . 583653
appY
b0564
regulatory protein affecting appA and other







genes


MDS12
CDS
complement(583903 . . . 584856)
ompT
b0565
outer membrane protein 3b (a), protease







VII


MDS13
CDS
15445 . . . 16557
yi81—
b0016
IS186 hypothetical protein 1


MDS13
CDS
complement(15869 . . . 16177)
yi82_1
b0017



MDS13
CDS
complement(16751 . . . 16960)
mokC
b0018
regulatory peptide whose translation







enables hokC (gef) expression


MDS13
CDS
17489 . . . 18655
nhaA
b0019
Na+/H antiporter, pH dependent


MDS13
CDS
18715 . . . 19620
nhaR
b0020
transcriptional activator of nhaA


MDS13
CDS
complement(19811 . . . 20314)
insB_1
b0021
IS1 protein InsB


MDS13
CDS
complement(20233 . . . 20508)
insA
b0022
IS1 protein InsA_1


MDS13
CDS
complement(16751 . . . 16903)
hokC
b4412
small toxic membrane polypeptide


MDS13
misc_RNA
16952 . . . 17006
sokC
b4413
antisense RNA blocking mokC (orf69) and







hokC (gef) translation


MDS14
CDS
complement(602639 . . . 603886)
ybdG
b0577
putative transport


MDS14
CDS
complement(603994 . . . 604647)
nfnB
b0578
oxygen-insensitive NAD(P)H nitroreductase


MDS14
CDS
complement(604741 . . . 605109)
ybdF
b0579
orf, hypothetical protein


MDS14
CDS
complement(605174 . . . 605422)
ybdJ
b0580
orf, hypothetical protein


MDS14
CDS
complement(605488 . . . 606606)
ybdK
b0581
orf, hypothetical protein


MDS14
CDS
607288 . . . 608400
yi81_2
b0582
IS186 hypothetical protein


MDS14
CDS
607059 . . . 607211
hokE
b4415
small toxic membrane polypeptide


MDS15
CDS
2507652 . . . 2508908

b2389
orf, hypothetical protein


MDS15
CDS
2509023 . . . 2509349
ypeC
b2390
orf, hypothetical protein


MDS15
CDS
complement(2509490 . . . 2510728)

b2392



MDS15
CDS
2511064 . . . 2512266
nupC
b2393
permease of transport system for3







nucleosides


MDS15
CDS
2512347 . . . 2513465
yi81_3
b2394



MDS15
CDS
complement(2513665 . . . 2515971)
yfeA
b2395
orf, hypothetical protein


MDS16
CDS
complement(379293 . . . 380066)
yaiO
b0358
orf, hypothetical protein


MDS16
CDS
complement(380068 . . . 380511)

b0359
putative transferase


MDS16
CDS
380530 . . . 380940
yi21_1
b0360
IS2 hypothetical protein


MDS16
CDS
380898 . . . 381803
yi22_1
b0361
IS2 hypothetical protein


MDS16
CDS
complement(381728 . . . 382114)

b0362
orf, hypothetical protein


MDS16
CDS
complement(381963 . . . 383159)
yaiP
b0363
polysaccharide metabolism


MDS16
CDS
complement(383283 . . . 383693)
yaiS
b0364
orf, hypothetical protein


MDS16
CDS
384399 . . . 385418
tauA
b0365
taurine transport system periplasmic protein


MDS16
CDS
385431 . . . 386198
tauB
b0366
taurine ATP-binding component of a







transport system


MDS16
CDS
386195 . . . 387022
tauC
b0367
taurine transport system permease protein


MDS16
CDS
387019 . . . 387870
tauD
b0368
taurine dioxygenase, 2-oxoglutarate-







dependent


MDS17
CDS
complement(389121 . . . 389390)

b0370
orf, hypothetical protein


MDS17
CDS
389475 . . . 390935
yaiT
b0371
orf, hypothetical protein


MDS17
CDS
complement(390963 . . . 391829)
tra5_1
b0372



MDS17
CDS
complement(391826 . . . 392134)

