Mutation Rate and Effective Population Size of the Model Cooperative Bacterium Myxococcus xanthus.

Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include effective population size (Ne), genome size, and gametic multicellularity, with the latter being associated with both increased mutation rates and decreased effective population sizes. However, data sufficient to test for possible relationships between microbial multicellularity and mutation rate (µ) are lacking. Here, we report estimates of two key population-genetic parameters, Ne and µ, for Myxococcus xanthus, a bacterial model organism for the study of aggregative multicellular development, predation, and social swarming. To estimate µ, we conducted an ∼400-day mutation accumulation experiment with 46 lineages subjected to regular single colony bottlenecks prior to clonal regrowth. Upon conclusion, we sequenced one clonal-isolate genome per lineage. Given collective evolution for 85,323 generations across all lines, we calculate a per base-pair mutation rate of ∼5.5 × 10-10 per site per generation, one of the highest mutation rates among free-living eubacteria. Given our estimate of µ, we derived Ne at ∼107 from neutral diversity at four-fold degenerate sites across two dozen M. xanthus natural isolates. This estimate is below average for eubacteria and strengthens an already clear negative correlation between µ and Ne in prokaryotes. The higher and lower than average mutation rate and Ne for M. xanthus, respectively, amplify the question of whether any features of its multicellular life cycle-such as group-size reduction during fruiting-body development-or its highly structured spatial distribution have significantly influenced how these parameters have evolved.

Whole-Genome Sequence of Myxococcus Phage Mx9.

Here, we characterize the genome of Myxococcus phage Mx9, a lysogenic, short-tailed phage (genus Lederbergvirus) phage infecting the bacterial host Myxococcus xanthus, a model for bacterial evolution and development. The 53.5-kb genome has a GC content of 67.5% and contains 98 predicted protein-coding genes, including the previously characterized site-specific integrase gene (int).

Regulation of sedimentation rate shapes the evolution of multicellularity in a close unicellular relative of animals.

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma-close unicellular relatives of animals with brief transient multicellular life stages-we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least 2 distinct physical mechanisms. First, we find extensive (>300×) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular Sphaeroforma arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in 4 additional species from the Sphaeroforma genus, suggesting that variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodeling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.

Complete Genome Sequence of Myxococcus Phage Mx4.

Myxococcus xanthus is a bacterial model in microbial developmental biology and social evolution. Here, I present the 57.0-kb circular genomic sequence of the wild-type Myxococcus phage Mx4, with a GC content of 70.1%. Annotation predicted 97 protein-coding genes. Head-neck-tail protein classification assigns Mx4 to the tailed, Mu-like members of the family Myoviridae of group type 1 (cluster 8).

Bacterial predator-prey coevolution accelerates genome evolution and selects on virulence-associated prey defences.

Generalist bacterial predators are likely to strongly shape many important ecological and evolutionary features of microbial communities, for example by altering the character and pace of molecular evolution, but investigations of such effects are scarce. Here we report how predator-prey interactions alter the evolution of fitness, genomes and phenotypic diversity in coevolving bacterial communities composed of Myxococcus xanthus as predator and Escherichia coli as prey, relative to single-species controls. We show evidence of reciprocal adaptation and demonstrate accelerated genomic evolution specific to coevolving communities, including the rapid appearance of mutator genotypes. Strong parallel evolution unique to the predator-prey communities occurs in both parties, with predators driving adaptation at two prey traits associated with virulence in bacterial pathogens-mucoidy and the outer-membrane protease OmpT. Our results suggest that generalist predatory bacteria are important determinants of how complex microbial communities and their interaction networks evolve in natural habitats.

Social genes are selection hotspots in kin groups of a soil microbe.

