Bradyrhizobium commune sp. nov., isolated from nodules of a wide range of native legumes across the Australian continent.

Bradyrhizobia are particularly abundant in Australia, where they nodulate native legumes growing in the acidic and seasonally dry soils that predominate in these environments. They are essential to Australian ecosystems by helping legumes to compensate for nutrient deficiencies and the low fertility of Australian soils. During a survey of Australian native rhizobial communities in 1994-1995, several Bradyrhizobium genospecies were identified, among which genospecies B appeared to be present in various edaphic and climatic conditions and associate with a large range of leguminous hosts across the whole continent. We took advantage of the recent sequencing of the genome of strain BDV5040T, representative of Bradyrhizobium genospecies B, to re-evaluate the taxonomic status of this lineage. We further characterized strain BDV5040T based on morpho-physiological traits and determined its phylogenetic relationships with the type strains of all currently described Bradyrhizobium species using both small subunit (SSU) rRNA gene and complete genome sequences. The digital DNA-DNA hybridization relatedness with any type strain was less than 35 % and both SSU rRNA gene and genome phylogenies confirmed the initial observation that this strain does not belong to any formerly described species within the genus Bradyrhizobium. All data thus support the description of the novel species Bradyrhizobium commune sp. nov. for which the type strain is BDV5040T (=CFBP 9110T=LMG 32898T), isolated from a nodule of Bossiaea ensata in Ben Boyd National Park in New South Wales, Australia.

Complete Genome Sequence of Bradyrhizobium sp. Strain BDV5419, Representative of Australian Genospecies L.

We report the complete genome sequence of Bradyrhizobium sp. strain BDV5419, representative of Bradyrhizobium genospecies L, which symbiotically associates with the Australian native legume Hardenbergia violaceae and is expected to represent a novel Bradyrhizobium species. The complete genome sequence provides a genetic reference for this Australian genospecies.

Complete Genome Sequence of Bradyrhizobium sp. Strain BDV5040, Representative of Widespread Genospecies B in Australia.

We report the complete genome sequence of Bradyrhizobium sp. strain BDV5040, representative of Bradyrhizobium genospecies B, which symbiotically associates with legume hosts belonging to all three Fabaceae subfamilies across the Australian continent. The complete genome sequence provides a genetic reference for this Australian genospecies.

Metapopulation ecology links antibiotic resistance, consumption, and patient transfers in a network of hospital wards.

Antimicrobial resistance (AMR) is a global threat. A better understanding of how antibiotic use and between-ward patient transfers (or connectivity) impact population-level AMR in hospital networks can help optimize antibiotic stewardship and infection control strategies. Here, we used a metapopulation framework to explain variations in the incidence of infections caused by seven major bacterial species and their drug-resistant variants in a network of 357 hospital wards. We found that ward-level antibiotic consumption volume had a stronger influence on the incidence of the more resistant pathogens, while connectivity had the most influence on hospital-endemic species and carbapenem-resistant pathogens. Piperacillin-tazobactam consumption was the strongest predictor of the cumulative incidence of infections resistant to empirical sepsis therapy. Our data provide evidence that both antibiotic use and connectivity measurably influence hospital AMR. Finally, we provide a ranking of key antibiotics by their estimated population-level impact on AMR that might help inform antimicrobial stewardship strategies.

Ail and PagC-related proteins in the entomopathogenic bacteria of Photorhabdus genus.

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

Modeling of 2-D DNA display.

2-D display is a fast and economical way of visualizing polymorphism and comparing genomes, which is based on the separation of DNA fragments in two steps, first according to their size and then to their sequence composition. In this article, we present an exhaustive study of the numerical issues associated with a model aimed at predicting the final absolute locations of DNA fragments in 2-D display experiments. We show that simple expressions for the mobility of DNA fragments in both dimensions allow one to reproduce experimental final absolute locations better than experimental uncertainties. However, our simulations also point out that the results of 2-D display experiments are not sufficient to determine the best set of parameters for the modeling of fragments separation in the second dimension and that additional detailed measurements of the mobility of a few sequences are necessary to achieve this goal. We hope that this work will help in establishing simulations as a powerful tool to optimize experimental conditions without having to perform a large number of preliminary experiments and to estimate whether 2-D DNA display is suited to identify a mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.

Full-length genome characterization of a novel simian immunodeficiency virus lineage (SIVolc) from olive Colobus (Procolobus verus) and new SIVwrcPbb strains from Western Red Colobus (Piliocolobus badius badius) from the Tai Forest in Ivory Coast.