b0373
putative flagellin structural protein


MDS17
CDS
392239 . . . 393642
yaiU
b0374
putative flagellin structural protein


MDS17
CDS
393685 . . . 394353
yaiV
b0375
orf, hypothetical protein


MDS17
CDS
complement(394354 . . . 395511)
yaiH
b0376



MDS17
CDS
395863 . . . 397083
sbmA
b0377
sensitivity to microcin B17, possibly







envelope protein


MDS17
CDS
397096 . . . 398190
yaiW
b0378
orf, hypothetical protein


MDS17
CDS
complement(398249 . . . 398557)
yaiY
b0379
orf, hypothetical protein


MDS17
CDS
398685 . . . 399029

b0380
orf, hypothetical protein


MDS18
CDS
complement(2992959 . . . 2993114)

b2856
orf, hypothetical protein


MDS18
CDS
complement(2993336 . . . 2993767)

b2857
orf, hypothetical protein


MDS18
CDS
complement(2993770 . . . 2993991)

b2858
orf, hypothetical protein


MDS18
CDS
complement(2993984 . . . 2994409)

b2859
orf, hypothetical protein


MDS18
CDS
complement(2994394 . . . 2995299)
yi22_4
b2860
IS2 hypothetical protein


MDS18
CDS
complement(2995257 . . . 2995622)
yi21_4
b2861
IS2 hypothetical protein


MDS18
CDS
complement(2995711 . . . 2996010)

b2862
orf, hypothetical protein


MDS18
CDS
complement(2996056 . . . 2996892)

b2863
orf, hypothetical protein


MDS19
CDS
3182802 . . . 3183152

b3042
orf, hypothetical protein


MDS19
CDS
3183436 . . . 3183987
ygiL
b3043
putative fimbrial-like protein


MDS19
CDS
3184209 . . . 3184574
yi21_5
b3044
IS2 hypothetical protein


MDS19
CDS
3184532 . . . 3185437
yi22_5
b3045
IS2 hypothetical protein


MDS19
CDS
3185422 . . . 3187887
yqiG
b3046
putative membrane protein


MDS19
CDS
3187894 . . . 3188652
yqiH
b3047
putative membrane protein


MDS19
CDS
3188654 . . . 3189718
yqiI
b3048
orf, hypothetical protein


MDS20
CDS
complement(687220 . . . 688236)
trs5_3
b0656
IS5 transposase


MDS21
CDS
1386912 . . . 1387919
ycjG
b1325
putative muconate cycloisomerase I (EC







5.5


MDS21
CDS
complement(1387894 . . . 1388682)
ycjI
b1326
putative carboxypeptidase


MDS21
CDS
complement(1388957 . . . 1389889)

b1327
orf, hypothetical protein


MDS21
CDS
1390015 . . . 1390914
ycjZ
b1328
putative transcriptional regulator LYSR-type


MDS21
CDS
1391230 . . . 1392864

b1329



MDS21
CDS
complement(1392915 . . . 1393946)

b1330
orf, hypothetical protein


MDS21
CDS
1394100 . . . 1395116
trs5_4
b1331
IS5 transposase


MDS21
CDS
1395389 . . . 1395646
ynaJ
b1332
orf, hypothetical protein


MDS21
CDS
complement(1395696 . . . 1396646)
ydaA
b1333
orf, hypothetical protein


MDS22
CDS
complement(2099919 . . . 2100935)
trs5_7
b2030



MDS22
CDS
complement(2100940 . . . 2101413)
yefJ
b2031



MDS22
CDS
complement(2101415 . . . 2102533)
wbbK
b2032
putative glucose transferase


MDS22
CDS
complement(2102518 . . . 2103108)
wbbJ
b2033
putative O-acetyl transferase


MDS22
CDS
complement(2103089 . . . 2104081)
wbbI
b2034
putative Galf transferase


MDS22
CDS
complement(2104084 . . . 2105250)
wbbH
b2035
O-antigen polymerase


MDS22
CDS
complement(2105250 . . . 2106353)
glf
b2036
UDP-galactopyranose mutase


MDS22
CDS
complement(2106361 . . . 2107608)
rfbX
b2037
putative O-antigen transporter


MDS22
CDS
complement(2107605 . . . 2108162)
rfbC
b2038
dTDP-6-deoxy-D-glucose-3,5 epimerase


MDS22
CDS
complement(2108162 . . . 2109043)
rfbA
b2039
glucose-1-phosphate thymidylyltransferase


MDS22
CDS
complement(2109101 . . . 2110000)
rfbD
b2040
dTDP-6-deoxy-L-mannose-dehydrogenase


MDS22
CDS
complement(2110000 . . . 2111085)
rfbB
b2041
dTDP-glucose 4,6 dehydratase