The composition of cooperative systems, including animal societies, organismal bodies, and microbial groups, reflects their past and shapes their future evolution. However, genomic diversity within many multiunit systems remains uncharacterized, limiting our ability to understand and compare their evolutionary character. We have analyzed genomic and social-phenotype variation among 120 natural isolates of the cooperative bacterium Myxococcus xanthus derived from six multicellular fruiting bodies. Each fruiting body was composed of multiple lineages radiating from a unique recent ancestor. Genomic evolution was concentrated in selection hotspots associated with evolutionary change in social phenotypes. Synonymous mutations indicated that kin lineages within the same fruiting body often first diverged from a common ancestor more than 100 generations ago. Thus, selection appears to promote endemic diversification of kin lineages that remain together over long histories of local interaction, thereby potentiating social coevolution.

Bacteriophages of Myxococcus xanthus, a Social Bacterium.

Bacteriophages have been used as molecular tools in fundamental biology investigations for decades. Beyond this, however, they play a crucial role in the eco-evolutionary dynamics of bacterial communities through their demographic impact and the source of genetic information they represent. The increasing interest in describing ecological and evolutionary aspects of bacteria⁻phage interactions has led to major insights into their fundamental characteristics, including arms race dynamics and acquired bacterial immunity. Here, we review knowledge on the phages of the myxobacteria with a major focus on phages infecting Myxococcus xanthus, a bacterial model system widely used to study developmental biology and social evolution. In particular, we focus upon the isolation of myxophages from natural sources and describe the morphology and life cycle parameters, as well as the molecular genetics and genomics of the major groups of myxophages. Finally, we propose several interesting research directions which focus on the interplay between myxobacterial host sociality and bacteria⁻phage interactions.

Kin discrimination and outer membrane exchange in Myxococcus xanthus: A comparative analysis among natural isolates.

Genetically similar cells of the soil bacterium Myxococcus xanthus cooperate at multiple social behaviours, including motility and multicellular development. Another social interaction in this species is outer membrane exchange (OME), a behaviour of unknown primary benefit in which cells displaying closely related variants of the outer membrane protein TraA transiently fuse and exchange membrane contents. Functionally incompatible TraA variants do not mediate OME, which led to the proposal that TraA incompatibilities determine patterns of intercellular cooperation in nature, but how this might occur remains unclear. Using natural isolates from a centimetre-scale patch of soil, we analyse patterns of TraA diversity and ask whether relatedness at TraA is causally related to patterns of kin discrimination in the form of both colony-merger incompatibilities (CMIs) and interstrain antagonisms. A large proportion of TraA functional diversity documented among global isolates is predicted to be contained within this cm-scale population. We find evidence of balancing selection on the highly variable PA14-portion of TraA and extensive transfer of traA alleles across genomic backgrounds. CMIs are shown to be common among strains identical at TraA, suggesting that CMIs are not generally caused by TraA dissimilarity. Finally, it has been proposed that interstrain antagonisms might be caused by OME-mediated toxin transfer. However, we predict that most strain pairs previously shown to exhibit strong antagonisms are incapable of OME due to TraA dissimilarity. Overall, our results suggest that most documented patterns of kin discrimination in a natural population of M. xanthus are not causally related to the TraA sequences of interactants.

The biogeography of kin discrimination across microbial neighbourhoods.

The spatial distribution of potential interactants is critical to social evolution in all cooperative organisms. Yet the biogeography of microbial kin discrimination at the scales most relevant to social interactions is poorly understood. Here we resolve the microbiogeography of social identity and genetic relatedness in local populations of the model cooperative bacterium Myxococcus xanthus at small spatial scales, across which the potential for dispersal is high. Using two criteria of relatedness-colony-merger compatibility during cooperative motility and DNA-sequence similarity at highly polymorphic loci-we find that relatedness decreases greatly with spatial distance even across the smallest scale transition. Both social relatedness and genetic relatedness are maximal within individual fruiting bodies at the micrometre scale but are much lower already across adjacent fruiting bodies at the millimetre scale. Genetic relatedness was found to be yet lower among centimetre-scale samples, whereas social allotype relatedness decreased further only at the metre scale, at and beyond which the probability of social or genetic identity among randomly sampled isolates is effectively zero. Thus, in M. xanthus, high-relatedness patches form a rich mosaic of diverse social allotypes across fruiting body neighbourhoods at the millimetre scale and beyond. Individuals that migrate even short distances across adjacent groups will frequently encounter allotypic conspecifics and territorial kin discrimination may profoundly influence the spatial dynamics of local migration. Finally, we also found that the phylogenetic scope of intraspecific biogeographic analysis can affect the detection of spatial structure, as some patterns evident in clade-specific analysis were masked by simultaneous analysis of all strains.