Simian immunodeficiency viruses (SIVs) are found in an extensive number of African primates and humans continue to be exposed to these viruses by hunting and handling of primate bushmeat. Full-length genome sequences were obtained from SIVs derived from two Colobinae species inhabiting the Taï forest, Ivory Coast, each belonging to a different genus: SIVwrc from western red colobus (Piliocolobus badius badius) (SIVwrcPbb-98CI04 and SIVwrcPbb-97CI14) and SIVolc (SIVolc-97CI12) from olive colobus (Procolobus verus). Phylogenetic analysis showed that western red colobus are the natural hosts of SIVwrc, and SIVolc is also a distinct species-specific lineage, although distantly related to the SIVwrc lineage across the entire length of its genome. Overall, both SIVwrc and SIVolc, are also distantly related to the SIVlho/sun lineage across the whole genome. Similar to the group of SIVs (SIVsyk, SIVdeb, SIVden, SIVgsn, SIVmus, and SIVmon) infecting members of the Cercopithecus genus, SIVs derived from western red and olive colobus, L'Hoest and suntailed monkeys, and SIVmnd-1 from mandrills form a second group of viruses that cluster consistently together in phylogenetic trees. Interestingly, the divergent SIVcol lineage, from mantled guerezas (Colobus guereza) in Cameroon, is also closely related to SIVwrc, SIVolc, and the SIVlho/sun lineage in the 5' part of Pol. Overall, these results suggest an ancestral link between these different lentiviruses and highlight once more the complexity of the natural history and evolution of primate lentiviruses.

Full molecular characterization of a simian immunodeficiency virus, SIVwrcpbt from Temminck's red colobus (Piliocolobus badius temminckii) from Abuko Nature Reserve, The Gambia.

Simian immunodeficiency viruses (SIVs) are found in an extensive number of African primates, and humans continue to be exposed to these viruses by hunting and handling of primate bushmeat. The purpose of our study was to examine to what extent Piliocolobus badius subspecies are infected with SIV in order to better characterize SIVwrc in general and to gain further insight into the impact of geographic barriers and subspeciation on the evolution of SIVwrc. We analysed sixteen faecal samples and two tissue samples of the P. b. temminckii subspecies collected in the Abuko Nature Reserve (The Gambia, West Africa). SIV infection could only be identified in one tissue sample, and phylogenetic tree analyses of partial pol and env sequences showed that the new SIVwrcPbt virus is closely related to SIVwrcPbb strains from P. b. badius in the Taï forest (Côte d'Ivoire), thus suggesting that geographically separated subspecies are infected with a closely related virus. Molecular characterization and phylogenetic analysis of the full-length genome sequence confirmed that SIVwrcPbt is a species-specific SIV lineage, although it is distantly related to the SIVlho and SIVsun lineages across its entire genome. Characterization of additional SIVwrc viruses is needed to understand the ancestral phylogenetic relation to SIVs from l'Hoest and sun-tailed monkeys and whether recombination occurred between ancestors of the SIVwrc and SIVlho/sun lineages.

Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage of Trypanosoma cruzi.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.

Quantitative predictions for DNA two-dimensional display according to size and nucleotide sequence composition.

2-D DNA display is a simple separation method that provides a fast and economical way of visualizing polymorphism and comparing genomes. The DNA fragments are separated first according to their size by standard gel electrophoresis and then according to their sequence composition using denaturing gradient gel electrophoresis. First developed by Fischer and Lerman (Cell 1979, 16, 191-200), this method has recently been used to distinguish strains within a bacterial species. The genomic restriction fragments are displayed as spots on a 2-D surface. Although most of the relevant physical mechanisms are understood, this technique is mostly empirical and remains essentially qualitative. In view of optimizing this procedure, we combine our understanding of the different physical mechanisms at play to develop a complete numerical model to predict the relative coordinates of the spots as a function of the corresponding DNA sequence and of the experimental conditions. We experimentally validate our model by predicting the outcome of a 2-D display of the lambda phage genome. It thus becomes possible to optimize in silico the experimental parameters, to predict whether specific mutations as well as yet undescribed genetic polymorphisms can be resolved, and to assist in interpreting the experimental data.

Prevalence and genetic diversity of simian immunodeficiency virus infection in wild-living red colobus monkeys (Piliocolobus badius badius) from the Taï forest, Côte d'Ivoire SIVwrc in wild-living western red colobus monkeys.

