First observation of low-lying excited states in the very neutron-rich 95Kr^{95}Kr

Microsecond isomers of neutron-rich nuclei in the mass A=95 chain were investigated at the ILL reactor, Grenoble. These nuclei were produced by thermal-neutron induced fission of $^{241}Pu$. The detection is based on time correlation between fission fragments selected by the Lohengrin mass spectrometer and the $\gamma$-rays from the isomers. In this paper the decay scheme of an isomer of half life 1.4(2) micro second in $^{95}Kr$ is reported. It is the first time that excited states of this nucleus have been measured. These data show that this neutron rich N = 59 isotone is spherical at low excitation energy

Population gene introgression and high genome plasticity for the zoonotic pathogen Streptococcus agalactiae

The influence that bacterial adaptation (or niche partitioning) within species has on gene spillover and transmission among bacteria populations occupying different niches is not well understood. Streptococcus agalactiae is an important bacterial pathogen that has a taxonomically diverse host range making it an excellent model system to study these processes. Here we analyze a global set of 901 genome sequences from nine diverse host species to advance our understanding of these processes. Bayesian clustering analysis delineated twelve major populations that closely aligned with niches. Comparative genomics revealed extensive gene gain/loss among populations and a large pan-genome of 9,527 genes, which remained open and was strongly partitioned among niches. As a result, the biochemical characteristics of eleven populations were highly distinctive (significantly enriched). Positive selection was detected and biochemical characteristics of the dispensable genes under selection were enriched in ten populations. Despite the strong gene partitioning, phylogenomics detected gene spillover. In particular, tetracycline resistance (which likely evolved in the human-associated population) from humans to bovine, canines, seals, and fish, demonstrating how a gene selected in one host can ultimately be transmitted into another, and biased transmission from humans to bovines was confirmed with a Bayesian migration analysis. Our findings show high bacterial genome plasticity acting in balance with selection pressure from distinct functional requirements of niches that is associated with an extensive and highly partitioned dispensable genome, likely facilitating continued and expansive adaptation

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively

Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study

The epidemic history of HCV genotype 5a is poorly documented in France, where its prevalence is very low, except in a small central area, where it accounts for 14.2% of chronic hepatitis C cases. A Bayesian coalescent phylogenetic investigation based on the E1 envelope gene and a non-structural genomic segment (NS3/4) was carried out to trace the origin of this epidemic using a large sample of genotype 5a isolates collected throughout France. The dates of documented transmissions by blood transfusion were used to calibrate five nodes in the phylogeny. The results of the E1 gene analysis showed that the best-fitting population dynamic model was the expansion growth model under a relaxed molecular clock. The rate of nucleotide substitutions and time to the most recent common ancestors (tMRCA) of genotype 5a isolates were estimated. The divergence of all the French HCV genotype 5a strains included in this study was dated to 1939 [95% HPD: 1921–1956], and the tMRCA of isolates from central France was dated to 1954 [1942–1967], which is in agreement with epidemiological data. NS3/4 analysis provided similar estimates with strongly overlapping HPD values. Phylodynamic analyses give a plausible reconstruction of the evolutionary history of HCV genotype 5a in France, suggesting the concomitant roles of transfusion, iatrogenic route and intra-familial transmission in viral diffusion

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.United States. Public Health Service (Grant GM050895

Genomics of high molecular weight plasmids isolated from an on-farm biopurification system

The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.Acknowledgements: This work was supported by the European Commission’s 7th Framework Programme (project Metaexplore 222625), the National Scientific and Technical Research Council of Argentina (Consejo Nacional de Investigaciones Científicas y Técnicas—CONICET, Argentina) and Ministry of Science Technology and Productive Innovation (Ministerio de Ciencia Tecnolología e Innovación Productiva—MinCyT, Argentina), projects PICT2013-0113, PICT2012-518 and PICT 2012-1719). MCM, FJA were supported by fellowships from CONICET. MFDP, MP, ML, GTT and AL are researchers at CONICET. The bioinformatics support of the BMBF-funded project (grant 031A533) within the German Network for Bioinformatics Infrastructure (de.NBI) is gratefully acknowledged. Work in FdlC group was supported by grant “Plasmid Offensive” BFU2014-55534-C2-1-P from Ministerio de Economía y Competitividad (MINECO, Spain), and Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015/0019) from Instituto de Salud Carlos III (Spain)-co-financed by European Development Regional Fund. The authors are grateful to Paula Giménez and Silvana Tongiani for excellent technical assistance