Tempo and mode of genome evolution in a 50,000-generation experiment.

Adaptation by natural selection depends on the rates, effects and interactions of many mutations, making it difficult to determine what proportion of mutations in an evolving lineage are beneficial. Here we analysed 264 complete genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The populations that retained the ancestral mutation rate support a model in which most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate. We also compared these populations to mutation-accumulation lines evolved under a bottlenecking regime that minimizes selection. Nonsynonymous mutations, intergenic mutations, insertions and deletions are overrepresented in the long-term populations, further supporting the inference that most mutations that reached high frequency were favoured by selection. These results illuminate the shifting balance of forces that govern genome evolution in populations adapting to a new environment.

A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus.

The bacterium Myxococcus xanthus glides through soil in search of prey microbes, but when food sources run out, cells cooperatively construct and sporulate within multicellular fruiting bodies. M. xanthus strains isolated from a 16 × 16-cm-scale patch of soil were previously shown to have diversified into many distinct compatibility types that are distinguished by the failure of swarming colonies to merge upon encounter. We sequenced the genomes of 22 isolates from this population belonging to the two most frequently occurring multilocus sequence type (MLST) clades to trace patterns of incipient genomic divergence, specifically related to social divergence. Although homologous recombination occurs frequently within the two MLST clades, we find an almost complete absence of recombination events between them. As the two clades are very closely related and live in sympatry, either ecological or genetic barriers must reduce genetic exchange between them. We find that the rate of change in the accessory genome is greater than the rate of amino-acid substitution in the core genome. We identify a large genomic tract that consistently differs between isolates that do not freely merge and therefore is a candidate region for harbouring gene(s) responsible for self/non-self discrimination.

Adaptation to Parasites and Costs of Parasite Resistance in Mutator and Nonmutator Bacteria.

Parasitism creates selection for resistance mechanisms in host populations and is hypothesized to promote increased host evolvability. However, the influence of these traits on host evolution when parasites are no longer present is unclear. We used experimental evolution and whole-genome sequencing of Escherichia coli to determine the effects of past and present exposure to parasitic viruses (phages) on the spread of mutator alleles, resistance, and bacterial competitive fitness. We found that mutator alleles spread rapidly during adaptation to any of four different phage species, and this pattern was even more pronounced with multiple phages present simultaneously. However, hypermutability did not detectably accelerate adaptation in the absence of phages and recovery of fitness costs associated with resistance. Several lineages evolved phage resistance through elevated mucoidy, and during subsequent evolution in phage-free conditions they rapidly reverted to nonmucoid, phage-susceptible phenotypes. Genome sequencing revealed that this phenotypic reversion was achieved by additional genetic changes rather than by genotypic reversion of the initial resistance mutations. Insertion sequence (IS) elements played a key role in both the acquisition of resistance and adaptation in the absence of parasites; unlike single nucleotide polymorphisms, IS insertions were not more frequent in mutator lineages. Our results provide a genetic explanation for rapid reversion of mucoidy, a phenotype observed in other bacterial species including human pathogens. Moreover, this demonstrates that the types of genetic change underlying adaptation to fitness costs, and consequently the impact of evolvability mechanisms such as increased point-mutation rates, depend critically on the mechanism of resistance.

Rapid and widespread de novo evolution of kin discrimination.

