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.     

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.     

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.     

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.     

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.     

Impact of mutS inactivation on foreign DNA acquisition by natural transformation in Pseudomonas stutzeri

In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.     

THE POTENTIAL FOR GENETIC EXCHANGE BY TRANSFORMATION WITHIN A NATURAL POPULATION OF BACILLUS SUBTILIS

We have investigated the potential for genetic exchange by transformation within a Mojave Desert population of Bacillus subtilis. Almost all strains surveyed were competent for transformation, and the strains varied over almost three orders of magnitude in their levels of competence. This high degree of variation suggests that natural selection toward an optimal level of competence is, at most, very weak in this population. Six of 24 competent strains showed sexual isolation from laboratory strain 168 (i.e., heterogamic transformation was reduced). Direct crosses between selected pairs of Mojave strains indicated sexual isolation within the Mojave population. Levels of sexual isolation observed within this population of B. subtilis were much less than those previously observed for transformation between named Bacillus species. Sexual isolation between 168 and Mojave strains, and among Mojave strains, was due to differences in restriction-modification systems and to DNA sequence divergence.     

Homologous recombination in Agrobacterium: potential implications for the genomic species concept in bacteria

According to current taxonomical rules, a bona fide bacterialspecies is a genomic species characterized by the genomic similarityof its members. It has been proposed that the genomic cohesionof such clusters may be related to sexual isolation, which limitsgene flow between too divergent bacteria. Homologous recombinationis one of the most studied mechanisms responsible for this geneticisolation. Previous studies on several bacterial models showedthat recombination frequencies decreased exponentially withincreasing DNA sequence divergence. In the present study, weinvestigated this relationship in the Agrobacterium tumefaciensspecies complex, which allowed us to focus on sequence divergencein the vicinity of the genetic boundaries of genomic species.We observed that the sensitivity of the recombination frequencyto DNA divergence fitted a log-linear function until approximately10% sequence divergence. The results clearly revealed that therewas no sharp drop in recombination frequencies at the pointwhere the sequence divergence distribution showed a "gap" delineatinggenomic species. The ratio of the recombination frequency inhomogamic conditions relative to this frequency in heterogamicconditions, that is, sexual isolation, was found to decreasefrom 8 between the most distant strains within a species to9 between the most closely related species, for respective increasesfrom 4.3% to 6.4% mismatches in the marker gene chvA. This meansthat there was only a 1.13-fold decrease in recombination frequenciesfor recombination events at both edges of the species border.Hence, from the findings of this investigation, we concludethat—at least in this taxon—sexual isolation basedon homologous recombination is likely not high enough to stronglyhamper gene flow between species as compared with gene flowbetween distantly related members of the same species. The 70%relative binding ratio cutoff used to define bacterial speciesis likely correlated to only minor declines in homologous recombinationfrequencies. Consequently, the sequence diversity, as a mechanisticfactor for the efficiency of recombination (as assayed in thelaboratory), appears to play little role in the genetic cohesionof bacterial species, and thus, the genomic species definitionfor prokaryotes is definitively not reconcilable with the biologicalspecies concept for eukaryotes.     

Gene Flow and Natural Selection in the Origin of Drosophila Pseudoobscura and Close Relatives

The divergence of Drosophila pseudoobscura and close relatives D. persimilis and D. pseudoobscura bogotana has been studied using comparative DNA sequence data from multiple nuclear loci. New data from the Hsp82 and Adh regions, in conjunction with existing data from Adh and the Period locus, are examined in the light of various models of speciation. The principal finding is that the three loci present very different histories, with Adh indicating large amounts of recent gene flow among the taxa, while little or no gene flow is apparent in the data from the other loci. The data were compared with predictions from several isolation models of divergence. These models include no gene flow, and they were found to be incompatible with the data. Instead the DNA data, taken together with other evidence, seem consistent with divergence models in which natural selection acts against gene flow at some loci more than at others. This family of models includes some sympatric and parapatric speciation models, as well as models of secondary contact and subsequent reinforcement of sexual isolation.     

Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae

In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbK-psbI, trnH-psbA, and rpoB.The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter- and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belongingto 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.     

