Genome sequencing of environmental Escherichia coli expands understanding of the ecology and speciation of the model bacterial species

Environmentally Adapted E. coli Lineages.

We recently described five Escherichia clades (C-I to C-V) that were recovered primarily from environmental sources and are indistinguishable from typical E. coli based on traditional phenotypic tests included in either the API20E Identification System (bioMerieux, Inc.) or the BBL Crystal Identification System (Becton, Dickinson and Company) (9). To provide genomic insights into the phylogenetic diversity and metabolic potential of these clades, we sequenced the genome of nine representatives from clades C-I, -III, -IV, and -V (Table 1, Table S1, and Fig. S1). Whole-genome phylogenetic analysis confirmed our earlier observations based on multilocus sequence typing that the clades span the phylogenetic tree between E. coli and E. albertii, forming a genetic continuum within the Escherichia genus. In particular, C-I appears to be a sister clade of typical E. coli, being only slightly more divergent than the B2 phylogenetic lineage that includes uropathogenic E. coli (UPEC). The remaining clades are more divergent from typical E. coli (Fig. 1). In agreement with previous phenotypic testing, the genomes of the strains of the four clades encode all genes shared by the available E. coli genomes (i.e., the E. coli core gene set) (Fig. 2A and Fig. S2). Thus, the clades appear to be phenotypically and genetically (e.g., in gene content) indistinguishable from typical E. coli. Based on this information and the current genomic standards for species demarcation (12), these clades would be justifiably classified as E. coli.

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Fig. 1.

Whole-genome phylogeny of the Escherichia genomes used in the study. The phylogenetic network shown was constructed with the SplitsTree software (27), using as input the concatenated alignment of 1,910 single-copy core genes. (Inset) The graph represents the amount of recent horizontal transfer of core genes between the genomes of the clades. The thickness of the line is proportional to the number of genes transferred (scale at upper left in figure).

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Fig. 2.

Gene-content signatures of Escherichia clades. Heatmap of gene presence (yellow) and absence (blue) in 20 selected genomes, using all nonredundant genes that were found in at least two of the genomes as reference. (A) Genomes were clustered based on the presence/absence of genes; values in red represent bootstrap support from Jackknifing resampling with 100 replicates. (B) Genes and pathways distinguishing enteric and environmental genomes were expanded (underlying data are provided in Table S2). 1, acetylglucosamine transporter; 2, fructose transporter; 3, diol utilization operon; 4, lysozyme production. (C) Occurrence of the genes composing the Escherichia pangenome in the 20 genomes ranges from one (a genome-specific gene) to 20 (a core gene).

The orders-of-magnitude higher abundances of these clades in environmental samples relative to those in human feces and the clinic (9) indicate that they represent truly environmentally adapted organisms (meaning that they are not associated primarily with mammal hosts). Consistent with this interpretation, a recent study found that strains of clades C-III, -IV, and -V form biofilms more readily, outcompete typical E. coli strains at low temperatures (which characterize the environment compared with the gastrointestinal tract of warm-blooded hosts), and are nonpathogenic in a mouse model of septicemia (13). Furthermore, screening of 2,701 strains from humans, animals, and the environment identified an additional 57 environmental clade strains, and these strains were found more often in environmental and bird samples than in human samples (9). These studies consistently support the hypothesis that the environmental clades substantially expand the known ecological niche of E. coli.

Functions Important in the Gut.