Diverse forms of kin discrimination, broadly defined as alteration of social behavior as a function of genetic relatedness among interactants, are common among social organisms from microbes to humans. However, the evolutionary origins and causes of kin-discriminatory behavior remain largely obscure. One form of kin discrimination observed in microbes is the failure of genetically distinct colonies to merge freely upon encounter. Here, we first use natural isolates of the highly social bacterium Myxococcus xanthus to show that colony-merger incompatibilities can be strong barriers to social interaction, particularly by reducing chimerism in multicellular fruiting bodies that develop near colony-territory borders. We then use experimental laboratory populations to test hypotheses regarding the evolutionary origins of kin discrimination. We show that the generic process of adaptation, irrespective of selective environment, is sufficient to repeatedly generate kin-discriminatory behaviors between evolved populations and their common ancestor. Further, we find that kin discrimination pervasively evolves indirectly between allopatric replicate populations that adapt to the same ecological habitat and that this occurs generically in many distinct habitats. Patterns of interpopulation discrimination imply that kin discrimination phenotypes evolved via many diverse genetic mechanisms and mutation-accumulation patterns support this inference. Strong incompatibility phenotypes emerged abruptly in some populations but strengthened gradually in others. The indirect evolution of kin discrimination in an asexual microbe is analogous to the indirect evolution of reproductive incompatibility in sexual eukaryotes and linguistic incompatibility among human cultures, the commonality being indirect, noncoordinated divergence of complex systems evolving in isolation.

devI is an evolutionarily young negative regulator of Myxococcus xanthus development.

UNLABELLED: During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation. IMPORTANCE: Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.

Introgressive hybridization and latitudinal admixture clines in North Atlantic eels.

BACKGROUND: Hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. Eels, both from North America and Europe migrate through the Atlantic to mate in a vast, overlapping area in the Sargasso Sea. Due to the lack of direct observation, it is unknown how these species remain reproductively isolated. The detection of inter-species hybrids in Iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in North Atlantic eels. RESULTS: Here, we show that while mitochondrial lineages remain completely distinct on both sides of the Atlantic, limited hybridization is detectable with nuclear DNA markers. The nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to Bayesian assignment analyses. By simulating increasing proportions of both F1 hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the European eel. Finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism. CONCLUSIONS: Fluctuating degrees of introgressive hybridization between Atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the European eel.

Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load.

Mutations are the ultimate source of heritable variation for evolution. Understanding how mutation rates themselves evolve is thus essential for quantitatively understanding many evolutionary processes. According to theory, mutation rates should be minimized for well-adapted populations living in stable environments, whereas hypermutators may evolve if conditions change. However, the long-term fate of hypermutators is unknown. Using a phylogenomic approach, we found that an adapting Escherichia coli population that first evolved a mutT hypermutator phenotype was later invaded by two independent lineages with mutY mutations that reduced genome-wide mutation rates. Applying neutral theory to synonymous substitutions, we dated the emergence of these mutations and inferred that the mutT mutation increased the point-mutation rate by ∼150-fold, whereas the mutY mutations reduced the rate by ∼40-60%, with a corresponding decrease in the genetic load. Thus, the long-term fate of the hypermutators was governed by the selective advantage arising from a reduced mutation rate as the potential for further adaptation declined.

Mutation Rate Inferred From Synonymous Substitutions in a Long-Term Evolution Experiment With Escherichia coli.

The quantification of spontaneous mutation rates is crucial for a mechanistic understanding of the evolutionary process. In bacteria, traditional estimates using experimental or comparative genetic methods are prone to statistical uncertainty and consequently estimates vary by over one order of magnitude. With the advent of next-generation sequencing, more accurate estimates are now possible. We sequenced 19 Escherichia coli genomes from a 40,000-generation evolution experiment and directly inferred the point-mutation rate based on the accumulation of synonymous substitutions. The resulting estimate was 8.9 × 10(-11) per base-pair per generation, and there was a significant bias toward increased AT-content. We also compared our results with published genome sequence datasets for other bacterial evolution experiments. Given the power of our approach, our estimate represents the most accurate measure of bacterial base-substitution rates available to date.