MDS22
CDS
complement(2111458 . . . 2112351)
galF
b2042
homolog of Salmonella UTP—glucose-1-P







uridyltransferase, probably a UDP-gal







transferase


MDS22
CDS
complement(2112526 . . . 2113920)
wcaM
b2043
orf, hypothetical protein


MDS22
CDS
complement(2113931 . . . 2115151)
wcaL
b2044
putative colanic acid biosynthesis glycosyl







transferase


MDS22
CDS
complement(2115148 . . . 2116428)
wcaK
b2045
putative galactokinase (EC 2.7.1.6


MDS22
CDS
complement(2116704 . . . 2118182)
wzxC
b2046
probable export protein


MDS22
CDS
complement(2118184 . . . 2119578)
wcaJ
b2047
putative colanic acid biosynthsis UDP-







glucose lipid carrier transferase


MDS22
CDS
complement(2119633 . . . 2121003)
cpsG
b2048
phosphomannomutase


MDS22
CDS
complement(2121108 . . . 2122544)
cpsB
b2049
mannose-1-phosphate guanyltransferase


MDS22
CDS
complement(2122547 . . . 2123770)
wcaI
b2050
putative colanic biosynthesis glycosyl







transferase


MDS22
CDS
complement(2123767 . . . 2124249)
wcaH
b2051
GDP-mannose mannosyl hydrolase


MDS22
CDS
complement(2124249 . . . 2125214)
wcaG
b2052
putative nucleotide di-P-sugar epimerase or







dehydratase


MDS22
CDS
complement(2125217 . . . 2126338)
gmd
b2053
GDP-D-mannose dehydratase


MDS22
CDS
complement(2126364 . . . 2126912)
wcaF
b2054
putative transferase


MDS22
CDS
complement(2126928 . . . 2127674)
wcaE
b2055
putative colanic acid biosynthesis glycosyl







transferase


MDS22
CDS
complement(2127685 . . . 2128902)
wcaD
b2056
putative colanic acid polymerase


MDS22
CDS
complement(2128877 . . . 2130094)
wcaC
b2057
putative glycosyl transferase


MDS22
CDS
complement(2130091 . . . 2130579)
wcaB
b2058
putative transferase


MDS22
CDS
complement(2130582 . . . 2131421)
wcaA
b2059
putative regulator


MDS22
CDS
complement(2131514 . . . 2133712)

b2060



MDS22
CDS
complement(2133679 . . . 2134122)
wzb
b2061
low molecular weight protein-tyrosine-







phosphatase


MDS22
CDS
complement(2134128 . . . 2135267)
wza
b2062
putative polysaccharide export protein


MDS23
CDS
complement(2284412 . . . 2286922)
yejO
b2190
putative ATP-binding component of a







transport system


MDS23
CDS
2286927 . . . 2287049

b2191
orf, hypothetical protein


MDS23
CDS
complement(2287087 . . . 2288103)
trs5_8
b2192
IS5 transposase


MDS24
CDS
3360134 . . . 3360808
yhcA
b3215
putative chaperone


MDS24
CDS
3360829 . . . 3363210
yhcD
b3216
putative outer membrane protein


MDS24
CDS
3363207 . . . 3363686
yhcE
b3217
orf, hypothetical protein


MDS24
CDS
complement(3363724 . . . 3364740)
trs5_10
b3218
IS5 transposase


MDS24
CDS
3364948 . . . 3365664
yhcF
b3219
putative transcriptional regulator


MDS25
CDS
3649314 . . . 3650096
yhiS
b3504
orf, hypothetical protein


MDS25
CDS
complement(3650205 . . . 3651221)
trs5_11
b3505
IS5 transposase


MDS26
CDS
complement(1128637 . . . 1129053)
flgN
b1070
protein of flagellar biosynthesis


MDS26
CDS
complement(1129058 . . . 1129351)
flgM
b1071
anti-FliA (anti-sigma) factor; also known as