Comparisons between the environmental genomes and their commensal or pathogenic (enteric) counterparts provided insights into the functional differentiation of E. coli strains. Consistent with the core gene results described above, we found almost no genes specific to enterics when queried against all genomes of environmental clades (Fig. S2). However, when the C-I clade was included in the enteric group (strains of C-I have been isolated from humans, and this clade does not appear to be overrepresented in environmental samples) and the stringency of the comparisons was relaxed to allow one or two genomes in each group not to encode the gene in question, we identified 84 and 120 genes as being specific to or highly enriched in the environmental and enteric groups, respectively (Fig. 2B and Table S2). The environment-specific gene set included several genes of unknown function as well as the complete pathway for diol utilization (energy substrate) and the gene for lysozyme production (hydrolysis of bacterial cell walls). These functions apparently are important for resource acquisition and survival in the environment. In contrast, the enteric-specific functions included genes involved in the transport and use of several nutrients that are thought to be abundant in the gut, such as N-acetylglucosamine, gluconate, and 5-C and 6-C sugars such as fucose (14). The latter genes were significantly enriched in the recently determined human microbiome (15), further corroborating their importance for colonization of the gut. Therefore, these genes characterize enteric E. coli strains relative to their environmental counterparts and may represent robust biomarkers for the development of molecular assays to count commensal E. coli cells in environmental samples more accurately than done by current methods. The enteric gene set also includes several prophage genes, consistent with recent findings from metagenomic studies indicating that the human virome is highly specialized to its host and differs from viromes of environmental ecosystems (16).

Ecologic Barriers to Gene Flow Within Escherichia.

The availability of several genome sequences that span the Escherichia tree provided the opportunity to evaluate the importance of interclade genetic flow for E. coli evolution with greater phylogenetic coverage than previously achieved (7, 17). To this end, we devised a strategy to assess recent genetic exchange events based on embedded quartet decomposition analysis (EDCA) (details are given in Materials and Methods and Figs. S3 and S4). We focused on recent events only because historic genetic exchange of core genes (mediated by homologous recombination) frequently was impossible to detect robustly because of multiple (old) recombination events on the same segment of the genome and the process of amelioration of the newly introduced DNA sequence into the recipient cell (18).

We observed detectable genetic exchange of core genes within the environmental clades, within enterics, and between C-I and enterics but not between enterics and the remaining environmental clades or E. albertii (Fig. 1 Inset and Fig. S5). The core genes exchanged were distributed randomly in the genome and did not show any strong biases in terms of function when compared with the rest of the genome (Fig. S6 and Table S3). These findings are consistent with a generalized mechanism for the transfer of genetic material (e.g., transformation or conjugation) and incorporation into the recipient genome via homologous recombination. They also confirm the closer affiliation of C-I with typical E. coli relative to the other clades and reveal reduced genetic flow between environmental and enteric genomes, presumably because of ecological barriers.

Nonetheless, the number of core genes exchanged within the evolutionary time that corresponded to 0.02 synonymous substitutions per site (the divergence time typically separating the genomes of the same clade) accounted for only a small portion of the total core genes in the genome (0.06–2.33%). We also observed that noncore (auxiliary) genes were exchanged among the clades less frequently than core genes (Fig. S5 and Table S3). Given also that more than 50% of the total unique genes of the E. coli pangenome are genome or clade specific (Fig. 2C), our observations suggest that asexual divergence coupled with clade-specific gene acquisition or deletion dominates interclade recombination in driving Escherichia evolution.

Test of the Fragmented Speciation Model.

It has been proposed recently that organisms of the Escherichia genus evolve according to a fragmented speciation model (19) and that the model may be applicable to additional bacterial groups (20). If the model were true, one would expect that genomic islands that differentiate two ecologically distinct populations to be flanked by regions of increased nucleotide divergence, because such population-specific islands are free from the homogenizing effects of recombination. In other words, because interpopulation homologous recombination is halted around the genomic island (the sequence is not conserved in the population that does not carry the island), the genetic variation of the flanking DNA would be increased between the two populations compared to within either of the individual populations (Fig. 3 gives a graphical representation of the expected signature of the model).

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Fig. 3.

Lack of evidence in support of the fragmented speciation model. A representative example of the nucleotide divergence patterns, measured as the number of SNPs (y axis) observed in the flanking regions of a genomic island (x axis) that differentiates environmental from enteric genomes. Note the difference between the SNP level expected for the environmental genomes according to the fragmented speciation model (ecologically distinct population) relative to the observed level (for the enteric group, the genome average SNP level represents the expected SNP level according to the model). The island shown encodes the genes for utilization of fucose, a sugar commonly found in the glycan structures of the cell wall of animals.

