Mutational meltdown of putative microbial altruists in Streptomyces coelicolor colonies.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a subpopulation of cells arises that hyperproduces metabolically costly antibiotics, resulting in a division of labor that increases colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is similar to altruistic worker castes in social insects or somatic cells in multicellular organisms. To understand these mutant cells' reproductive and genomic fate after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that in contrast to wild-type cells, putatively altruistic mutant cells continue to decline in fitness during transfer while they lose more fragments from their chromosome ends. In addition, the base-substitution rate in mutants increases roughly 10-fold, possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest the cells arising in the S. coelicolor division of labor are analogous to altruistic reproductively sterile castes of social insects.

First-Principles Study on III-Nitride Polymorphs: AlN/GaN/InN in the Pmn21 Phase.

The structural, mechanical, and electronic properties, as well as stability, elastic anisotropy and effective mass of AlN/GaN/InN in the Pmn21 phase were determined using density functional theory (DFT). The phonon dispersion spectra and elastic constants certify the dynamic and mechanical stability at ambient pressure, and the relative enthalpies were lower than those of most proposed III-nitride polymorphs. The mechanical properties reveal that Pmn21-AlN and Pmn21-GaN possess a high Vickers hardness of 16.3 GPa and 12.8 GPa. Pmn21-AlN, Pmn21-GaN and Pmn21-InN are all direct semiconductor materials within the HSE06 hybrid functional, and their calculated energy band gaps are 5.17 eV, 2.77 eV and 0.47 eV, respectively. The calculated direct energy band gaps and mechanical properties of AlN/GaN/InN in the Pmn21 phase reveal that these three polymorphs may possess great potential for industrial applications in the future.

Genome rearrangements and megaplasmid loss in the filamentous bacterium Kitasatospora viridifaciens are associated with protoplast formation and regeneration.

Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.

Antibiotic production in Streptomyces is organized by a division of labor through terminal genomic differentiation.

One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of Streptomyces coelicolor is coordinated by a division of labor. We show that S. coelicolor colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

The evolution of no-cost resistance at sub-MIC concentrations of streptomycin in Streptomyces coelicolor.

At the high concentrations used in medicine, antibiotics exert strong selection on bacterial populations for the evolution of resistance. However, these lethal concentrations may not be representative of the concentrations bacteria face in soil, a recognition that has led to questions of the role of antibiotics in soil environments as well as the dynamics of resistance evolution during sublethal challenge. Here we examine the evolution of resistance to sub-minimal inhibitory concentrations (sub-MIC) of streptomycin in the filamentous soil bacterium Streptomyces coelicolor. First, we show that spontaneous resistance to streptomycin causes an average fitness deficit of ~21% in the absence of drugs; however, these costs are eliminated at concentrations as low as 1/10 the MIC of susceptible strains. Using experimental evolution, we next show that resistance to >MIC levels of streptomycin readily evolves when bacteria are exposed to sub-MIC doses for 500 generations. Furthermore, the resistant clones that evolved at sub-MIC streptomycin concentrations carry no fitness cost. Whole-genome analyses reveal that evolved resistant clones fixed some of the same mutations as those isolated at high drug concentrations; however, all evolved clones carry additional mutations and some fixed mutations that either compensate for costly resistance or have no associated fitness costs. Our results broaden the conditions under which resistance can evolve in nature and suggest that rather than low-concentration antibiotics acting as signals, resistance evolves in response to antibiotics used as weapons.

Understanding Microbial Divisions of Labor.

Divisions of labor are ubiquitous in nature and can be found at nearly every level of biological organization, from the individuals of a shared society to the cells of a single multicellular organism. Many different types of microbes have also evolved a division of labor among its colony members. Here we review several examples of microbial divisions of labor, including cases from both multicellular and unicellular microbes. We first discuss evolutionary arguments, derived from kin selection, that allow divisions of labor to be maintained in the face of non-cooperative cheater cells. Next we examine the widespread natural variation within species in their expression of divisions of labor and compare this to the idea of optimal caste ratios in social insects. We highlight gaps in our understanding of microbial caste ratios and argue for a shift in emphasis from understanding the maintenance of divisions of labor, generally, to instead focusing on its specific ecological benefits for microbial genotypes and colonies. Thus, in addition to the canonical divisions of labor between, e.g., reproductive and vegetative tasks, we may also anticipate divisions of labor to evolve to reduce the costly production of secondary metabolites or secreted enzymes, ideas we consider in the context of streptomycetes. The study of microbial divisions of labor offers opportunities for new experimental and molecular insights across both well-studied and novel model systems.

Parallel Analysis of 124 Universal SNPs for Human Identification by Targeted Semiconductor Sequencing.