RflB protein


MDS26
CDS
complement(1129427 . . . 1130086)
flgA
b1072
flagellar biosynthesis; assembly of basal-







body periplasmic P ring


MDS26
CDS
1130241 . . . 1130657
flgB
b1073
flagellar biosynthesis, cell-proximal portion







of basal-body rod


MDS26
CDS
1130661 . . . 1131065
flgC
b1074
flagellar biosynthesis, cell-proximal portion







of basal-body rod


MDS26
CDS
1131077 . . . 1131772
flgD
b1075
flagellar biosynthesis, initiation of hook







assembly


MDS26
CDS
1131797 . . . 1133005
flgE
b1076
flagellar biosynthesis, hook protein


MDS26
CDS
1133025 . . . 1133780
flgF
b1077
flagellar biosynthesis, cell-proximal portion







of basal-body rod


MDS26
CDS
1133952 . . . 1134734
flgG
b1078
flagellar biosynthesis, cell-distal portion of







basal-body rod


MDS26
CDS
1134787 . . . 1135485
flgH
b1079
flagellar biosynthesis, basal-body outer-







membrane L (lipopolysaccharide layer) ring







protein


MDS26
CDS
1135497 . . . 1136594
flgI
b1080
homolog of Salmonella P-ring of flagella







basal body


MDS26
CDS
1136594 . . . 1137535
flgJ
b1081
flagellar biosynthesis


MDS26
CDS
1137601 . . . 1139244
flgK
b1082
flagellar biosynthesis, hook-filament







junction protein 1


MDS26
CDS
1139256 . . . 1140209
flgL
b1083
flagellar biosynthesis; hook-filament







junction protein


MDS27
CDS
complement(1960604 . . . 1960996)
flhE
b1878
flagellar protein


MDS27
CDS
complement(1960996 . . . 1963074)
flhA
b1879
flagellar biosynthesis; possible export of







flagellar proteins


MDS27
CDS
complement(1963067 . . . 1964215)
flhB
b1880
putative part of export apparatus for







flagellar proteins


MDS27
CDS
complement(1964417 . . . 1965061)
cheZ
b1881
chemotactic response; CheY protein







phophatase; antagonist of CheY as switch







regulator


MDS27
CDS
complement(1965072 . . . 1965461)
cheY
b1882
chemotaxis regulator transmits







chemoreceptor signals to flagelllar motor







components


MDS27
CDS
complement(1965476 . . . 1966525)
cheB
b1883
response regulator for chemotaxis (cheA







sensor); protein methylesterase


MDS27
CDS
complement(1966528 . . . 1967388)
cheR
b1884
response regulator for chemotaxis protein







glutamate methyltransferase


MDS27
CDS
complement(1967407 . . . 1969008)
tap
b1885
methyl-accepting chemotaxis protein IV







peptide sensor receptor


MDS27
CDS
complement(1969054 . . . 1970715)
tar
b1886
methyl-accepting chemotaxis protein II







aspartate sensor receptor


MDS27
CDS
complement(1970860 . . . 1971363)
cheW
b1887
positive regulator of CheA protein activity


MDS27
CDS
complement(1971384 . . . 1973348)
cheA
b1888
sensory transducer kinase between chemosignal







receptors and CheB and CheY


MDS27
CDS
complement(1973353 . . . 1974279)
motB
b1889
enables flagellar motor rotation linking







torque machinery to cell wall


MDS27
CDS
complement(1974276 . . . 1975163)
motA
b1890
proton conductor component of motor; no







effect on switching


MDS27
CDS
complement(1975290 . . . 1975868)
flhC
b1891
regulator of flagellar biosynthesis acting on







class 2 operons; transcription initiation







factor


MDS27
CDS
complement(1975871 . . . 1976230)
flhD
b1892
regulator of flagellar biosynthesis acting on







class 2 operons; transcription initiation







factor


MDS27
CDS
complement(1976542 . . . 1977045)
insB_5
b1893
IS1 protein InsB


MDS27
CDS
complement(1976964 . . . 1977239)
insA_5
b1894
IS1 protein InsA


MDS28
CDS
complement(1995086 . . . 1995838)
yecC
b1917
putative ATP-binding component of a







transport system


MDS28
CDS
complement(1995835 . . . 1996503)
yecS
b1918
putative transport system permease protein







(former yecC)


MDS28
CDS
complement(1996518 . . . 1997600)
yedO
b1919
putative 1-aminocyclopropane-1-







carboxylate deaminase


MDS28
CDS
complement(1997609 . . . 1998409)
fliY
b1920
putative periplasmic binding transport







protein


MDS28
CDS
complement(1998497 . . . 1999084)
fliZ
b1921
orf, hypothetical protein


MDS28
CDS
complement(1999094 . . . 1999813)
fliA
b1922
flagellar biosynthesis; alternative sigma







factor 28; regulation of flagellar operons


MDS28
CDS
complement(2000134 . . . 2001630)
fliC
b1923
flagellar biosynthesis; flagellin filament







structural protein


MDS28
CDS
2001896 . . . 2003302
fliD
b1924
flagellar biosynthesis; filament capping







protein; enables filament assembly


MDS28
CDS
2003327 . . . 2003737
fliS
b1925
flagellar biosynthesis; repressor of class 3a







and 3b operons (RflA activity)