Population structure of the parasitic nematode Anguillicola crassus, an invader of declining North Atlantic eel stocks.

Probably half of all animal species exhibit a parasitic lifestyle and numerous parasites have recently expanded their distribution and host ranges due to anthropogenic activities. Here, we report on the population genetic structure of the invasive nematode Anguillicola crassus, a parasite in freshwater eels, which recently spread from Asia to Europe and North America. Samples were collected from the newly colonized naive host species Anguilla anguilla (Europe) and Anguilla rostrata (North America), and from indigenous Anguilla japonica in Taiwan and Japan. Using seven microsatellite loci and one mitochondrial marker, we show that the parasite's population structure in Europe mirrors the zoogeographic Boreal-Lusitanian break along the English Channel. Both the north-to-south decline of nuclear allelic diversity and the loss of private alleles in the same direction are consistent with a significant isolation-by-distance pattern based on rho(ST) values. In combination with the specific topology of the distance tree among nematode populations, our data suggest that Europe was invaded only once from Taiwan, and that subsequently, genetic diversity was lost due to random drift. On the contrary, the North American sample shares distinct nuclear and mitochondrial signatures with Japanese specimens. We propose that the genetic structure in Europe was shaped by long-range anthropogenic eel host transfers in the north and a single dispersal event into the southwest. The genetically distinct Brittany sample at the edge of the Boreal-Lusitanian boundary is indicative of natural dispersal of fish hosts since recruitment occurs naturally there and invertebrate host dissemination is interrupted due to oceanic currents.

Parasite communities in eels of the Island of Reunion (Indian Ocean): a lesson in parasite introduction.

Eel populations from the small rivers on the Island of Reunion (French Overseas Department in the Indian Ocean) were investigated with respect to the occurrence and abundance of helminths during the autumn of 2005. The native species Anguilla marmorata (n = 80), Anguilla bicolor (n = 23), and Anguilla mossambica (n = 15) were studied. Six species of helminths were identified, four of them having a definitely nonnative status. Furthermore, unidentified intra-intestinal juvenile cestodes and extra-intestinal encapsulated anisakid nematode larvae were present in a few eels. We found that the invasive swim bladder nematode Anguillicoloides (Anguillicola) crassus had been introduced into the island. Six specimens were collected, four from A. marmorata, one from A. bicolor and one from A. mossambica. The maximum intensity of infection was two worms. The other helminths also showed a low abundance. These species were the monogenean gill worms Pseudodactylogyrus anguillae and Pseudodactylogyrus bini and the intestinal parasites Bothriocephalus claviceps (Cestodes), Paraquimperia africana (Nematodes), and the acanthocephalan Acanthocephalus reunionensis Warner, Sasal, and Taraschewski, 2007. The latter species, found as intra-intestinal immatures, is thought to utilize amphibians as required hosts; its status, introduced or native, could not be determined. P. africana was described from A. mossambica in South Africa and has not been recorded outside Africa. The other species are known from populations of European and American eels. However, A. crassus and the two Pseudodactylogyrus species originate from East Asia, where they are indigenous parasites of Anguilla japonica. Both an assignment test based on seven specific microsatellite loci and subsequent sequencing of mitochondrial haplotypes of a partial fragment of cytochrome c oxidase 1 strongly suggest that the A. crassus may originated around the Baltic Sea. According to the results presented here, populations of the indigenous eel species from Reunion can be considered to harbor extremely isolationist alien parasite communities. Our findings support the hypothesis that during the present time of global biological change, invasion by a nonnative species into a target island is more likely to reflect the political affiliation of the colonized environment and the pathways of trade and tourism than geographic proximity between donor and recipient areas or other natural circumstances.