Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.     

How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains

Uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31) is one of the model organisms of extraintestinal pathogenic E. coli (ExPEC). To analyze this strain's genetic basis of urovirulence, we sequenced the entire genome and compared the data with the genome sequence of UPEC strain CFT073 (O6:K2:H1) and to the available genomes of nonpathogenic E. coli strain MG1655 (K-12) and enterohemorrhagic E. coli. The genome of strain 536 is approximately 292 kb smaller than that of strain CFT073. Genomic differences between both UPEC are mainly restricted to large pathogenicity islands, parts of which are unique to strain 536 or CFT073. Genome comparison underlines that repeated insertions and deletions in certain parts of the genome contribute to genome evolution. Furthermore, 427 and 432 genes are only present in strain 536 or in both UPEC, respectively. The majority of the latter genes is encoded within smaller horizontally acquired DNA regions scattered all over the genome. Several of these genes are involved in increasing the pathogens' fitness and adaptability. Analysis of virulence-associated traits expressed in the two UPEC O6 strains, together with genome comparison, demonstrate the marked genetic and phenotypic variability among UPEC. The ability to accumulate and express a variety of virulence-associated genes distinguishes ExPEC from many commensals and forms the basis for the individual virulence potential of ExPEC. Accordingly, instead of a common virulence mechanism, different ways exist among ExPEC to cause disease.     

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.     

Physical organization of the ilvEDAC genes of Escherichia coli strain K-12

We have determined the physical location of the ilvEDAC genes on the restriction cleavage map of the ilv region of Escherichia coli K-12 by two methods: (i) heteroduplex and endonuclease cleavage analysis of hybrid phages carrying genetically defined parts of the ilv cluster and (ii) complementation analysis and enzyme assays to determine ilv gene expression from hybrid plasmids containing DNA restriction fragments of the transducing phage lambdah80dilv. The ilvEDA and ilvC operons occupy 2.4 and 0.9 megadalton sequences of DNA, respectively, and are separated by a region of 0.6-0.75 megadalton. The ilvD region, specifying dihydroxy acid dehydrase, has a maximum coding capacity of about 55,000 daltons of polypeptide. Our results confirm that ilvC is transcribed clockwise on the E. coli K-12 map, in the same direction as ilaEDA. A secondary lambda attachment site within ilvC has been located on a small (0.45 megadalton) EcoRI fragment. Our results are compared to other physical studies of ilv DNA.     

Phylogenetic and pathotypic similarities between Escherichia coli isolates from urinary tract infections in dogs and extraintestinal infections in humans

Seventeen Escherichia coli isolates from dogs with urinary tract infection (UTI) were characterized with respect to phylogenetic background and virulence genotype and were compared with the E. coli reference (ECOR) collection and with human clinical isolates with similar serotypes from patients with diverse extraintestinal infections. Most of the canine urine isolates were from (virulence-associated) E. coli phylogenetic groups B2 or D, expressed papG allele III, and exhibited numerous other putative virulence genes that are characteristic of human extraintestinal pathogenic E. coli (ExPEC). Close phylogenetic and pathotypic correspondence was documented within 5 clonal groups among individual canine and human isolates, including archetypal human ExPEC strains CFT073 (O6:K2:H1), 536 (O6:K15:H31), and J96 (O4:K-:H5). These findings suggest that canine UTI isolates, rather than being dog-specific pathogens, as previously suspected, may pose an infectious threat to humans. Commonality between canine and human ExPEC has potentially important implications for disease prevention, antibiotic resistance avoidance, and studies of pathogenesis.     

Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors

We reported recently that the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans and that many P. aeruginosa virulence factors (genes) required for maximum virulence in mouse pathogenicity are also required for maximum killing of C. elegans. Here we report that among eight P. aeruginosa PA14 TnphoA mutants isolated that exhibited reduced killing of C. elegans, at least five also exhibited reduced virulence in mice. Three of the TnphoA mutants corresponded to the known virulence-related genes lasR, gacA, and lemA. Three of the mutants corresponded to known genes (aefA from Escherichia coli, pstP from Azotobacter vinelandii, and mtrR from Neisseria gonorrhoeae) that had not been shown previously to play a role in pathogenesis, and two of the mutants contained TnphoA inserted into novel sequences. These data indicate that the killing of C. elegans by P. aeruginosa can be exploited to identify novel P. aeruginosa virulence factors important for mammalian pathogenesis.     

