Coli K12 Strain Research Articles

Escherichia coli K1 RS218 Interacts with Human Brain Microvascular Endothelial Cells via Type 1 Fimbria Bacteria in the Fimbriated State

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monophosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour

The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, D- and L-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached. In the downshift experiment, the adenylate energy charge behaved inversely; no by-products were formed, indicating a tight coupling of anabolism and catabolism. Both dilution rate shifts were accompanied by an instantaneous increase of cAMP, presaging the subsequent changes in metabolic pathway utilization. Intracellular key metabolites of the Embden-Meyerhof-Parnas (EMP) pathway were measured to evaluate the metabolic perturbation during the upshift. Fructose 1,6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP) increased rapidly after the upshift, while glyceraldehyde 3-phosphate decreased. It is concluded that this imbalance at the branch-point of FDP induces the methylglyoxal (MG) pathway, a low-energy-yielding bypass of the lower EMP pathway, through the increasing level of DHAP. MG pathway activation after the upshift was simulated by restricting anabolic rates using a stoichiometry-based metabolic model. The metabolic model predicted that low-energy-yielding catabolic pathways are utilized preferentially in the transient after the upshift. Upon severe dilution rate upshifts, an oscillatory behaviour occurred, apparent through long-term oscillations of respiratory activity, which started when the cytotoxic compound MG reached a threshold concentration of 1.5 mg l(-1) in the medium.

Global Expression of Prophage Genes in Escherichia coli O157:H7 Strain EDL933 in Response to Norfloxacin

We investigated the influence of a low concentration of the gyrase inhibitor norfloxacin on the transcriptome of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. For this purpose, we used a commercial DNA microarray containing oligonucleotides specific for E. coli O157:H7 strains EDL933 and RIMD0509952 and E. coli K-12 strain MG1655. Under the conditions applied, 5,963 spots (94% of all spots) could be analyzed. Among these, 118 spots (P < 0.05) indicated transcriptional upregulation and 122 spots (P < 0.05) indicated transcriptional downregulation of the E. coli genes present on the array. Eighty-five upregulated EDL933 genes were phage borne. Fifty-two of them could be ascribed to the Shiga toxin-encoding phages (Stx phages) BP-933W and CP-933V; the other 33 genes belonged to non-Stx prophage elements in the EDL933 genome. Genes present in the BP-933W prophage genome were induced most strongly up to 158-fold in the case of stxA(2) upon induction with norfloxacin. Twenty-two additional upregulated genes appeared to be E. coli O157:H7 strain RIMD0509952-specific phage elements, and the remaining 11 genes were related mainly to recombination and stress functions. Downregulation was indicated predominantly for genes responsible for bacterial primary metabolism, such as energy production, cell division, and amino acid biosynthesis. Interestingly, some genes present in the locus of enterocyte effacement appeared to be downregulated. The results of the study have shown that a low concentration of norfloxacin has profound effects on the transcriptome of E. coli O157:H7.

Production of H2 from sucrose by Escherichia coli strains carrying the pUR400 plasmid, which encodes invertase activity

Escherichia coli HD701, a hydrogenase-upregulated strain, has the potential for industrial-scale H2 production but is unable to metabolise sucrose, which is a major constituent of many waste materials that could be used as feedstocks for H2 production processes. A 70 kb plasmid (pUR400), which carries the genes necessary for sucrose transport into the cell and its metabolism, was conjugated into E. coli strains HD701 and FTD701 [a derivative of HD701 which has a deletion of the tatC gene of the twin arginine transport (Tat) protein system] from an E. coli K12 strain. Comparative studies on H2 evolution by FTD701 and HD701, with and without the pUR400 plasmid, were made using sucrose as substrate. The parental strains did not evolve H2, although HD701/pUR400 and FTD701/pUR400 evolved 1.27 +/- 0.09 and 1.38 +/- 0.05 ml H2 mg dry wt(-1) l culture(-1), respectively over 10 h. This work provides the choice for using a recombinant E. coli strain, which produces H2 from sucrose, as an alternative to coupling-in an upstream invertase, and hence this provides a simpler method for the bioproduction of H2 from sucrose.

