The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

Assembly of CS1 pili: the role of specific residues of the major pilin, CooA

CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA(-)) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA(-) cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif "AGxYxG(x(6))T," which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.     

The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin In the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coii K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K-12 and a virulence locus for provoking keratocon-junctivitis in the eyes of guinea-pigs, named kcpA is located near purE in S. fiexnerl, and the two loci are involved in VirG expression, the KcpA～ mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompr gene from E. coli K-12, giving rise to a KcpA phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.     

Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli

Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1. For the assembly of the major-pilin subunit, CooA, into pili, each of four linked genes, cooB,A,C, and D, is required. In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera. CooD appears to be an integral pilus protein because it co-purifies with, and is strongly associated with, CS1 pili. In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1-piliated strains) was detected in periplasmic inter-molecular complexes with the major-pilin subunit CooA. CooB is an assembly protein found exclusively in the periplasm of CS1-piliated strains. CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure. Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1-piliated E. coli. Based on this information, we have proposed a model for CS1 -pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E. coli. As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution.     

Cloning of the Genes That Control Formation of the Fimbrial Colonization Factor Antigen III (CFA/III) from an Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strain 260-1 produces colonization factor antigen III and heat-labile enterotoxin. A 55-kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K-12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260-1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4-kb region was found to be responsible for the expression of the pili.     

Effect of Sucrose Concentration on the Infectivity of ϕX 174 DNA to Protoplast of Escherichia coli

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS–PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg⁺², ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 μM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.     

The frequency of expression of pyelonephritis-associated pili is under regulatory control

The Bscherfchia coli urinary tract Isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pllin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pill composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the ceil surface. These results Indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of piiin-17 expression increased about 15-foid when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.     

omp T: Escherichia coli K-12 structural gene for protein a (3b)

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.     

Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera-like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH₂-terminal amino acid sequence revealed homology with the toxin-coregulated pilus of Vibrio cholerae, the bundle-forming pilus of enteropathogenic E. coli and type IV pilins of some Gram-negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K-12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno-prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat-stable toxin only.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.     

Mechanism of conjugation

Summary A new conditional thermosensitive Hfr mutant of Escherichia coli K-12 was isolated. The ts mutation is cotransducible with purE and tsx loci on the E. coli chromosome. Upon temperature shift to 42° C the DNA synthesis and transfer of chromosome is stopped immediately and RNA, protein synthesis in about ten minutes.     

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2

We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I-CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BinI sites in glt-tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E. coli K-12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.     

Functional analysis of the minor subunits of S fimbrial adhesion (SfaI) in pathogenic Escherichia coli

S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and -H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site-directed mutagenesis. Received: 15 March 1999 / Accepted: 2 November 1999     

Ectopic Gene Conversions in Four <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Genomes: Increased Recombination in Pathogenic Strains

We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli. Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent. Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain. Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12. This higher recombination level might be due to mutations in some of their mismatch-repair genes. In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E. coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes. In E. coli, gene conversions are not more frequent near the origin of replication. However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant. This suggests that, in E. coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E. coli cells.     

Strain Engineering by Genome Mass Transfer: Efficient Chromosomal Trait Transfer Method Utilizing Donor Genomic DNA and Recipient Recombineering Hosts

Strain engineering, like cloning, is a fundamental technology used to confer new traits onto existing strains. While effective methods for trait development through gene modification within strains have been developed, methods for trait transfer between Escherichia coli strains to create complex strains are needed. We report herein the development of genome mass transfer (GMT), a broadly applicable new strain engineering methodology enabling rapid trait transfer from a donor strain into a recombineering gene-expressing recipient strain. GMT utilizes electroporation of donor chromosomal DNA into a recombineering recipient cell for precise trait transfer. GMT transfer of traits between E. coli strains can be used to rapidly assemble new strains incorporating combinations of marked gene knockouts, for example, utilizing the existing E. coli K-12 Keio gene knockout collection as source target genes. Optional use of random primed isothermal amplified DNA eliminates the need for initial DNA purification, affording high throughput application. This allows unprecedented simplicity and speed for rational design engineering of complex phenotypes in industrial strains.     

Sensitivity to x-radiation of strains of Escherichia coli K-12 which lack DNA polymerase II

Summary The polB1 and polA1 polB1 strains of E. coli K-12, wihch are deficient in DNA polymerase II and in DNA polymerases I and II, respectively, were found to have essentially the same sensitivity to anoxic or aerobic X-irradiation as their related wild-type and polA1 strains, respectively. Thus, DNA polymerase II appears to play no major role in the repair of X-ray damage.