Isolation and identification of Escherichia albertii from a patient in an outbreak of gastroenteritis

A microbial strain harboring the eae gene, which is known as the virulence gene of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli, was isolated from a patient in a gastroenteritis outbreak that occurred in 22 patients in Akita Prefecture, Japan, in November 2011. The biochemical characteristics of the isolate were more similar to those of a novel Escherichia sp., E. albertii than E. coli. Partial 16S rRNA gene sequences of the isolate were identical to those of a certain E. albertii strain, but also showed a high degree of similarity to those of E. coli strains. Finally, we identified this isolate as E. albertii by performing PCR analysis that targeted the uidA, lysP, mdh, and cdtB genes in addition to stx and eae genes to differentiate between the EPEC and E. albertii strains.     

Genetic recombination can generate altered restriction specificity.

A recombinant strain, isolated following the transduction of an Escherichia coli recipient carrying the Salmonella typhimurium (SB) specificity genes with DNA from a donor having the Salmonella potsdam (SP) specificity, was shown [Bullas, L.R., Colson, C. & Van Pel, A. (1976) J. Gen. Microbiol. 95, 166-172] to have neither SB nor SP specificity but to encode a novel restriction specificity, SQ. The heteroduplex analysis of the hsdS (specificity) genes of the SB and SP restriction and modification systems described here identifies a conserved sequence of around 100 base pairs flanked by two nonhomologous regions each of approximately 500 base pairs. This organization parallels that previously deduced from the DNA sequences of the hsdS genes of the related E. coli K-12, B, and D restriction systems. The present heteroduplex analyses further show that the hsdS gene conferring the SQ specificity derives one nonhomologous region from the SB gene and the other from the SP gene, as predicted from genetic exchange within the conserved sequence. This finding supports the idea that two domains of an hsdS polypeptide, which are different for each specificity, may correlate with two regions of the DNA sequence recognized. It has been shown that the recognition sequences for E. coli K-12 and B each consist of two short oligonucleotide sequences interrupted by a nonspecific sequence. A similar organization is suggested for the Salmonella specificity systems, providing the potential for evolutionary diversification of restriction specificities as a result of recombination within the conserved sequence of the hsdS gene.     

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.     

Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract.

Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces and inflammatory cells in vitro, but their contribution to virulence has not been defined. This study presents evidence that type 1 fimbriae increase the virulence of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory response to infection. In a clinical study, we observed that disease severity was greater in children infected with E. coli O1:K1:H7 isolates expressing type 1 fimbriae than in those infected with type 1 negative isolates of the same serotype. The E. coli O1:K1:H7 isolates had the same electrophoretic type, were hemolysin-negative, expressed P fimbriae, and carried the fim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:K1:H7 isolates survived in higher numbers, and induced a greater neutrophil influx into the urine, than O1:K1:H7 type 1-negative isolates. To confirm a role of type 1 fimbriae, a fimH null mutant (CN1016) was constructed from an O1:K1:H7 type 1-positive parent. E. coli CN1016 had reduced survival and inflammatogenicity in the mouse urinary tract infection model. E. coli CN1016 reconstituted with type 1 fimbriae (E. coli CN1018) had restored virulence similar to that of the wild-type parent strain. These results show that type 1 fimbriae in the genetic background of a uropathogenic strain contribute to the pathogenesis of E. coli in the urinary tract.     

Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria.

Restriction-modification systems can protect bacteria against viral infection. Sequences of the hsdM gene, encoding one of the three subunits of type I restriction-modification systems, have been determined for four strains of enterobacteria. Comparison with the known sequences of EcoK and EcoR124 indicates that all are homologous, though they fall into three families (exemplified by EcoK, EcoA, and EcoR124), the first two of which are apparently allelic. The extent of amino acid sequence identity between EcoK and EcoA is so low that the genes encoding them might be better termed pseudoalleles; this almost certainly reflects genetic exchange among highly divergent species. Within the EcoK family the ratio of intra- to interspecific divergence is very high. The extent of divergence between the genes from Escherichia coli K-12 and Salmonella typhimurium LT2 is similar to that for other genes with the same level of codon usage bias. In contrast, intraspecific divergence (between E. coli strains B and K-12) is extremely high and may reflect the action of frequency-dependent selection mediated by bacteriophages. There is also evidence of lateral transfer of a short sequence between E. coli and S. typhimurium.     