MDS28
CDS
2003737 . . . 2004102
fliT
b1926
flagellar biosynthesis; repressor of class 3a







and 3b operons (RflA activity)


MDS28
CDS
2004180 . . . 2005667
amyA
b1927
cytoplasmic alpha-amylase


MDS28
CDS
complement(2005701 . . . 2006114)
yedD
b1928
orf, hypothetical protein


MDS28
CDS
2006301 . . . 2007506
yedE
b1929
putative transport system permease protein


MDS28
CDS
2007503 . . . 2007736
yedF
b1930
orf, hypothetical protein


MDS28
CDS
2007845 . . . 2008513
yedK
b1931
orf, hypothetical protein


MDS28
CDS
2008624 . . . 2009103
yedL
b1932
orf, hypothetical protein


MDS28
CDS
complement(2009372 . . . 2009563)

b1933
orf, hypothetical protein


MDS28
CDS
complement(2009573 . . . 2009893)
yedN
b1934
orf, hypothetical protein


MDS28
CDS
complement(2010025 . . . 2010375)
yedM
b1935
orf, hypothetical protein


MDS28
CDS
2010526 . . . 2010804

b1936
orf, hypothetical protein


MDS28
CDS
complement(2010724 . . . 2011038)
fliE
b1937
flagellar biosynthesis; basal-body







component, possibly at (MS-ring)-rod







junction


MDS28
CDS
2011253 . . . 2012911
fliF
b1938
flagellar biosynthesis; basal-body







MS(membrane and supramembrane)-ring







and collar protein


MDS28
CDS
2012904 . . . 2013899
fliG
b1939
flagellar biosynthesis, component of motor







switching and energizing, enabling rotation







and determinin its direction


MDS28
CDS
2013871 . . . 2014578
fliH
b1940
flagellar biosynthesis; export of flagellar







proteins


MDS28
CDS
2014578 . . . 2015951
fliI
b1941
flagellum-specific ATP synthase


MDS28
CDS
2015970 . . . 2016413
fliJ
b1942
flagellar fliJ protein


MDS28
CDS
2016410 . . . 2017537
fliK
b1943
flagellar hook-length control protein


MDS28
CDS
2017642 . . . 2018106
fliL
b1944
flagellar biosynthesis


MDS28
CDS
2018111 . . . 2019115
fliM
b1945
flagellar biosynthesis, component of motor







switch and energizing, enabling rotation







and determining its direction


MDS28
CDS
2019112 . . . 2019525
fliN
b1946
flagellar biosynthesis, component of motor







switch and energizing, enabling rotation







and determining its direction


MDS28
CDS
2019588 . . . 2019893
fliO
b1947
flagellar biosynthesis


MDS28
CDS
2019893 . . . 2020630
fliP
b1948
flagellar biosynthesis


MDS28
CDS
2020640 . . . 2020909
fliQ
b1949
flagellar biosynthesis


MDS28
CDS
2020917 . . . 2021702
fliR
b1950
flagellar biosynthesis


MDS29
CDS
4552599 . . . 4553372
uxuR
b4324
regulator for uxu operon


MDS29
CDS
complement(4553513 . . . 4554343)
yjiC
b4325
orf, hypothetical protein


MDS29
CDS
4555007 . . . 4555408
yjiD
b4326
orf, hypothetical protein


MDS29
CDS
complement(4555401 . . . 4556312)
yjiE
b4327
putative transcriptional regulator LYSR-type


MDS29
CDS
complement(4556377 . . . 4557549)
iadA
b4328
isoaspartyl dipeptidase


MDS29
CDS
complement(4557562 . . . 4558023)
yjiG
b4329
orf, hypothetical protein


MDS29
CDS
complement(4558020 . . . 4558715)
yjiH
b4330
orf, hypothetical protein


MDS29
CDS
4558851 . . . 4559507
yjiI
b4331
orf, hypothetical protein


MDS29
CDS
complement(4559520 . . . 4560698)
yjiJ
b4332
putative transport protein