Phylogenomics of Unusual Histone H2A Variants in Bdelloid Rotifers

Rotifers of Class Bdelloidea are remarkable in having evolved for millions of years, apparently without males and meiosis. In addition, they are unusually resistant to desiccation and ionizing radiation and are able to repair hundreds of radiation-induced DNA double-strand breaks per genome with little effect on viability or reproduction. Because specific histone H2A variants are involved in DSB repair and certain meiotic processes in other eukaryotes, we investigated the histone H2A genes and proteins of two bdelloid species. Genomic libraries were built and probed to identify histone H2A genes in Adineta vaga and Philodina roseola, species representing two different bdelloid families. The expressed H2A proteins were visualized on SDS-PAGE gels and identified by tandem mass spectrometry. We find that neither the core histone H2A, present in nearly all other eukaryotes, nor the H2AX variant, a ubiquitous component of the eukaryotic DSB repair machinery, are present in bdelloid rotifers. Instead, they are replaced by unusual histone H2A variants of higher mass. In contrast, a species of rotifer belonging to the facultatively sexual, desiccation- and radiation-intolerant sister class of bdelloid rotifers, the monogononts, contains a canonical core histone H2A and appears to lack the bdelloid H2A variant genes. Applying phylogenetic tools, we demonstrate that the bdelloid-specific H2A variants arose as distinct lineages from canonical H2A separate from those leading to the H2AX and H2AZ variants. The replacement of core H2A and H2AX in bdelloid rotifers by previously uncharacterized H2A variants with extended carboxy-terminal tails is further evidence for evolutionary diversity within this class of histone H2A genes and may represent adaptation to unusual features specific to bdelloid rotifers

A Toxin-Antitoxin Module in Bacillus subtilis Can Both Mitigate and Amplify Effects of Lethal Stress

Bacterial type-2 (protein-protein) toxin-antitoxin (TA) modules are two-gene operons that are thought to participate in the response to stress. Previous work with Escherichia coli has led to a debate in which some investigators conclude that the modules protect from stress, while others argue that they amplify lethal stress and lead to programmed cell death. To avoid ambiguity arising from the presence of multiple TA modules in E. coli, the effect of the sole type-2 toxin-antitoxin module of Bacillus subtilis was examined for several types of lethal stress.Genetic knockout of the toxin gene, ndoA (ydcE), conferred protection to lethal stressors that included kanamycin, moxifloxacin, hydrogen peroxide, and UV irradiation. However, at low doses of UV irradiation the ndoA deficiency increased lethality. Indeed, gradually increasing UV dose with the ndoA mutant revealed a crossover response--from the mutant being more sensitive than wild-type cells to being less sensitive. For high temperature and nutrient starvation, the toxin deficiency rendered cells hypersensitive. The ndoA deficiency also reduced sporulation frequency, indicating a role for toxin-antitoxin modules in this developmental process. In the case of lethal antimicrobial treatment, deletion of the toxin eliminated a surge in hydrogen peroxide accumulation observed in wild-type cells.A single toxin-antitoxin module can mediate two opposing effects of stress, one that lowers lethality and another that raises it. Protective effects are thought to arise from toxin-mediated inhibition of translation based on published work. The enhanced, stress-mediated killing probably involves toxin-dependent accumulation of reactive oxygen species, since a deficiency in the NdoA toxin suppressed peroxide accumulation following antimicrobial treatment. The type and perhaps the level of stress appear to be important for determining whether this toxin will have a protective or detrimental effect