致病性大肠埃希菌O2、O（78）及融合双价弱毒菌株O（2,78）（Chl^rNor^r）ompA基因的克隆及序列分析

目的 应用PCR扩增大肠埃希菌O2、O78及融合双价弱毒菌株O2,78的外膜蛋白A（ompA）基因,并进行克隆。方法 根据GenBank中人源大肠埃希菌K-12的ompA核苷酸序列设计特异引物,应用PCR对大肠埃希菌O2、O78及融合双价弱毒菌株O2,78的ompA基因进行体外扩增、克隆和序列分析。结果 O2、O78和O2,78经PCR获得的ompA基因片段大小为1 041 bp,与理论值相符;经过克隆和测序分析,该基因的核苷酸序列均由2 271 nt组成,编码1个由346个氨基酸组成的前外膜蛋白A,三菌株ompA基因核苷酸序列与GenBank提供的已知基因序列比较,同源性均为100%。结论 本研究成功克隆了大肠埃希菌ompA基因全长。     

A virulence plasmid in Escherichia coli enterotoxigenic for humans: intergenetic transfer and expression

We studied the colonization-factor antigen I (CFA/I) fimbriae- and heat-stable enterotoxin (ST)-coding plasmid of enterotoxigenic Escherichia coli (strain H10407, serotype O78:H11) pathogenic for humans. With use of conjugal-transfer system of E. coli H10407 and transposon-labeling techniques, the virulence plasmid was shown to be transferable to many species of the family Enterobacteriaceae, including the enteropathogens, Shigella and Salmonella species, and the opportunistic pathogens, Klebsiella, Enterobacter, Citrobacter, Edwardsiella, Serratia, and Proteus species. The virulence plasmid-carrying transconjugants produced both CFA/I fimbriae and ST (exotoxin). Moreover, most of the transconjugants stably inherited the virulence plasmid, although plasmid stability was greatly dependent on culture temperature. Finally, administration of the virulence plasmid-carrying living bacterial cells of Klebsiella, Enterobacter, and Citrobacter species and E. coli K12 resulted in fluid accumulation in both infant-mouse and rabbit ileal-loop tests.     

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.     

Challenge studies in volunteers using Escherichia coli strains with diffuse adherence to HEp-2 cells

Diffusely adherent (DA) Escherichia coli, a newly described category, has been associated with diarrhea in children in some but not all epidemiologic studies. To investigate its diarrheagenic potential, two strains of DA E. coli were fed to adult volunteers in doses of 10(5)-10(10) colony-forming units. None of 22 volunteers who received strain 57-1 or of 21 who received strain C1845 developed symptoms of diarrhea as defined for this study. All volunteers shed the challenge DA E. coli in their stools. Duodenal string cultures were positive in some volunteers only at the highest doses. Only 6 developed IgG or IgA antibodies to whole DA E. coli. Thus, the pathogenic potential of DA E. coli could not be shown in this model. Further studies are needed to characterize potential virulence factors of DA E. coli that might distinguish a pathogenic subset.     

Regulated covalent modifications of lipid A

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella PhoP/PhoQ-activated gene pagP is required for resistance to cationic antimicrobial peptides and for biosynthesis of hepta-acylated lipid A species containing palmitate. Interestingly, pagP encodes an unusual enzyme of lipid A biosynthesis localized in the outer membrane, whereas all previously characterized lipid A enzymes are cytoplasmic or associated with the inner membrane. PagP is not unique, however, as pagL encodes another outer membrane enzyme in Salmonella that deacylates the 3 position of lipid A.S. typhimurium also synthesizes S-2-hydroxymyristate modified lipid A in a PhoP/PhoQ-dependent manner. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Using well-characterized dioxygenase sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A. The S. typhimurium aspartyl beta-hydroxylase homologue (lpxO) was cloned and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression induces O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO may be an inner membrane enzyme that catalyzes Fe(2+)/ascorbate/alpha-ketoglutarate dependent hydroxylation of lipid A. We propose that 2-hydroxymyristate released from LPS inside infected animal cells might be converted to 2-hydroxymyristoyl coenzyme A, a potent inhibitor of protein N-myristoyl transferase.