MDS29
CDS
complement(4560766 . . . 4561737)
yjiK
b4333
orf, hypothetical protein


MDS29
CDS
complement(4561945 . . . 4562718)
yjiL
b4334
putative enzyme


MDS29
CDS
complement(4562722 . . . 4563894)
yjiM
b4335
orf, hypothetical protein


MDS29
CDS
complement(4563989 . . . 4565269)
yjiN
b4336
orf, hypothetical protein


MDS29
CDS
complement(4565310 . . . 4566542)
yjiO
p4337
putative transport protein


MDS29
CDS
4567021 . . . 4567332
yjiP
b4338
orf, hypothetical protein


MDS29
CDS
4567381 . . . 4567941
yjiQ
b4339
orf, hypothetical protein


MDS29
CDS
complement(4568185 . . . 4569597)
yjiR
b4340
putative regulator


MDS29
CDS
4569774 . . . 4569938
yjiS
b4341
orf, hypothetical protein


MDS29
CDS
4570389 . . . 4571954
yjiT
b4342
orf, hypothetical protein


MDS29
CDS
complement(4574935 . . . 4575981)
mcrC
b4345
component of McrBC 5-methylcytosine







restriction system


MDS29
CDS
complement(4575981 . . . 4577378)
mcrB
b4346
component of McrBC 5-methylcytosine







restriction system


MDS29
CDS
complement(4577522 . . . 4577920)
yjiW
b4347
orf, hypothetical protein


MDS29
CDS
complement(4578091 . . . 4579485)
hsdS
b4348
specificity determinant for hsdM and hsdR


MDS29
CDS
complement(4579482 . . . 4581071)
hsdM
b4349
host modification; DNA methylase M


MDS29
CDS
complement(4581272 . . . 4584838)
hsdR
b4350
host restriction; endonuclease R


MDS29
CDS
4584972 . . . 4585886
mrr
b4351
restriction of methylated adenine


MDS29
CDS
complement(4585932 . . . 4586786)
yjiA
b4352
orf, hypothetical protein


MDS29
CDS
complement(4586899 . . . 4587102)
yjiX
b4353
orf, hypothetical protein


MDS29
CDS
complement(4587152 . . . 4589317)
yjiY
b4354
putative carbon starvation protein


MDS29
CDS
4589680 . . . 4591335
tsr
b4355
methyl-accepting chemotaxis protein I







serine sensor receptor


MDS29
CDS
complement(4591384 . . . 4592745)
yjiZ
b4356
putative transport protein, cryptic orf, joins







former yjiZ and yjjL


MDS29
CDS
complement(4592960 . . . 4593874)
yjjM
b4357
orf, hypothetical protein


MDS29
CDS
4593998 . . . 4595035
yjjN
b4358
putative oxidoreductase


MDS29
CDS
4572158 . . . 4574878
yjiV
b4486
conserved hypothetical protein


MDS30
CDS
522485 . . . 526765
rhsD
b0497
rhsD protein in rhs element


MDS30
CDS
526805 . . . 527173

b0498
orf, hypothetical protein


MDS30
CDS
527173 . . . 527883

b0499
orf, hypothetical protein


MDS30
CDS
527864 . . . 528124
ybbD
b0500
orf, hypothetical protein


MDS30
CDS
528163 . . . 528354

b0501
orf, hypothetical protein


MDS30
CDS
complement(528869 . . . 529276)

b0502
orf, hypothetical protein


MDS31
CDS
728806 . . . 732999
rhsC
b0700



MDS31
CDS
732593 . . . 732814

b0701



MDS31
CDS
732999 . . . 733325
ybfB
b0702
orf, hypothetical protein


MDS31
CDS
733443 . . . 734876

b0703
orf, hypothetical protein


MDS31
CDS
734873 . . . 735442
ybfC
b0704
orf, hypothetical protein


MDS31
CDS
736327 . . . 737184
ybfL
b0705
putative receptor protein


MDS31
CDS
737315 . . . 738076
ybfD
b0706
putative DNA ligase


MDS32
CDS
1525914 . . . 1527962
rhsE
b1456



MDS32
CDS
1527946 . . . 1528428
ydcD
b1457
orf, hypothetical protein


MDS32
CDS
1528610 . . . 1529356

b1458
orf, hypothetical protein


MDS32
CDS
1529400 . . . 1529600

b1459
orf, hypothetical protein


MDS32
CDS
1529840 . . . 1530976
ydcC
b1460
H repeat-associated protein (ORF-H)