Numerous African primates are infected with simian immunodeficiency viruses (SIVs). It is now well established that the clade of SIVs infecting west-central African chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla gorilla) represent the progenitors of human immunodeficiency virus type 1 (HIV-1), whereas HIV-2 results from different cross-species transmissions of SIVsmm from sooty mangabeys (Cercocebus atys atys). We present here the first molecular epidemiological survey of simian immunodeficiency virus (SIVwrc) in wild-living western red colobus monkeys (Piliocolobus badius badius) which are frequently hunted by the human population and represent a favourite prey of western chimpanzees (Pan troglodytes verus). We collected faecal samples (n=88) and we assessed individual discrimination by microsatellite analyses and visual observation. We tested the inferred 53 adult individuals belonging to two neighbouring habituated groups for presence of SIVwrc infection by viral RNA (vRNA) detection. We amplified viral polymerase (pol) (650 bp) and/or envelope (env) (570 bp) sequences in 14 individuals, resulting in a minimal prevalence of 26% among the individuals sampled, possibly reaching 50% when considering the relatively low sensitivity of viral RNA detection in faecal samples. With a few exceptions, phylogenetic analysis of pol and env sequences revealed a low degree of intragroup genetic diversity and a general viral clustering related to the social group of origin. However, we found a higher intergroup diversity. Behavioural and demographic data collected previously from these communities indicate that red colobus monkeys live in promiscuous multi-male societies, where females leave their natal group at the sub-adult stage of their lives and where extra-group copulations or male immigration have been rarely observed. The phylogenetic data we obtained seem to reflect these behavioural characteristics. Overall, our results indicate that wild-living red colobus represent a substantial reservoir of SIVwrc. Moreover, because of their frequent association with other monkey species, the predation pressure exerted by chimpanzees (Pan troglodytes verus) and by poachers around and inside the park, simian to simian and simian to human SIVwrc cross-species transmission cannot be excluded.

Identification and molecular characterization of new STLV-1 and STLV-3 strains in wild-caught nonhuman primates in Cameroon.

Humans and simian species are infected by deltaretroviruses (HTLV and STLV respectively), which are collectively called primate T-cell lymphotropic viruses (PTLVs). In humans, four types of HTLV have been described (HTLV-1 to -4) with three of them having closely related simian virus analogues named STLV-1, 2 and 3. In this study, our aim was to search for a simian HTLV-4-related virus and to document and characterize further the diversity of STLV infections in wild primate populations. We screened 1297 whole blood samples from 13 different primate species from southern Cameroon. Overall, 93 samples gave HTLV-1, HTLV-2 or dual HTLV-1/-2 INNOLIA profiles, 12 were HTLV positive but untypeable and 14 were indeterminate. Subsequently, we performed generic and specific (STLV-1 to -3) tax-rex PCRs to discriminate the different PTLV types, completed with phylogenetic analysis of 450-bp LTR sequences for STLV-1 and 900 bp pX-LTR sequences for STLV-3. We show for the first time that Lophocebus albigena and Cercopithecus cephus carry both STLV-1 and a divergent STLV-3. We also identified a new STLV-1 lineage in one C. cephus. Finally, we identify relative divergence levels in the tax/rex phylogeny suggesting that additional types of PTLV should be defined, particularly for the highly divergent STLV-1(MarB43) strain that we provisionally name STLV-5.

Genetic diversity and phylogeographic clustering of SIVcpzPtt in wild chimpanzees in Cameroon.

It is now well established that the clade of simian immunodeficiency viruses (SIVs) infecting west central African chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla gorilla) comprises the progenitors of human immunodeficiency virus type 1 (HIV-1). In this study, we have greatly expanded our previous molecular epidemiological survey of SIVcpz in wild chimpanzees in Cameroon. The new results confirm a wide but uneven distribution of SIVcpzPtt in P. t. troglodytes throughout southern Cameroon and indicate the absence of SIVcpz infection in Pan troglodytes vellerosus. Analyzing 725 fecal samples from 15 field sites, we obtained partial nucleotide sequences from 16 new SIVcpzPtt strains and determined full-length sequences for two of these. Phylogenetic analyses of these new viruses confirmed the previously reported phylogeographic clustering of SIVcpzPtt lineages, with viruses related to the ancestors of HIV-1 groups M and N circulating exclusively in southeastern and south central P. t. troglodytes communities, respectively. Importantly, the SIVcpzPtt strains from the southeastern corner of Cameroon represent a relatively isolated clade indicating a defined geographic origin of the chimpanzee precursor of HIV-1 group M. Since contacts between humans and apes continue, the possibility of ongoing transmissions of SIV from chimpanzees (or gorillas) to humans has to be considered. In this context, our finding of distinct SIVcpzPtt envelope V3 sequence clades suggests that these peptides may be useful for the serological differentiation of SIVcpzPtt and HIV-1 infections, and thus the diagnosis of new cross-species transmissions if they occurred.

Molecular diversity of legume root-nodule bacteria in Kakadu National Park, Northern Territory, Australia.