Exploration of Biases That Affect the Interpretation of Restriction Fragment Patterns Produced by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) has been used extensively in epidemiological investigations of bacteria, especially during food-borne outbreaks or nosocomial infections. The relationship between similarities in PFGE patterns and true genetic relatedness is poorly understood. In this study, computer-simulated populations of Escherichia coli isolates were created by mutating the sequence of E. coli K-12 strain MG1655. The simulated populations of isolates were then digested, again through simulation, with different restriction enzymes and were analyzed for their relatedness by different techniques. Errors associated with band determination and band matching were incorporated into the analyses, as both of these error types have been shown to affect PFGE interpretations. These errors increased the apparent similarities of the isolates. The use of multiple enzymes improved the fidelity between the results of PFGE analyses and the true sequence similarities. These findings, when they are combined with results from laboratory studies, emphasize the need for the inclusion of multiple enzymes and additional epidemiological data in order to make more accurate interpretations.

First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx(2c) gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx(2c) gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

Inactivation of the Mycobacterial Rhamnosyltransferase, Which Is Needed for the Formation of the Arabinogalactan-Peptidoglycan Linker, Leads to Irreversible Loss of Viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.

The pathogenicity island-associated K15 capsule determinant exhibits a novel genetic structure and correlates with virulence in uropathogenic Escherichia coli strain 536.

The K15 capsule determinant of uropathogenic Escherichia coli strain 536 (O6:K15:H31) is part of a novel 79.6-kb pathogenicity island (PAI) designated PAI V536 that is absent from the genome of nonpathogenic E. coli K-12 strain MG1655. PAI V536 shows typical characteristics of a composite PAI that is associated with the pheV tRNA gene and contains the pix fimbriae determinant as well as genes coding for a putative phosphoglycerate transport system, an autotransporter protein, and hypothetical open reading frames. A gene cluster coding for a putative general secretion pathway system, together with a kps(K15) determinant, is localized downstream of a truncated pheV gene ('pheV) also present in this chromosomal region. The distribution of genes present on PAI V536 was studied by PCR in different pathogenic and nonpathogenic E. coli isolates of various sources. Analysis of the 20-kb kps locus revealed a so far unknown genetic organization. Generally, the kps(K15) gene cluster resembles that of group 2 and 3 capsules, where two conserved regions (regions 1 and 3) are located up- or downstream of a highly variable serotype-specific region (region 2). Interestingly, recombination of a group 2 and 3 determinant may have been involved in the evolution of the K15 capsule-encoding gene cluster. Expression of the K15 capsule is important for virulence in a murine model of ascending urinary tract infection but not for serum resistance of E. coli strain 536.

The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli.

DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

A Salmonella fim homologue in Citrobacter freundii mediates invasion in vitro and crossing of the blood-brain barrier in the rat pup model.

From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed. Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium. The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim(Cf) genes. Expression of the Fim(Cf) proteins was shown by (35)S labeling and/or Western blotting. In the infant rat model of experimental hematogenous meningitis, C. freundii strain 3009 and the in vitro invasive recombinant E. coli K-12 strain harboring the fim(Cf) determinant reached the cerebrospinal fluid, in contrast to the case for the control strain. The fim determinant was also necessary for efficient in vitro invasion by C. freundii, because a deletion mutant was strongly reduced in its invasion efficiency. The mutation could be complemented in trans by the corresponding genes. Invasion by C. freundii could be blocked only by d-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested. In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate. This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion. These results showed the fim determinant of C. freundii to be responsible for d-mannose- and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.

Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.

Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 micro g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.