MDS32
CDS
1531076 . . . 1531309
ydcE
b1461
orf, hypothetical protein


MDS32
CDS
complement(1531306 . . . 1531923)

b1462
orf, hypothetical protein


MDS33
CDS
3616611 . . . 3617012
yhhG
b3481



MDS33
CDS
3617215 . . . 3621450
rhsB
b3482
rhsB protein in rhs element


MDS33
CDS
3621437 . . . 3621805
yhhH
b3483
orf, hypothetical protein


MDS33
CDS
3622401 . . . 3623537
yhhI
b3484
putative receptor


MDS34
CDS
complement(3759370 . . . 3759978)
yibF
b3592
putative S-transferase


MDS34
CDS
3760206 . . . 3764339
rhsA
b3593
rhsA protein in rhs element


MDS34
CDS
3764360 . . . 3765202
yibA
b3594
orf, hypothetical protein


MDS34
CDS
3765244 . . . 3765945
yibJ
b3595
orf, hypothetical protein


MDS34
CDS
3766200 . . . 3766661
yibG
b3596
orf, hypothetical protein


MDS35
CDS
complement(1041253 . . . 1043433)
yccC
b0981
orf, hypothetical protein


MDS35
CDS
complement(1043453 . . . 1043911)
yccY
b0982
yccY putative phosphatase


MDS35
CDS
complement(1043887 . . . 1045026)
yccZ
b0983
putative function in exopolysaccharide







production


MDS35
CDS
complement(1045072 . . . 1047168)
ymcA
b0984
orf, hypothetical protein


MDS35
CDS
complement(1047168 . . . 1047914)
ymcB
b0985
orf, hypothetical protein


MDS35
CDS
complement(1047911 . . . 1048555)
ymcC
b0986
putative regulator


MDS35
CDS
complement(1048662 . . . 1048985)
ymcD
b0987
orf, hypothetical protein


MDS35
CDS
1049250 . . . 1049753
insB_4
b0988
IS1 protein InsB


MDS36
CDS
complement(1085329 . . . 1085742)
ycdP
b1021
orf, hypothetical protein


MDS36
CDS
complement(1085744 . . . 1087069)
ycdQ
b1022
orf, hypothetical protein


MDS36
CDS
complement(1087062 . . . 1089080)
ycdR
b1023
orf, hypothetical protein


MDS36
CDS
complement(1089089 . . . 1091512)
ycdS
b1024
putative outer membrane protein


MDS36
CDS
1092099 . . . 1093457
ycdT
b1025
orf, hypothetical protein


MDS36
CDS
complement(1093498 . . . 1094364)
tra5_3
b1026



MDS36
CDS
complement(1094361 . . . 1094669)

b1027



MDS36
CDS
1094746 . . . 1095069

b1028
orf, hypothetical protein


MDS36
CDS
1095066 . . . 1096052
ycdU
b1029
orf, hypothetical protein


MDS37
CDS
2163174 . . . 2163545

b2080
orf, hypothetical protein


MDS37
CDS
2163692 . . . 2165053
yegQ
b2081
orf, hypothetical protein


MDS37
CDS
complement(2165326 . . . 2165544)
ogrK
b2082
prophage P2 ogr protein


MDS37
CDS
complement(2165626 . . . 2165772)

b2083
orf, hypothetical protein


MDS37
CDS
complement(2165759 . . . 2166025)

b2084
orf, hypothetical protein


MDS37
CDS
complement(2166013 . . . 2166390)
yegR
b2085
orf, hypothetical protein


MDS37
CDS
2166736 . . . 2167635

b2086
orf, hypothetical protein


MDS37
CDS
complement(2167717 . . . 2168163)
gatR_1
b2087
split galactitol utilization operon repressor


MDS37
CDS
2168251 . . . 2168559

b2088



MDS37
CDS
2168556 . . . 2169422
tra5_4
b2089



MDS37
CDS
complement(2169419 . . . 2169757)
gatR_2
b2090



MDS37
CDS
complement(2169857 . . . 2170897)
gatD
b2091
galactitol-1-phosphate dehydrogenase


MDS37
CDS
complement(2170945 . . . 2172300)
gatC
b2092
PTS system galactitol-specific enzyme IIC