SNPs, abundant in human genome with lower mutation rate, are attractive to genetic application like forensic, anthropological and evolutionary studies. Universal SNPs showing little allelic frequency variation among populations while remaining highly informative for human identification were obtained from previous studies. However, genotyping tools target only dozens of markers simultaneously, limiting their applications. Here, 124 SNPs were simultaneous tested using Ampliseq technology with Ion Torrent PGM platform. Concordance study was performed with 2 reference samples of 9947A and 9948 between NGS and Sanger sequencing. Full concordance were obtained except genotype of rs576261 with 9947A. Parameter of FMAR (%) was introduced for NGS data analysis for the first time, evaluating allelic performance, sensitivity testing and mixture testing. FMAR values for accurate heterozygotes should be range from 50% to 60%, for homozygotes or Y-SNP should be above 90%. SNPs of rs7520386, rs4530059, rs214955, rs1523537, rs2342747, rs576261 and rs12997453 were recognized as poorly performing loci, either with allelic imbalance or with lower coverage. Sensitivity testing demonstrated that with DNA range from 10 ng-0.5 ng, all correct genotypes were obtained. For mixture testing, a clear linear correlation (R(2) = 0.9429) between the excepted FMAR and observed FMAR values of mixtures was observed.

Cloning and functional characterization of the pig (Sus scrofa) organic anion transporting polypeptide 1a2.

1. Organic anion transporting polypeptides (OATPs) are a family of transporter proteins that have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion of various drugs. Human OATP1A2 has been demonstrated to transport wide spectrum of endogenous and exogenous compounds. Study on OATP1A2 orthologues of other species, however, is still limited. 2. Here, we described the cloning and functional characterization of a member of the OATP/Oatp family member obtained from pig (Sus scrofa) liver. Sequence analysis suggested that it has a high homology with human OATP1A2 and bovine Oatp1a2. Prototypic substrates estrone-3-sulfate (E-3-S) and taurocholic acid were transported by the protein. The transport of these two substrates is pH-dependent, with lower pH showing higher uptake function. Kinetic study showed the transport of these two substrates have a Km of 42.5 ± 12.1 and 33.1 ± 8.7 µM, respectively. Pig Slco1a2 has the highest expression level in the liver, and to a less extend in the brain and small intestine. 3. In conclusion, an OATP member was cloned from pig liver. Sequence analysis and phylogenic study revealed it as an orthologue of human OATP1A2. Its kinetic characteristic for prototypic substrates and organ distribution are similar with that of OATP1A2.

Identification of multiple binding sites for substrate transport in bovine organic anion transporting polypeptide 1a2.

Organic anion transporting polypeptides (OATP) have been extensively recognized as key determinants of absorption, distribution, metabolism, and excretion of various drugs because of their broad substrate specificity, wide tissue distribution, and the involvement of drug-drug interaction. As the first cloned human OATP, OATP1A2 has been found to transport a wide spectrum of endogenous and exogenous compounds. Bovine Oapt1a2 shared high homology with the human transporter and is considered as its functional ortholog. In the present study, we expressed bovine Oatp1a2 in human embryonic kidney 293 cells and found that, unlike human OATP1A2, the transport of estrone-3-sulfate (E-3-S) exhibited biphasic saturation kinetics. The K(m) values are 0.25 ± 0.08 and 46.6 ± 18.5 µM, and V(max) values were 24.5 ±4.4 and 375 ± 142 pmol/mg protein/min for high- and low-affinity sites, respectively, suggesting the presence of multiple binding sites. Further study on other Oatp1a2 substrates showed that the high affinity component for E-3-S is responsible for the interaction with taurocholate, bromsulphthalein, and rifampicin and is sensitive to proton concentration change, whereas the low affinity binding site is only involved in the binding of the antitumor drug methotrexate and had no response to change of pH.

Genetic polymorphisms in 12 autosomal STRs in a Shanghai Han population from China.

In this study, a total of 484 unrelated healthy individuals and 359 two-generation families in the Han population in Shanghai, China were successfully analyzed with the Investigator HDplex Kit (Qiagen, Hilden, Germany). Hardy-Weinberg equilibrium tests demonstrated no significant deviation from expected values (p > 0.05) for the 12 autosomal STRs included in this kit. A total of 173 alleles were detected in 12 autosomal STR loci; the largest number of alleles (32) was detected for the SE33 locus. The combined power of discrimination was 0.99999999992. The combined mean exclusion chance in duo cases was 0.99982, whereas the combined mean exclusion chance in trio cases was 0.9999986. The average mutation rate across all loci was 0.0023 (95% confidence intervals: 0.0013-0.0039). These results suggest that the 12 autosomal STR loci can provide highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China.

[Forensic application of investigator HDplex kit in Han nationality of Eastern China].

OBJECTIVE: To investigate the genetic data of 12 autosomal STR loci included in Investigator HDplex kit and to evaluate its forensic application in Han nationality of Eastern China. METHODS: A total of 484 unrelated healthy individuals in Han nationality of Eastern China were investigated with Investigator HDplex kit. Allele frequencies, population genetics parameters and linkage disequilibrium information of the 12 autosomal STR loci were statistically analyzed. RESULTS: No deviations from Hardy-Weinberg equilibrium were detected and all loci were independent form each other within the studied 484 unrelated healthy individuals. DP values of the 12 autosomal STR loci were all above 0.8, and CDP was 0.999 999 999 92. The cumulative probability of paternity exclusion in duo and in trio were 0.999 82 and 0.999 998 6, respectively. CONCLUSION: Investigator HDplex kit with 12 highly polymorphic STR loci in Han nationality of Eastern China could be used effectively for forensic DNA genotyping.