BACKGROUND: Symbiotic relationships between leguminous plants (family Fabaceae) and nodule-forming bacteria in Australia native ecosystems remain poorly characterized despite their importance. Most studies have focused on temperate parts of the country, where the use of molecular approaches have already revealed the presence of Bradyrhizobium, Ensifer (formerly Sinorhizobium), Mesorhizobium and Rhizobium genera of legume root-nodule bacteria. We here provide the first molecular characterization of nodulating bacteria from tropical Australia. METHODOLOGY/PRINCIPAL FINDINGS: 45 nodule-forming bacterial strains, isolated from eight native legume hosts at eight locations in Kakadu National Park, Northern Territory, Australia, were examined for their genetic diversity and phylogenetic position. Using SSU rDNA PCR-RFLPs and phylogenetic analyses, our survey identified nine genospecies, two of which, Bradyrhizobium genospp. B and P, had been previously identified in south-eastern Australia and one, Mesorhizobium genospecies AA, in southern France. Three of the five newly characterized Bradyrhizobium genospecies were more closely related to B. japonicum USDA110, whereas the other two belonged to the B. elkanii group. All five were each more closely related to strains sampled in various tropical areas outside Australia than to strains known to occur in Australia. We also characterized an entirely novel nodule-forming lineage, phylogenetically distant from any previously described rhizobial and non-rhizobial legume-nodulating lineage within the Rhizobiales. CONCLUSIONS/SIGNIFICANCE: Overall, the present results support the hypothesis of tropical areas being centres of biodiversity and diversification for legume root-nodule bacteria and confirm the widespread occurrence of Bradyrhizobium genosp. B in continental Australia.

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles.

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.

Bradyrhizobia isolated from root nodules of Parasponia (Ulmaceae) do not constitute a separate coherent lineage.

Rhizobial bacteria almost exclusively nodulate members of the families Fabaceae, Mimosaceae and Caesalpiniaceae, but are found on a single non-legume taxon, Parasponia (Ulmaceae). Based on their host-range, their nitrogen-fixing ability and strain competition experiments, bacterial strains isolated from Parasponia were thought to constitute a separate lineage that would account for their exceptional host affinity. This hypothesis was investigated by focusing on four isolates that are representative of the morphological and cultural types of Parasponia-nodulating bradyrhizobia. Their evolutionary relationships with other rhizobia were analysed using 16S rRNA gene sequences and their nodulation properties were explored using the nodA gene as a proxy for host-range specificity. Phylogenetic analyses of the 16S rRNA and nodA gene sequences revealed that bacterial isolates from Parasponia species are embedded among other bradyrhizobia. They did not cluster together in topologies based on the 16S rRNA or nodA gene sequences, but were scattered among other bradyrhizobia belonging to either the Bradyrhizobium japonicum or the Bradyrhizobium elkanii lineages. These data suggest that the ability of some bradyrhizobia to nodulate species of the genus Parasponia does not represent a historical relationship that predates the relationship between rhizobia and legumes, but is probably a more recent host switch for some rhizobia.

MgtC as a horizontally-acquired virulence factor of intracellular bacterial pathogens: evidence from molecular phylogeny and comparative genomics.

MgtC is a virulence factor required for intramacrophage survival and growth in low Mg2+ medium in two pathogens that are not phylogenetically related, Salmonella typhimurium and Mycobacterium tuberculosis. In S. typhimurium, mgtC is carried by the SPI-3 pathogenicity island and hybridization studies have suggested that the distribution of mgtC among enterobacteria is limited. In the present study, we searched for the presence of mgtC-like sequences in eubacterial genomes. Analyses of MgtC-like proteins phylogeny and mgtC-like chromosomal context support the hypothesis that mgtC has been acquired by horizontal gene transfer repeatedly throughout bacterial evolution. In addition, the phylogenetic analysis revealed the existence of a subgroup of proteins, that includes the S. typhimurium and M. tuberculosis MgtC proteins, as well as MgtC-related proteins from other pathogens that are able to survive in macrophages, B. melitensis and Y. pestis. We propose that MgtC has a similar function in all these distantly related pathogens, most likely providing the ability to grow in a low Mg2+ environment.

Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium.

Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.

Absence of translationally selected synonymous codon usage bias in Helicobacter pylori.

Synonymous codon usage in the complete genome of Helicobacter pylori was investigated. The moderate A+T-richness of the genome (G+C=39 mol%) is reflected in the overall synonymous codon usage but the frequencies of some codons cannot be explained by simple mutational biases. A low level of heterogeneity among genes was observed, but this does not appear to be due to varying mutational bias or translational selection. Some of the heterogeneity was due to amino acid composition variation among the encoded proteins, and some may be attributable to recent acquisition of genes from other species. Since Hel. pylori codon usage is not dominated by biased mutation patterns, the absence of evidence for translationally mediated selection among synonymous codons is striking. This has implications with regard to the life history of this species, and in particular suggests that Hel. pylori strains are not subject to periods of competitive exponential growth. Despite the lack of selected codon usage, base composition immediately after the translation initiation site is skewed, consistent with selection against secondary structure formation in this region.