Mapping of insertion elements IS1, IS2 and IS3 on the Escherichia coli K-12 chromosome. Role of the insertion elements in formation of Hfrs and F' factors and in rearrangement of bacterial chromosomes

The chromosome of an Escherichia coli K-12 strain W3110 contains seven copies of insertion element IS1, 12 copies of IS2 and six copies of IS3. We determined the approximate locations of six copies of IS1 (named is1A to is1F), ten copies of IS2 (named is2A to is2J), and five copies of IS3 (named is3A to is3E) on the W3110 chromosome by plaque hybridization using the "mini-set" of the lambda phage library that includes 476 clones carrying chromosomal segments that cover the W3110 chromosome almost entirely. Cleavage maps of the W3110 chromosome and cleavage analysis of phage DNAs carrying insertion elements allowed us to assign more precise locations to most of the insertion elements and to determine their orientations. Insertion elements were distributed randomly along the W3110 chromosome in one or other orientation. Several of these were located at the same positions on the chromosome of another E. coli K-12 strain, JE5519, and they were assumed to be the original complement of insertion elements in E. coli K-12 wild-type. Locations and orientations of such insertion elements were correlated well with Hfr points of origin and with crossover points for excision of some F' factors derived from several Hfrs. Insertion elements may be involved also in rearrangement of bacterial chromosomes.     

Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5

We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110. Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them. We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome. Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E. coli K-12 strain, JE5519. While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5. Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present. In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E. coli K-12 derivatives.     

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation

Escherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

Comparative proteomic and genetic analyses reveal unidentified mutations in Escherichia coli XL1-Blue and DH5α

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.     

A rapid screening procedure to identify mini-Tn10 insertion mutants of Escherichia coli K-12 with altered adhesion properties

Abstract A rapid screening procedure was developed for detection of Escherichia coli mutants with altered adhesion abilities using polystyrene 96-well microtiter plates as attachment surfaces. During this assay, bacterial strains grew and adhered simultaneously, and attached cells were measured after crystal violet staining. Starting with a total of 7000 W3110::Tn10 insertion mutants of E. coli K-12 W3110, 50 adhesion-deficient mutants were isolated which showed less than 40% attachment, and 22 mutants were found with an attachment of 40–75%. Motility assays were performed on these 72 mutants, and 34 displayed altered motility.     

Extensive genomic diversity in pathogenic Escherichia coli and Shigella Strains revealed by comparative genomic hybridization microarray

Escherichia coli, including the closely related genus Shigella, is a highly diverse species in terms of genome structure. Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of E. coli K-12 with the gene contents of pathogenic strains. Missing genes in a pathogen were detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of W3110, a commonly used wild-type K-12 strain. For 22 strains subjected to the CGH microarray analyses 1,424 ORFs were found to be absent in at least one strain. The common backbone of the E. coli genome was estimated to contain about 2,800 ORFs. The mosaic distribution of absent regions indicated that the genomes of pathogenic strains were highly diversified because of insertions and deletions. Prophages, cell envelope genes, transporter genes, and regulator genes in the K-12 genome often were not present in pathogens. The gene contents of the strains tested were recognized as a matrix for a neighbor-joining analysis. The phylogenic tree obtained was consistent with the results of previous studies. However, unique relationships between enteroinvasive strains and Shigella, uropathogenic, and some enteropathogenic strains were suggested by the results of this study. The data demonstrated that the CGH microarray technique is useful not only for genomic comparisons but also for phylogenic analysis of E. coli at the strain level.     

Potential for gene transfer from recombinantEscherichia coli K-12 used in bovine somatotropin production to indigenous bacteria in river water

Summary This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), fromEscherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×10⁷ CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated withE. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated withE. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study.     

Precise mapping of the rnpB gene encoding the RNA component of RNase P in Escherichia coli K-12.

In Kohara's library derived from Escherichia coli K-12 W3110 (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), multiple copies of chromosomal sequence are found at 68 and at 64 to 65 min (M. Umeda and E. Ohtsubo, J. Mol. Biol. 213:229-237, 1990). We have determined that the rnpB gene (previously mapped at 70 min [B. J. Bachmann, Microbiol. Rev. 54:130-197, 1990]) is located within these segments of repeated sequences as five separate copies, together with tdcA, B, C, and R (mapped at 68 min [Bachmann, 1990]) and six unidentified open reading frames. Since close linkage of rnpB and tdc is found in various strains of E. coli K-12, the rnpB gene should be mapped at 68 min rather than 70 min.     