Escherichia coli rpoS gene has an internal secondary translation initiation region

Sigma S (σ s) encoded by rpoS in Escherichia coli is a stationary phase specific sigma subunit of the RNA polymerase holoenzyme. Widespread among the E. coli K12 strains is an amber mutation that prematurely terminates σ s. These rpoSAm mutants would be expected to show no σ s activity. However, suppressor free rpoSAm mutants retain an intermediate catalase activity, a sigma S controlled function. By analyzing the sequence of the rpoS gene we hypothesize that a 277 amino acids long Δ1-53σ s of about 30 kDa can be translated from an internal secondary translation initiation region (STIR, AGGGAGN 11GUG) that is located downstream of the amber codon. By cloning this rpoSAm gene, following the expression, function, and N-terminal sequence of this mutant protein, we report the presence of a functional internal STIR in E. coli rpoS, from where a truncated but nevertheless functional form of σ s can be synthesized.

EHP002 Plasmid-encoded fimbrial genes as a specific feature and diagnostic target for sorbitol-fermenting enterohaemorrhagic Escherichia coli O157:H−

The presence of virulence genes, encoding enterohemorrhagic Escherichia coli (EHEC)-hemolysin (EHEC-hlyA), intimin (eae), and Shiga toxins 1 (stx1) and 2 (stx2), in 178 isolates of pathogenic E. coli, was determined using the polymerase chain reaction with primers specific for each virulence gene. The tested organisms were 120 isolates of E. coli O157:H7 from human patients, cattle, sheep and foods, 16 non-O157:H7 EHEC isolates from patients suffering from hemorrhagic colitis or hemolytic uremic syndrome, 15 non-O157:H7 Shiga toxin-producing E. coli (STEC) isolates from cattle and foods, 26 isolates of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC), and an E. coli K12 strain. Results revealed that all isolates of O157:H7 carried EHEC-hlyA, eae, and one or both stx genes; 15 of the 16 non-O157:H7 EHEC isolates had EHEC-hlyA, but all possessed eae and one or both stx genes; only seven of the 15 non-O157 STEC isolated from cattle and foods contained both EHEC-hlyA and eae genes. The EPEC, EIEC, ETEC, and the E. coli K12 strain did not carry these virulence genes, except eight EPEC isolates were positive for eae. Results suggest that a combination of EHEC-hlyA and eae genes could serve as markers to differentiate EHEC from less pathogenic STEC, and other pathogenic or non-pathogenic E. coli.

Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli.

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains of Escherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogen E. coli O157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuated E. coli K-12 strain. In the case of stable mutators, mutS defects in strains identified among natural isolates were analysed, including two E. coli O157 : H7 strains, a diarrhoeagenic E. coli O55 : H7 strain, and a uropathogenic strain from the E. coli reference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient in mutS transcripts or produced transcripts truncated at the 3' end. Nucleotide sequence analysis revealed extensive deletions in the mutS region of three strains, ranging from 7.5 to 17.3 kb relative to E. coli K-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in the mutS coding sequence. These results provide insights into the status of the mutS gene and its product in pathogenic strains of E. coli.

Definition of the Escherichia coli MC4100 genome by use of a DNA array.

We have used an Escherichia coli K-12 whole-genome array based on the DNA sequence of strain MG1655 as a tool to identify deletions in another E. coli K-12 strain, MC4100, by probing the array with labeled chromosomal DNA. Despite the continued widespread use of MC4100 as an experimental system, the specific genetic relationship of this strain to the sequenced K-12 derivative MG1655 has not been resolved. MC4100 was found to contain four deletions, ranging from 1 to 97 kb in size. The exact nature of three of the deletions was previously unresolved, and the fourth deletion was altogether unknown.

Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.

Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain.

Escherichia coli is a diverse bacterial species that comprises commensal nonpathogenic strains such as E. coli K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic E. coli strain chi7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-E. coli K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and E. coli iro locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain chi7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in E. coli K-12. These E. coli conserved genes are likely to be highly expressed in vivo and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression and physiology of a pathogenic E. coli strain in a natural animal host during the infectious process.

Identification and characterization of lpfABCC'DE, a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7.

The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC'DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC'DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim(-)) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12.

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.