Detecting Selective Sweeps in Naturally Occurring Escherichia Coli

The nucleotide sequences of the gapA and pabB genes (separated by approximately 32.5 kb) were determined in 12 natural isolates of Escherichia coli. Three analyses were performed on the data. First, the levels of polymorphism at the loci were compared within and between E. coli and Salmonella strains relative to their degrees of constraint. Second, the gapA and pabB loci were analyzed by the Hudson-Kreitman-Aguade (HKA) test for selective neutrality. Four additional dispersed genes (crr, putP, trp and gnd) were added to the analysis to provide the necessary frame of reference. Finally, the gene genealogies of gapA and pabB were examined for topological consistency within and between the loci. These lines of evidence indicate that some evolutionary event has recently purged the variability in the region surrounding the gapA and pabB loci in E. coli. This can best be explained by the spread of a selected allele through the global E. coli population by directional selection and the resulting loss in variability in the surrounding regions due to genetic hitchhiking.     

Conservation and variation of nucleotide sequences within related bacterial genomes: enterobacteria

We have assessed the degree of relatedness of several portions of the Escherichia coli genome to the corresponding portions of the genomes of representative enteric bacteria, using the Southern transfer and hybridization technique (E. Southern, J. Mol. Biol. 98:503-517, 1975). The degree of relatedness varied among the regions examined. Judging both by the relative amounts of deoxyribonucleic acid in the various enteric genomes that are highly homologous and by the conservation of positions of restriction enzyme cleavage sites in these regions, the enteric genomes have diverged to greater extents in some parts of the genomes than in others. Portions of the genomes (including the tnaA and thyA genes, the trp operon, and one other unassigned segment) appear to have evolved in concert with the genome as a whole. By contrast, the lacZ gene and portions of the genome that are homologous to phage lambda vary more widely, perhaps reflecting a separate evolutionary origin for these segments of deoxyribonucleic acid.     

Conservation and variation of nucleotide sequences within related bacterial genomes: Escherichia coli strains.

Changes in the patterns produced by annealing restriction endonuclease digests of bacterial genomes with probe deoxyribonucleic acids (DNAs) containing small portions of a bacterial genome provide sensitive indicator of the degree of nucleotide sequence relatedness that exists in localized regions of the genomes of closely related bacteria. We have used five probe DNAs to explore the relatedness of parts of the genomes of six laboratory Escherichi coli strains. A range in in the amount of variability in the positions of restriction enzyme cleavage sites in the selected portions of the genomes was found. Portions of the genome that are believed to be inacative were more variable than portions that contained functional genes: the sites in and near regions of homology to phage lambda DNA in the genome showed the greatest variability. These regions probably represent remnants of cryptic prophages. Variability was assessed pairwise among four of the E. coli strains and ranged from 5 to > 25% base pair substitutions in the lambda-related regions. In contrast, the endonuclease cleavage sites in the trp, tna, lac, thy regions, and one other as-yet-unidentified segment of the genome were more highly conserved. It seems likely that these sites lie in genetic locations that are subject to functional constraints.     

Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes

The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical sigma S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of sigma S sequence conservation to include other Gram-negative bacteria, the E. coli sigma S sequences were compared with those from diverse Gram-negative organisms; sigma S sequence identities ranged from 50.2 to 99.7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates > 10% after 2 h exposure to pH 2.5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.     

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.     

Heterogeneity of genome sizes among natural isolates of Escherichia coli.

Comparisons of the genetic maps of Escherichia coli K-12 and Salmonella typhimurium LT2 suggest that the size and organization of bacterial chromosomes are highly conserved. Employing pulsed-field gel electrophoresis, we have estimated the extent of variation in genome size among 14 natural isolates of E. coli. The BlnI and NotI restriction fragment patterns were highly variable among isolates, and genome sizes ranged from 4,660 to 5,300 kb, which is several hundred kilobases larger than the variation detected between enteric species. Genome size differences increase with the evolutionary genetic distance between lineages of E. coli, and there are differences in genome size among the major subgroups of E. coli. In general, the genomes of natural isolates are larger than those of laboratory strains, largely because of the fact that laboratory strains were derived from the subgroup of E. coli with the smallest genomes.     

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.     

Genetic Analysis of Recombination-Deficient Mutants of Escherichia coli K-12 Carrying rec Mutations Cotransducible with thyA

The rec mutations carried by 20 strains of Escherichia coli K-12 which are defective in genetic recombination and sensitive to ultraviolet light and X rays, and whose lambda lysogens show spontaneous phage production, have been mapped near thyA. In 15 of the strains, the rec mutation fails to complement recB21 but complements rec-22. The other five strains carry a rec mutation which complements recB21 but not rec-22. These mutations map closer to thyA than those which fail to complement recB21. They therefore appear to be defective in a different recombination gene, denoted recC. The order of recB and recC on the linkage map of E. coli K-12 is thyA-recC-recB-argA.     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.     

Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli.     

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.     

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.     

Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli.

Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.     

Evidence for convergent evolution of CTX-M-14 ESBL in Escherichia coli and its prevalence

A total of 2440 Escherichia coli strains isolated in 2003 at the Hospital de la Santa Creu i Sant Pau were evaluated for the presence of extended-spectrum beta-lactamases. Two different nucleotide sequences that encode the same beta-lactamase, CTX-M-14, were detected when the bla(CTX-M-14)-genes of 35 E. coli isolates were analysed. Thirty-two of the 35 had the previously described sequence of the bla(CTX-M-14) (AF252622), named bla(CTX-M-14a), and the remaining three isolates showed a nucleotide sequence identical to that of the bla(CTX-M-9) gene except for one nucleotide, named bla(CTX-M-14b). Characterisation of the regions surrounding the bla(CTX-M-14a) showed the ISEcp1 and the IS903 upstream and downstream, respectively, of the bla gene, whereas the regions surrounding the bla(CTX-M-14b) contained the genetic environment described for the bla(CTX-M-9) gene, the In60. Characterisation by hybridisation showed that the bla(CTX-M-14a) was present in IncK plasmids, whereas the bla(CTX-M-14b) was found in the HI2 Inc group. The CTX-M-14 ESBL in E. coli isolates is the result of the convergence of two different genes.     

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.     

Plasmid association and nucleotide sequence relationships of two genes encoding heat-stable enterotoxin production in Escherichia coli H-10407.

Plasmid DNA from enterotoxigenic Escherichia coli strains H-10407 and H-10407-P was examined for nucleotide sequence homology to two E. coli genes encoding infant mouse-active heat-stable enterotoxins (ST). A 62-megadalton plasmid of strain H-10407 contained sequences homologous to the gene encoding a toxin designated STIb, previously isolated from a human isolate of E. coli. A 42-megadalton plasmid of strains H-10407 and H-10407-P contained sequences homologous to the gene encoding a toxin designated STIa, previously isolated from bovine and porcine isolates of E. coli.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.