THE EVOLUTION OF ASYMMETRY IN SEXUAL ISOLATION: A MODEL AND A TEST CASE

We constructed a model for the evolution of sexual isolation by extending Lande's (1981) model of sexual selection. The model predicts that asymmetric sexual isolation is a transient phenomenon, characteristic of intermediate stages of divergence in sexually selected traits. Unlike the Kaneshiro (1976, 1980) proposal, our model does not depend upon drift and the loss of courtship elements to produce asymmetries in sexual isolation. According to our model, the direction of evolution cannot be predicted from asymmetry in sexual isolation. We tested some features of the model using data from an experimental study of sexual isolation in the salamander Desmognathus ochrophaeus. We tested for sexual isolation between 12 allopatric populations and found significant asymmetry in sexual isolation in about a quarter of the test cases. The highest degrees of asymmetry were associated with intermediate levels of divergence. A curvilinear relationship between isolation asymmetry and divergence was predicted by our model and was supported by statistical analysis of the salamander data.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

DOES RECOMBINATION CONSTRAIN NEUTRAL DIVERGENCE AMONG BACTERIAL TAXA?

A coalescence model for predicting the fate of neutral divergence among closely related taxa distinguishable as separate DNA sequence clusters is presented here. The model simulates iteratively the positive feedback between sequence divergence and sexual isolation among taxa, where increases in sequence divergence result in reduced recombination, and reduced recombination results in increased sequence divergence. Iteration of this feedback is continued until sequence divergence either converges on a steady state or reaches a runaway process. The eventual outcome of sequence divergence was shown to depend on four estimable population-genetic parameters: the expected intrataxon sequence diversity, the baseline rate of intertaxon recombination, the sensitivity of the recombination rate to sequence divergence, and the neutral mutation rate. The model can be used to determine whether neutral divergence among actual taxa is destined to stop at an equilibrium level, or whether neutral divergence will reach a runaway process. Application of the model to the group of taxa containing Bacillus subtilis and its closest relatives showed these taxa to be on a trajectory of unbounded neutral divergence from one another.     

Molecular correlates of reproductive isolation

Evolution of reproductive isolation as a byproduct of genetic divergence in isolated populations is the dominant (albeit not exclusive) mode of speciation in sexual animals. But little is known about the factors linking speciation to general divergence. Several authors have argued that allopatric speciation should proceed more rapidly if isolated populations also experience divergent selection. Reproductive isolation between allopatric populations is not subject to direct selection; it can accumulate only by random drift or as a fortuitous byproduct of selection on other traits. Here I present a novel analysis of published data, demonstrating that pre- and postmating isolation of Drosophila species are more tightly correlated with allozyme divergence than with silent DNA divergence. Inasmuch as proteins are more subject to the action of natural selection than are silent DNA polymorphisms, this result provides broad support for a model of selection-mediated allopatric speciation.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.     

DOES GENE FLOW CONSTRAIN ADAPTIVE DIVERGENCE OR VICE VERSA? A TEST USING ECOMORPHOLOGY AND SEXUAL ISOLATION IN TIMEMA CRISTINAE WALKING‐STICKS

Population differentiation often reflects a balance between divergent natural selection and the opportunity for homogenizing gene flow to erode the effects of selection. However, during ecological speciation, trait divergence results in reproductive isolation and becomes a cause, rather than a consequence, of reductions in gene flow. To assess both the causes and the reproductive consequences of morphological differentiation, we examined morphological divergence and sexual isolation among 17 populations of Timema cristinae walking-sticks. Individuals from populations adapted to using Adenostoma as a host plant tended to exhibit smaller overall body size, wide heads, and short legs relative to individuals using Ceonothus as a host. However, there was also significant variation in morphology among populations within host-plant species. Mean trait values for each single population could be reliably predicted based upon host-plant used and the potential for homogenizing gene flow, inferred from the size of the neighboring population using the alternate host and mitochondrial DNA estimates of gene flow. Morphology did not influence the probability of copulation in between-population mating trials. Thus, morphological divergence is facilitated by reductions in gene flow, but does not cause reductions in gene flow via the evolution of sexual isolation. Combined with rearing data indicating that size and shape have a partial genetic basis, evidence for parallel origins of the host-associated forms, and inferences from functional morphology, these results indicate that morphological divergence in T. cristinae reflects a balance between the effects of host-specific natural selection and gene flow. Our findings illustrate how data on mating preferences can help determine the causal associations between trait divergence and levels of gene flow.     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene ( rpoB  ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.