Frederick M. Cohan

Re-evaluating prokaryotic species

There is no widely accepted concept of species for prokaryotes, and assignment of isolates to species is based on measures of phenotypic or genome similarity. The current methods for defining prokaryotic species are inadequate and incapable of keeping pace with the levels of diversity that are being uncovered in nature. Prokaryotic taxonomy is being influenced by advances in microbial population genetics, ecology and genomics, and by the ease with which sequence data can be obtained. Here, we review the classical approaches to prokaryotic species definition and discuss the current and future impact of multilocus nucleotide-sequence-based approaches to prokaryotic systematics. We also consider the potential, and difficulties, of assigning species status to biologically or ecologically meaningful sequence clusters.

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.

Origins of bacterial diversity through horizontal genetic transfer and adaptation to new ecological niches

Horizontal genetic transfer (HGT) has played an important role in bacterial evolution at least since the origins of the bacterial divisions, and HGT still facilitates the origins of bacterial diversity, including diversity based on antibiotic resistance. Adaptive HGT is aided by unique features of genetic exchange in bacteria such as the promiscuity of genetic exchange and the shortness of segments transferred. Genetic exchange rates are limited by the genetic and ecological similarity of organisms. Adaptive transfer of genes is limited to those that can be transferred as a functional unit, provide a niche-transcending adaptation, and are compatible with the architecture and physiology of other organisms. Horizontally transferred adaptations may bring about fitness costs, and natural selection may ameliorate these costs. The origins of ecological diversity can be analyzed by comparing the genomes of recently divergent, ecologically distinct populations, which can be discovered as sequence clusters. Such genome comparisons demonstrate the importance of HGT in ecological diversification. Newly divergent populations cannot be discovered as sequence clusters when their ecological differences are coded by plasmids, as is often the case for antibiotic resistance; the discovery of such populations requires a screen for plasmid-coded functions. This paper reviews the features of bacterial genetics that allow HGT, the similarities between organisms that foster HGT between them, the limits to the kinds of adaptations that can be transferred, and amelioration of fitness costs associated with HGT; the paper also reviews approaches to discover the origins of new, ecologically distinct bacterial populations and the role that HGT plays in their founding.

Identifying the fundamental units of bacterial diversity: A paradigm shift to incorporate ecology into bacterial systematics

The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach (“ecotype simulation”) to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the “Evolution Canyons” of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.

A Systematics for Discovering the Fundamental Units of Bacterial Diversity

Bacterial systematists face unique challenges when trying to identify ecologically meaningful units of biological diversity. Whereas plant and animal systematists are guided by a theory-based concept of species, microbiologists have yet to agree upon a set of ecological and evolutionary properties that will serve to define a bacterial species. Advances in molecular techniques have given us a glimpse of the tremendous diversity present within the microbial world, but significant work remains to be done in order to understand the ecological and evolutionary dynamics that can account for the origin, maintenance, and distribution of that diversity. We have developed a conceptual framework that uses ecological and evolutionary theory to identify the DNA sequence clusters most likely corresponding to the fundamental units of bacterial diversity. Taking into account diverse models of bacterial evolution, we argue that bacterial systematics should seek to identify ecologically distinct groups with evidence of a history of coexistence, as based on interpretation of sequence clusters. This would establish a theory-based species unit that holds the dynamic properties broadly attributed to species outside of microbiology.

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.

Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses.

In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B′) isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B′, but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.

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.

RECOMBINATION AND MIGRATION RATES IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS MOJAVENSIS

We have investigated the rates of recombination and migration in native populations of two closely related, naturally competent Bacillus species. Native soil isolates of Bacillus subtilis and Bacillus mojavensis were obtained from three continents and, within North America, from populations at a range of geographical distances from one another. The rate of recombination within populations of each species was estimated from restriction‐site data for three genes. Recombination was shown to occur within each species at about the same rate as neutral mutation, whatever the geographical scale or phylogenetic scale over which strains were sampled. The rate of migration between populations was estimated by a cladistic analysis and was shown to be high (i.e., Nm > 1), even among populations on different continents. The level of migration within each species is sufficient to prevent neutral geographical divergence within species.

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.