Our results strongly indicate that the environmentally adapted genomes are ecologically differentiated as compared with their enteric counterparts and thus are more appropriate for testing the model directly than are the divergent Salmonella genomes used previously (19). Although several candidate (ecologically relevant) genomic islands were identified (such as the islands encoding the fucose and gluconate utilization operons), and these islands were flanked by DNA sequences that were conserved and syntenic in the environmental strains, no island showed the predicted signature of the fragmented speciation model. Instead, the level of nucleotide divergence in the flanking regions of the islands covaried between the environmental and enteric genome (Fig. 3). Similar patterns were observed when the analysis was restricted to commensal vs. pathogenic E. coli for the genomic islands that encode the known pathogenicity factors of the latter genomes (Fig. S7). Thus, the predicted signature of the model was not observed even in comparisons of genomes that show both higher genetic relatedness and genetic flow than observed between environmental and enteric strains. In a few of the genomic islands examined, the flanking genes did show increased nucleotide divergence between ecologically distinct genomes. However, this pattern typically was associated with genes that were interrupted by the insertion of mobile elements; because of relaxed functional constraints, the truncation of gene(s), rather than the action of recombination, presumably caused an increased accumulation of mutations. Such truncated genes or their remnants may underlie some of the incongruent phylogenetic signal observed previously (20).

Conclusions and Perspectives.

Our results collectively suggest that asexual divergence coupled with clade-specific gene acquisition or deletion has a much stronger influence on the evolution of the Escherichia genus than homologous recombination (sexual reproduction). These results differ quantitatively from those reported previously (7, 17). The difference is caused, at least in part, by the different genomes and methods used in the analysis. For instance, the previous studies evaluated the intraclade level, whereas our analysis was focused on more divergent genomes, an approach that is advantageous for unequivocally detecting recent gene exchange and recombination events (21, 22). Although our results do not rule out the existence of high levels of recombination within a clade, they do reveal that genetic exchange between incipient ecologically distinct clades of E. coli may not be as pronounced or prolonged as would be expected by the fragmented speciation model (19), and this reduced level of exchange probably accounts for the lack of evidence in support of the model.

Data described here concerning the environmental Escherichia clades show that justifiable species, which are ecologically distinct, sexually isolated, and phylogenetically tractable, may be identifiable even in cases of apparent phenotypic identity or a genetic continuum (such as revealed within the Escherichia genus in Fig. 1). These findings, which also are consistent with recent metagenomic studies of natural populations (22), suggest that a more ecologic definition for species is more appropriate than the current definition that is heavily based on genetic distinctiveness alone. Comparative genomic analyses linked a substantial fraction of the clade-specific gene acquisitions (and deletions) to the unique ecology of the clade (e.g., Fig. 2B). These findings further corroborate the notion that it is time to start replacing traditional approaches of defining diagnostic phenotypes for new species with omics-based procedures.

What the preferred ecological niche or host (if any) of clades II–V is and whether the clades actually can persist in the external environment in the absence of fecal inputs (i.e., represent truly free-living bacteria) remain elusive, and additional data need to be collected before more robust conclusions can emerge. For instance, strains of clades II–V have been recovered occasionally from birds and ruminant mammals (9), but the extent to which these results are influenced by the processes of strain migration and extinction (as opposed to persistence within the host) is unclear. What our genomic data as well as data from physiological studies and environmental surveys performed previously (9, 13) suggest is that clades II–V are better at surviving in the external environment than is commensal E. coli and are poor competitors in the human gastrointestinal tract relative to successful clonal complexes such as those represented by CFT073 and MG1555 strains. Therefore, clades II–V are highly unlikely to represent a risk to public health.

Of practical significance, the cryptic clades represent microorganisms that show worldwide distribution (Table S1) and have been readily identified as typical E. coli by expert microbiologists in the laboratory and by managers of water quality who use this organism to assess fecal pollution of surface waters. However, these organisms probably should not be considered E. coli and are highly unlikely to represent an environmental hazard, according to our analyses. These findings underscore the need to reevaluate coliform testing and the microbiologic dogma that the niche of enteric microbes, such as E. coli, is the mammalian intestinal tract.