Tracking bacterial responses to global warming with an ecotype-based systematics.

The broadly defined species of bacterial systematics frequently contain unnamed and unrecognized populations (ecotypes) differing in physiology, genome content, and ecology. Without formal recognition of such ecotypes, it is difficult for microbial ecologists to detect replacement of one ecotype by another in the face of global warming. The ecotype simulation algorithm has proved capable of supporting investigation of such replacements, as it has detected temperature-distinguished ecotypes that are invisible to the present bacterial systematics. Creating an ecotype-based systematics will help to identify the units of diversity that we will want to track as we seek to observe the early microbial responses to global warming.

Genomics, environmental genomics and the issue of microbial species.

A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.

Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona.

Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.

Bacterial species and speciation.

Bacteria are profoundly different from eukaryotes in their patterns of genetic exchange. Nevertheless, ecological diversity is organized in the same way across all of life: individual organisms fall into more less discrete clusters on the basis of their phenotypic, ecological, and DNA sequence characteristics. Each sequence cluster in the bacterial world appears to correspond to an "ecotype," defined as a population of cells in the same ecological niche, which would all be out-competed by any adaptive mutant coming from the population. Ecotypes, so defined, share many of the dynamic properties attributed to eukaryotic species: genetic diversity within an ecotype is limited by a force of cohesion (in this case, periodic selection); different ecotypes are free to diverge without constraint from one another; and ecotypes are ecologically distinct. Also, ecotypes can be discovered and classified as DNA sequence clusters, even when we are ignorant of their ecology. Owing to the rarity and promiscuity of bacterial genetic exchange, speciation in the bacterial world is expected to be much less constrained than in the world of animals and plants.

Protein-coding genes as molecular markers for ecologically distinct populations: the case of two Bacillus species.

Bacillus globisporus and Bacillus psychrophilus are one among many pairs of ecologically distinct taxa that are distinguished by very few nucleotide differences in 16S rRNA gene sequence. This study has investigated whether the lack of divergence in 16S rRNA between such species stems from the unusually slow rate of evolution of this molecule, or whether other factors might be preventing neutral sequence divergence at 16S rRNA as well as every other gene. B. globisporus and B. psychrophilus were each surveyed for restriction-site variation in two protein-coding genes. These species were easily distinguished as separate DNA sequence clusters for each gene. The limited ability of 16S rRNA to distinguish these species is therefore a consequence of the extremely slow rate of 16S rRNA evolution. The present results, and previous results involving two Mycobacterium species, demonstrate that there exist closely related species which have diverged long enough to have formed clearly separate sequence clusters for protein-coding genes, but not for 16S rRNA. These results support an earlier argument that sequence clustering in protein-coding genes could be a primary criterion for discovering and identifying ecologically distinct groups, and classifying them as separate species.

Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation.

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.

DNA sequence similarity requirements for interspecific recombination in Bacillus.

Gene transfer in bacteria is notoriously promiscuous. Genetic material is known to be transferred between groups as distantly related as the Gram positives and Gram negatives. However, the frequency of homologous recombination decreases sharply with the level of relatedness between the donor and recipient. Several studies show that this sexual isolation is an exponential function of DNA sequence divergence between recombining substrates. The two major factors implicated in producing the recombinational barrier are the mismatch repair system and the requirement for a short region of sequence identity to initiate strand exchange. Here we demonstrate that sexual isolation in Bacillus transformation results almost exclusively from the need for regions of identity at both the 5' and 3' ends of the donor DNA strand. We show that, by providing the essential identity, we can effectively eliminate sexual isolation between highly divergent sequences. We also present evidence that the potential of a donor sequence to act as a recombinogenic, invasive end is determined by the stability (melting point) of the donor-recipient complex. These results explain the exponential relationship between sexual isolation and sequence divergence observed in bacteria. They also suggest a model for rapid spread of novel adaptations, such as antibiotic resistance genes, among related species.

Adapt globally, act locally: the effect of selective sweeps on bacterial sequence diversity.