MDS37
CDS
complement(2172304 . . . 2172588)
gatB
b2093
galactitol-specific enzyme IIB of







phosphotransferase system


MDS37
CDS
complement(2172619 . . . 2173071)
gatA
b2094
galactitol-specific enzyme IIA of







phosphotransferase system


MDS37
CDS
complement(2173081 . . . 2174343)
gatZ
b2095
putative tagatose 6-phosphate kinase 1


MDS37
CDS
complement(2174372 . . . 2175232)
gatY
b2096
tagatose-bisphosphate aldolase 1


MDS37
misc_RNA
2165136 . . . 2165221
ryeE
b4438



MDS38
CDS
complement(3577791 . . . 3578828)
yhhX
b3440
putative regulator


MDS38
CDS
3579161 . . . 3579649
yhhY
b3441
orf, hypothetical protein


MDS38
CDS
3579886 . . . 3581064
yhhZ
b3442
orf, hypothetical protein


MDS38
CDS
3581061 . . . 3581477
yrhA
b3443
orf, hypothetical protein


MDS38
CDS
3581506 . . . 3581781
insA_6
b3444
IS1 protein InsA


MDS38
CDS
3581700 . . . 3582203
insB_6
b3445
IS1 protein InsB


MDS38
misc_RNA
complement(3578946 . . . 3579039)
ryhB
b4451
regulatory RNA mediating Fur regulon


MDS39
CDS
3718072 . . . 3718284
cspA
b3556
cold shock protein 7.4, transcriptional







activator of hns


MDS39
CDS
3718703 . . . 3719224
yi5A
b3557
IS150 hypothetical protein


MDS39
CDS
3719221 . . . 3720072
t150
b3558
IS150 putative transposase


MDS39
CDS
complement(3718471 . . . 3718623)
hokA
b4455
small toxic membrane polypeptide


MDS40
CDS
1869885 . . . 1871555
yeaJ
b1786
orf, hypothetical protein


MDS41
CDS
167484 . . . 169727
fhuA
b0150
outer membrane protein receptor for







ferrichrome, colicin M, and phages T1, T5,







and phi80


MDS41
CDS
169778 . . . 170575
fhuC
b0151
ATP-binding component of hydroxymate-







dependent iron transport


MDS41
CDS
170575 . . . 171465
fhuD
b0152
hydroxamate-dependent iron uptake







cytoplasmic membrane component


MDS41
CDS
171462 . . . 173444
fhuB
b0153
hydroxamate-dependent iron uptake







cytoplasmic membrane component

## Claims

1. A non-naturally occurring Escherichia coli bacterium lacking all genomic and non-genomic IS1, IS2, IS3, IS4, IS5, IS10, IS30, IS150, IS186, IS600 and IS911 insertion sequences, wherein the genome of said bacterium is between 4.41 Mb and 2.78 Mb.

2. The bacterium of claim 1, wherein its genome is between 4.27 Mb and 2.78 Mb.

3. The bacterium of claim 1, wherein its genome is between 4.00 Mb and 2.78 Mb.

4. The bacterium of claim 1, wherein its genome is between 3.71 Mb and 2.78 Mb.

5. The bacterium of claim 1, wherein the parent strain of said bacterium is selected from the group consisting of DH10B, DH5α, INVα, Top10, Top10F, JM103, JM105, JM109, MC1061, MC4100, XL1-Blue, EC100, and EC300.

6. The bacterium of claim 1, wherein the bacterium is competent to be transformed.

7. The bacterium of claim 1, wherein the bacterium lacks Insertion Sequence mini-circles.

8. The bacterium of claim 1, wherein the bacterium comprises a vector.

9. The bacterium of claim 8, wherein the vector lacks insertion sequences.

10. The bacterium of claim 9, wherein the vector comprises a nucleic acid encoding a polypeptide, and wherein the nucleic acid is operatively linked to an expression control sequence.

11. The bacterium of claim 9, wherein the vector is a plasmid.

12. A method of propagating a nucleic acid comprising:
(a) transforming a bacterium according to claim 1 with a nucleic acid to create a transformed bacterium; and
(b) propagating the transformed bacterium of step (a) under conditions that allow replication of said nucleic acid.

13. The method of claim 12, wherein the bacterium is transformed by electroporation.

14. A method of producing a polypeptide comprising:
(a) incubating a bacterium according to claim 10 under suitable nutrient conditions to allow expression of the polypeptide; and
(b) optionally isolating and purifying said polypeptide.