Genomic heterogeneity and ecological speciation within one subspecies of Bacillus subtilis

Abstract

Closely related bacterial genomes usually differ in gene content, suggesting that nearly every strain in nature may be ecologically unique. We have tested this hypothesis by sequencing the genomes of extremely close relatives within a recognized taxon and analyzing the genomes for evidence of ecological distinctness. We compared the genomes of four Death Valley isolates plus the laboratory strain W23, all previously classified as Bacillus subtilis subsp. spizizenii and hypothesized through multilocus analysis to be members of the same ecotype (an ecologically homogeneous population), named putative ecotype 15 (PE15). These strains showed a history of positive selection on amino acid sequences in 38 genes. Each of the strains was under a different regimen of positive selection, suggesting that each strain is ecologically unique and represents a distinct ecological speciation event. The rate of speciation appears to be much faster than can be resolved with multilocus sequencing. Each PE15 strain contained unique genes known to confer a function for bacteria. Remarkably, no unique gene conferred a metabolic system or subsystem function that was not already present in all the PE15 strains sampled. Thus, the origin of ecotypes within this clade shows no evidence of qualitative divergence in the set of resources utilized. Ecotype formation within this clade is consistent with the nanoniche model of bacterial speciation, in which ecotypes use the same set of resources but in different proportions, and genetic cohesion extends beyond a single ecotype to the set of ecotypes utilizing the same resources.

FIG 1

Models of bacterial speciation. Ecotypes are represented by different colors, periodic selection events are indicated by asterisks, and extinct lineages are represented by dashed lines. The letters at the top represent the resources that each group of organisms can utilize. In cases where ecotypes utilize the same set of resources but in different proportions, the predominant resource of each ecotype is noted by a capital letter. (A) Stable-ecotype model. In this model, each ecotype endures many periodic selection events during its long lifetime. The stable-ecotype model generally yields a one-to-one correspondence between ecotypes and sequence clusters. The ecotypes are able to coexist indefinitely because each has a resource not shared with the others (22). (Reprinted from reference [copyright 2011 Federation of European Microbiological Societies; published by Blackwell Publishing Ltd., all rights reserved].) (B) Speedy-speciation model. This model is much like the stable-ecotype model, except that speciation occurs so rapidly that most newly divergent ecotypes cannot be detected as sequence clusters in multilocus analyses (51). (Adapted from reference with permission of Elsevier.) (C) Nanoniche model. Three nanoniche ecotypes use the same set of resources but in different proportions (noted by Abc, aBc, and abC). Each nanoniche ecotype can coexist with the other two because they have partitioned their resources, at least quantitatively. However, because the ecotypes share all their resources, each is vulnerable to a possible speciation-quashing mutation that may arise in the other ecotypes. This could be a mutation that increases efficiency in utilization of all resources. These speciation-quashing mutations are indicated by a large asterisk; each of these extinguishes the other nanoniche ecotypes. Thus, in the nanoniche model, cohesion can cut across ecologically distinct populations, provided that they are only quantitatively different in their resource utilization (22). (Reprinted from reference [copyright 2011 Federation of European Microbiological Societies; published by Blackwell Publishing Ltd., all rights reserved].) (D) Speciesless model. Here the diversity within an ecotype is limited not by periodic selection but instead by the short time from the ecotype's invention as a single mutant until its extinction. The origination and extinction of each ecotype i are indicated by s_i and e_i, respectively. In the absence of periodic selection, each extant ecotype that has given rise to another ecotype is a paraphyletic group, and each recent ecotype that has not yet given rise to another ecotype is monophyletic (50). (Adapted from reference .) (E) Recurrent niche invasion model. Here a lineage may move, frequently and recurrently, from one ecotype to another, usually by acquisition and loss of niche-determining plasmids. Red lines indicate the times in which a lineage is in the plasmid-containing ecotype; blue lines indicate the times when the lineage is in the plasmid-absent ecotype. Periodic selection events within one ecotype extinguish only the lineages of the same ecotype. For example, in the most ancient periodic selection event shown, which is in the plasmid-absent (blue) ecotype, only the lineages missing the plasmid at the time of periodic selection are extinguished, while the plasmid-containing lineages (red) persist. Ecotypes determined by a plasmid are not likely to be discoverable as sequence clusters (22). (Reprinted from reference [copyright 2011 Federation of European Microbiological Societies; published by Blackwell Publishing Ltd., all rights reserved].)

FIG 2

Maximum likelihood core genome phylogeny of PE15 strains, rooted by strain 168 of PE10, with genome content comparisons (A) and positive selection analyses (B). In each internode, the unique genes are classified as follows: total genes/genes with known bacterial function/genes present in characterized functional subsystems. Each node was supported by 100% of 1,000 bootstrap replicates. In panel A, the left pie chart for each internode indicates the proportion of unique genes that are hypothetical, are phage or transposon related, or have a known bacterial function; the right pie chart indicates the proportion of unique genes in each of the RAST major functional systems. In the case of strain 168, the fraction of genes in five systems was too small to be visible, with each of the following at 1%: cell division and cell cycle, nitrogen metabolism, potassium metabolism, respiration, and membrane transport. In panel B, the pie charts indicate the functional classification of genes under positive selection for amino acid sequence.

FIG 3

Functional classification of gene content for strain G1A3 of PE15, by RAST functional systems (A) and subsystems (B) within the carbohydrate system. The number of genes in each system or subsystem is indicated in parentheses. The percentages of genes in each system category were very similar across the PE15 strains (with an average standard deviation of 0.20% across all system categories) (see Table S3 in the supplemental material). Likewise, the PE15 genomes were similar in the number of genes at the subsystem level. For example, in the carbohydrate subsystem, the average standard deviation of gene content was 0.66%.

FIG 4

Stationary-phase densities in monoculture with maltose, maltodextrin, inositol, or glucose. (A) Stationary-phase density (K) as estimated by absorbance; (B) stationary-phase density corrected by density in glucose.