Previous studies have shown that genetic exchange in bacteria is too rare to prevent neutral sequence divergence between ecological populations. That is, despite genetic exchange, each population should diverge into its own DNA sequence-similarity cluster. In those studies, each selective sweep was limited to acting within a single ecological population. Here we postulate the existence of globally adaptive mutations, which may confer a selective advantage to all ecological populations constituting a metapopulation. Such adaptations cause global selective sweeps, which purge the divergence both within and between populations. We found that the effect of recurrent global selective sweeps on neutral sequence divergence is highly dependent on the mechanism of genetic exchange. Global selective sweeps can prevent populations from reaching high levels of neutral sequence divergence, but they cannot cause two populations to become identical in neutral sequence characters. The model supports the earlier conclusion that each ecological population of bacteria should form its own distinct DNA sequence-similarity cluster.

Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov.

Earlier phylogenetic studies based on the inferred DNA sequences of the polC, rpoB and gyrA genes suggested that strains of the species Bacillus subtilis formed two clusters, indicating the presence two closely related taxa; one contained the laboratory strain 168 and the other the laboratory strain W23. Significant sexual isolation was found between strain 168 and members of the group containing W23, but no sexual isolation was observed between strain 168 and other members of the 168 group. DNA reassociation between the two groups ranged from 58 to 69% and intragroup DNA relatedness ranged from 82 to 100%. Because group 168 strains were highly related to the B. subtilis type strain, they were considered to be bona fide members of the species. About 99.5% sequence identity was observed between the 16S rRNA genes of the 168 and W23 groups. Ribitol and anhydroribitol were principal cell wall constituents of the W23 but not of the 168 group. These observations revealed two closely related but genetically and phenotypically distinct groups within B. subtilis that correspond to two historically important strains. Subspecies distinction is proposed for the 168 and W23 groups, with the names Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov., respectively. The type strain of the former is NRRL NRS-744T and the latter NRRL B-23049T.

The effect of mismatch repair and heteroduplex formation on sexual isolation in Bacillus.

In Bacillus transformation, sexual isolation is known to be an exponential function of the sequence divergence between donor and recipient. Here, we have investigated the mechanism under which sequence divergence results in sexual isolation. We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence. Mismatch repair was shown to contribute to sexual isolation but was responsible for only a small fraction of the sexual isolation observed. Another possible mechanism of sexual isolation is that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex. A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence. Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli. For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we conclude that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation.

Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data.

All living organisms fall into discrete clusters of closely related individuals on the basis of gene sequence similarity. Evolutionary genetic theory predicts that in the bacterial world, each sequence similarity cluster should correspond to an ecologically distinct population. Indeed, surveys of sequence diversity in protein-coding genes show that sequence clusters correspond to ecological populations. Future population surveys of protein-coding gene sequences can be expected to disclose many previously unknown ecological populations of bacteria. Sequence similarity clustering in protein-coding genes is recommended as a primary criterion for demarcating taxa.

Bacillus vallismortis sp. nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California.

Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis. The type strain is strain DV1-F-3 (= NRRL B-14890). On the basis of previously published restriction digestion data, B. vallismortis is most closely related to Bacillus subtilis. At this time B. vallismortis can be distinguished from B. subtilis only by differences in whole-cell fatty acid compositions, DNA sequences, and levels of reassociation of genomic DNA.

Homology among nearly all plasmids infecting three Bacillus species.

We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B. mojavensis and B. licheniformis. Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic. Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group. A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups. These groups differed in the sizes of their host ranges and geographical ranges. All but 1 of the 18 plasmids from these three host species are homologous with one another. The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts. The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible. Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.

The size and continuity of DNA segments integrated in Bacillus transformation.

We investigated the size and continuity of DNA segments integrated in Bacillus subtilis transformation. We transformed B. subtilis strain 1A2 toward rifampicin resistance (coded by rpoB) with genomic DNA and with a PCR-amplified 3.4-kb segment of the rpoB gene from several donors. Restriction analysis showed that smaller lengths of donor DNA integrated into the chromosome with transformation by PCR-amplified DNA than by genomic DNA. Nevertheless, integration of very short segments (< 2 kb) from large, genomic donor molecules was not a rare event. With PCR-amplified segments as donor DNA, smaller fragments were integrated when there was greater sequence divergence between donor and recipient. There was a large stochastic component to the pattern of recombination. We detected discontinuity in the integration of donor segments within the rpoB gene, probably due to multiple integration events involving a single donor molecule. The transfer of adaptations across Bacillus species may be facilitated by the small sizes of DNA segments integrated in transformation.

