Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti.

Toxin and antitoxin (TA) gene pairs are addiction systems that are present in many microbial genomes. Sinorhizobium meliloti is an N2-fixing bacterial symbiont of alfalfa and other leguminous plants, and its genome consists of three large replicons, a circular chromosome (3.7 Mb) and the megaplasmids pSymA (1.4 Mb) and pSymB (1.7 Mb). S. meliloti carries 211 predicted type II TA genes, each encoding a toxin or an antitoxin. We constructed defined deletion strains that collectively removed the entire pSymA and pSymB megaplasmids except for their oriV regions. Of approximately 100 TA genes on pSymA and pSymB, we identified four whose loss was associated with cell death or stasis unless copies of the genes were supplied in trans. Orthologs of three of these loci have been characterized in other organisms (relB/E [sma0471/sma0473], Fic [DOC] [sma2105], and VapC [PIN] [orf2230/sma2231]), and this report contains the first experimental proof that RES/Xre (smb21127/smb21128) loci can function as a TA system. Transcriptome sequencing (RNA-seq) analysis did not reveal transcriptional differences between the TA systems to account for why deletion of the four "active" systems resulted in cell toxicity. These data suggest that severe cell growth phenotypes result from the loss of a few TA systems and that loss of most TA systems may result in more subtle phenotypes. These four TA systems do not appear to play a direct role in the S. meliloti-alfalfa symbiosis, as strains lacking these TA systems had a symbiotic N2 fixation phenotype that was indistinguishable from the wild type.

Mate choice and the origin of menopause.

Human menopause is an unsolved evolutionary puzzle, and relationships among the factors that produced it remain understood poorly. Classic theory, involving a one-sex (female) model of human demography, suggests that genes imparting deleterious effects on post-reproductive survival will accumulate. Thus, a 'death barrier' should emerge beyond the maximum age for female reproduction. Under this scenario, few women would experience menopause (decreased fertility with continued survival) because few would survive much longer than they reproduced. However, no death barrier is observed in human populations. Subsequent theoretical research has shown that two-sex models, including male fertility at older ages, avoid the death barrier. Here we use a stochastic, two-sex computational model implemented by computer simulation to show how male mating preference for younger females could lead to the accumulation of mutations deleterious to female fertility and thus produce a menopausal period. Our model requires neither the initial assumption of a decline in older female fertility nor the effects of inclusive fitness through which older, non-reproducing women assist in the reproductive efforts of younger women. Our model helps to explain why such effects, observed in many societies, may be insufficient factors in elucidating the origin of menopause.

An ABC-type cobalt transport system is essential for growth of Sinorhizobium meliloti at trace metal concentrations.

We report expression and mutant phenotypes for a gene cluster in Sinorhizobium meliloti, designated cbtJKL, that has been shown to encode an ABC-type cobalt transport system. Transcription of cbtJKL initiated 384 nucleotides upstream from the cbtJ translation start codon, and the resulting 5' region contained a putative B(12)riboswitch. Expression of the cbtJKL genes appeared to be controlled by (cobalt-loaded) cobalamin interacting at the B(12)riboswitch, since (i) a putative B(12)riboswitch was located within this large upstream region, (ii) cbtJ transcription was repressed upon addition of cobalt or vitamin B(12), and (iii) deletions in the B(12)riboswitch resulted in constitutive cbtJKL transcription. Insertion mutants in cbtJKL failed to grow in LB medium, and growth was restored through the addition of cobalt but not other metals. This growth phenotype appeared to be due to the chelation of cobalt present in LB, and cbtJKL mutants also failed to grow in minimal medium containing the chelating agent EDTA unless the medium was supplemented with additional or excess cobalt. In uptake experiments, (57)Co(2+)accumulation was high in wild-type cells expressing the cbtJKL genes, whereas wild-type cells in which cbtJKL expression was repressed showed reduced accumulation. In cbtJKL mutant cells, (57)Co(2+)accumulation was reduced relative to that of the wild type, and presumably, this residual cobalt transport occurred via an alternate ion uptake system(s) that is not specific to cobalt. In symbiosis, the alternate system(s) appeared to mediate cobalt transport into bacteroid cells, as low cbtJKL expression was detected in bacteroids and cbtJKL mutants formed N(2)-fixing nodules on alfalfa.

Separating the effects of mutation and selection in producing DNA skew in bacterial chromosomes.

BACKGROUND: Many bacterial chromosomes display nucleotide asymmetry, or skew, between the leading and lagging strands of replication. Mutational differences between these strands result in an overall pattern of skew that is centered about the origin of replication. Such a pattern could also arise from selection coupled with a bias for genes coded on the leading strand. The relative contributions of selection and mutation in producing compositional skew are largely unknown. RESULTS: We describe a model to quantify the contribution of mutational differences between the leading and lagging strands in producing replication-induced skew. When the origin and terminus of replication are known, the model can be used to estimate the relative accumulation of G over C and of A over T on the leading strand due to replication effects in a chromosome with bidirectional replication arms. The model may also be implemented in a maximum likelihood framework to estimate the locations of origin and terminus. We find that our estimations for the origin and terminus agree very well with the location of genes that are thought to be associated with the replication origin. This indicates that our model provides an accurate, objective method of determining the replication arms and also provides support for the hypothesis that these genes represent an ancestral cluster of origin-associated genes. CONCLUSION: The model has several advantages over other methods of analyzing genome skew. First, it quantifies the role of mutation in generating skew so that its effect on composition, for example codon bias, can be assessed. Second, it provides an objective method for locating origin and terminus, one that is based on chromosome-wide accumulation of leading vs lagging strand nucleotide differences. Finally, the model has the potential to be utilized in a maximum likelihood framework in order to analyze the effect of chromosome rearrangements on nucleotide composition.

A reanalysis of protein polymorphism in Drosophila melanogaster, D. simulans, D. sechellia and D. mauritiana: effects of population size and selection.

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.

Rapid evolution of the sex-determining gene, transformer: structural diversity and rate heterogeneity among sibling species of Drosophila.

While developmentally regulated genes are generally conserved, transformer (tra), a key locus involved in the regulation of sexual differentiation, is highly diverged between species of Drosophila. With an aim to understand its divergence between sibling species, we investigated tra sequence variation among members of the Drosophila melanogaster species complex, D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In this species group, tra divergence is rapid yet clocklike and exhibits large differences in protein size. D. melanogaster contains a 13-amino acid tandem duplication, whereas D. sechellia possesses a 72-amino acid tandem duplication representing a 30% increase in total amino acid residues. We also found evidence of a nonrandom distribution of replacement substitutions and heterogeneity in substitution rates using clustering statistics and a codon substitution model. We show that tra's rapid divergence in this species complex is the result of generally lower selective constraints around regions that encode arginine-serine (RS) domains and a significantly higher rate of substitutions around the insertion site of D. sechellia's large duplication. The proximity of rapidly diverged regions to sites of nucleotide insertion suggests that higher local rates of mutation may provide a causal mechanism for TRA's rapid divergence in this subgroup. A comparison of tra orthologs across the genus Drosophila suggest that TRA maintains an assortment of RS domains for proper sex determining function while much of the protein evolves relatively unconstrained.