含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响

摘要：【目的】通过分子生物学手段构建重组质粒,将其转入野生型大肠杆菌W3110,分析含苏氨酸操纵子基因的质粒及质粒定点突变解除反馈抑制时,对L-苏氨酸积累的影响。【方法】以W3110染色体DNA为模板,PCR扩增苏氨酸操纵子基因,即启动子THrLp、编码前导肽基因thrL以及thrA、thrB、thrC基因,通过重叠延伸PCR的方法对thrA基因定点突变,解除苏氨酸对它的反馈抑制,构建出重组表达质粒WYE112和WYE134,5 L发酵实验测定L-苏氨酸的产量。【结果】经5 L发酵罐发酵产酸实验,W3110的L-苏氨酸产量为0.036 ± 0.004 g/L,携带含苏氨酸操纵子质粒的W3110菌株L-苏氨酸产量为2.590 ± 0.115 g/L,质粒上thrA解除反馈抑制后,L-苏氨酸的产量增加到9.223 ± 1.279 g/L。【结论】过表达苏氨酸操纵子基因可以使L-苏氨酸积累,进一步解除thrA基因的反馈抑制,可以增强L-苏氨酸积累的效果,为L-苏氨酸工程菌改造的进一步研究奠定了基础。     

Completion of the detailed restriction map of the E. coli genome by the isolation of overlapping cosmid clones.

Ordered sets of cosmids derived from E. coli K-12 803 overlap the 6 remaining gaps left in the physical map of strain W3110. We present detailed restriction maps of the gaps and surrounding regions, thus providing a comparison of about 30% of the genome of the two E. coli strains. Our analysis shows that there is a high degree of homology between the strains, with only occasional restriction fragment differences. However, the large inversion occurring between rrnD (72.1') and rrnE (90.4') in strain W3110 is absent in strain 803. Instead, a new inversion and adjacent deletion near argF is present in strain 803. The distribution of cosmid clones at, and adjacent to, the gaps shows that all gaps except one were difficult to clone in both lambda and cosmid clones. A low copy number cosmid vector, pOU61cos, developed previously, was essential for cloning 3 of the 8 gaps.     

Drosophila melanogaster protein phosphatase inhibitor-2: identification of a site important for PP1 inhibition

Two genome sequences of Escherichia coli K-12 substrains, one partial W3110 and one complete MG1655, have been determined by Japanese and American genome projects, respectively. In order to estimate the rate of nucleotide changes, we directly compared 2 Mb of the nucleotide sequences from these closely-related E. coli substrains. Given that the two substrains separated about 40 years ago, the rate of nucleotide changes was estimated to be less than 10(-7) per site per year. This rate was supported by a further comparison between partial genome sequences of E. coli and Shigella flexneri.     

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.     

Cell cycle parameters of Escherichia coli K-12.

A computer simulation routine was used to calculate the DNA distributions of exponentially growing cultures of Escherichia coli K-12. Simulated distributions were compared with distributions obtained experimentally by flow cytometry. Durations of the DNA replication period (C) and the postreplication period (D) were found by minimizing the difference between theoretical and experimental DNA histograms. It was demonstrated that the K-12 strains AB1157 and CM735 had C and D periods that differed widely from each other and from those of the previously measured strain B/rA, while strain MC1000 was shown to have the same durations of the C and D periods as strain B/rA. The variation between K-12 strains may explain the divergence in the literature regarding their C and D periods. Strains W3110 and AB1157 recA1 had DNA histograms that could not be adequately simulated by the classical Cooper-Helmstetter model, which is consistent with the asymmetrically located origin and terminus for W3110 and the asynchrony of initiation for AB1157 recA1.     

mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with ¹⁴C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions.     

High expression of the second lysine decarboxylase gene, ldc, in Escherichia coli WC196 due to the recognition of the stop codon (TAG), at a position which corresponds to the 33th amino acid residue of sigma38, as a serine residue by the amber suppressor, supD

Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, sigma38, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of sigma38 protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA (SerU), called, supD. Analysis of the amino acid sequence of a sigma38 preparation from WC196 showed that the 33th residue of sigma38 is a serine residue. The deltarpoS deltacadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of sigma38, expressed a significant amount of Ldc and accumulated a large amount of sigma38. However, the deltarpoS deltacadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither sigma38 nor Ldc significantly.     

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.