The log-linear relationship between sexual isolation and sequence divergence in Bacillus transformation is robust.

The relationship between sexual isolation and sequence divergence in Bacillus transformation was previously shown to be log linear. In the present study, we have shown that this relationship is robust with respect to naturally occurring genetic variation among recipient strains of Bacillus subtilis and B. mojavensis. Naturally occurring restriction endonuclease activity was shown not to affect this relationship. Also, seven out of eight recombination mutants tested for their sensitivity to sequence divergence have shown the same relationship between sequence divergence and sexual isolation; a mutant for recH was more sensitive to sequence divergence, suggesting that the product of this gene may be involved in resolution of mismatches in heterogamic transformation. We have also shown that the relationship between sexual isolation and sequence divergence is robust with respect to variation in the conditions of transformation, including variation in the length of donor DNA, the concentration of donor DNA, and intracellular competition between donor-derived and recipient-derived DNA. The robustness of the relationship between sexual isolation and sequence divergence among naturally occurring strains and across transformation conditions allows us to predict the eventual outcome of sequence divergence among B. subtilis and its closest relatives.

Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition.

A number of Bacillus strains isolated from desert soil samples were shown to belong to a previously unidentified species, for which we propose the name Bacillus mojavensis. The type strain is RO-H-1 (= NRRL B-14698). On the basis of restriction digest data, B. mojavensis is most closely related to Bacillus amyloliquefaciens, Bacillus atrophaeus, and Bacillus subtilis. So far, B. mojavensis can be distinguished from B. subtilis only by differences in whole-cell fatty acid composition, divergence in DNA sequence, and resistance to genetic transformation between taxa (in addition to reduced genome relatedness values). Sequence divergence and sexual isolation may prove to be more useful than metabolic characteristics for delimiting cryptic Bacillus species.

Genetic exchange and evolutionary divergence in prokaryotes.

Recent work shows that genetic exchange in prokaryotes is less frequent but more promiscuous than that in eukaryotes. As a result, genetic exchange plays very different roles in determining the patterns of evolutionary divergence in these major groups. Because sexual isolation is not a prerequisite for divergence in the prokaryotic world, the biological species concept is not appropriate for bacteria. However, there is a species concept that may apply universally.

The effect of DNA sequence divergence on sexual isolation in Bacillus.

We have investigated the relationship between sexual isolation and DNA sequence divergence in the transformation (at locus rpoB) of a naturally competent strain of Bacillus subtilis. Using both genomic DNA and a PCR-amplified segment of gene rpoB as donor, we found that the extent of sexual isolation at locus rpoB was closely predicted, over three orders of magnitude, as a log-linear function of sequence divergence at that locus. Because sexual isolation between a recipient and any potential donor may be determined as a general mathematical function of sequence divergence, transformation is perhaps the only sexual system, in either the prokaryotic or the eukaryotic world, in which sexual isolation can be predicted for a pair of species without having to perform the cross. These observations suggest the possibility of a general approach to the indirect prediction of sexual isolation in bacteria recombining principally by natural transformation.

Genetic divergence under uniform selection. III. Selection for knockdown resistance to ethanol in Drosophila pseudoobscura populations and their replicate lines.

Replicate D. pseudoobscura lines from populations collected at different geographic locations were selected for increased knockdown resistance to ethanol. Population background affected the initial rate of response but not the extent that lines responded. Lines were tested for physiological traits contributing to increased knockdown resistance. Populations showed different correlated responses for two traits (tolerance of ethanol, and of acetone), suggesting that they had responded to selection by different mechanisms. Replicate lines had diverged for most traits. The results indicate that drift and/or differences in genetic background can lead to divergence under uniform selection, even when fairly large population sizes are maintained.