Endemic Mimosa species from Mexico prefer alphaproteobacterial rhizobial symbionts.

The legume genus Mimosa has > 500 species, with two major centres of diversity, Brazil (c. 350 spp.) and Mexico (c. 100 spp.). In Brazil most species are nodulated by Burkholderia. Here we asked whether this is also true of native and endemic Mexican species. We have tested this apparent affinity for betaproteobacteria by examining the symbionts of native and endemic species of Mimosa in Mexico, especially from the central highlands where Mimosa spp. have diversified. Nodules were tested for betaproteobacteria using in situ immunolocalization. Rhizobia isolated from the nodules were genetically characterized and tested for their ability to nodulate Mimosa spp. Immunological analysis of 25 host taxa suggested that most (including all the highland endemics) were not nodulated by betaproteobacteria. Phylogenetic analyses of 16S rRNA, recA, nodA, nodC and nifH genes from 87 strains isolated from 20 taxa confirmed that the endemic Mexican Mimosa species favoured alphaproteobacteria in the genera Rhizobium and Ensifer: this was confirmed by nodulation tests. Host phylogeny, geographic isolation and coevolution with symbionts derived from very different soils have potentially contributed to the striking difference in the choice of symbiotic partners by Mexican and Brazilian Mimosa species.

High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.

Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine.

Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.

Burkholderia diazotrophica sp. nov., isolated from root nodules of Mimosa spp.

Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 °C (optimum, 30-37 °C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1 % (w/v) NaCl [optimum, 0 % (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2 % sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2 % to Burkholderia terrae KMY02(T), 97.1 % to Burkholderia phymatum STM815(T) and 97.1 % to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C(16 : 1) iso I and/or C(14 : 0) 3-OH), summed feature 3 (comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c), C(16 : 0) , C(16 : 0) 3-OH, C(17 : 0) cyclo, C(18 : 1)ω7c and C(19 : 0) cyclo ω8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54 %. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) ( = LMG 26031(T) = BCRC 80259(T) = KCTC 23308(T)).

Burkholderia symbiotica sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil.

Four strains, designated JPY-345(T), JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera, that are native to North East Brazil, and their taxonomic positions were investigated by using a polyphasic approach. All four strains grew at 15-43 °C (optimum 35 °C), at pH 4-7 (optimum pH 5) and with 0-2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene sequence analysis, strain JPY-345(T) showed 97.3 % sequence similarity to the closest related species Burkholderia soli GP25-8(T), 97.3 % sequence similarity to Burkholderia caryophylli ATCC25418(T) and 97.1 % sequence similarity to Burkholderia kururiensis KP23(T). The predominant fatty acids of the strains were C(18 : 1)ω7c (36.1 %), C(16 : 0) (19.8 %) and summed feature 3, comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c (11.5 %). The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 64.2-65.7 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. DNA-DNA hybridizations between the novel strain and recognized species of the genus Burkholderia yielded relatedness values of <51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is JPY-345(T) (= LMG 26032(T) = BCRC 80258(T) = KCTC 23309(T)).

Prevalence and mobility of integrative and conjugative elements within a Streptomyces natural population.

Horizontal Gene Transfer (HGT) is a powerful force generating genomic diversity in bacterial populations. HGT in Streptomyces is in large part driven by conjugation thanks to plasmids, Integrative and Conjugative elements (ICEs) and Actinomycete ICEs (AICEs). To investigate the impact of ICE and AICE conjugation on Streptomyces genome evolution, we used in silico and experimental approaches on a set of 11 very closely related strains isolated from a millimeter scale rhizosphere population. Through bioinformatic searches of canonical conjugation proteins, we showed that AICEs are the most frequent integrative conjugative elements, with the central chromosome region being a hotspot for integrative element insertion. Strains exhibited great variation in AICE composition consistent with frequent HGT and/or gene loss. We found that single insertion sites can be home to different elements in different strains (accretion) and conversely, elements belonging to the same family can be found at different insertion sites. A wide variety of cargo genes was present in the AICEs with the potential to mediate strain-specific adaptation (e.g., DNA metabolism and resistance genes to antibiotic and phages). However, a large proportion of AICE cargo genes showed hallmarks of pseudogenization, consistent with deleterious effects of cargo genes on fitness. Pock assays enabled the direct visualization of conjugal AICE transfer and demonstrated the transfer of AICEs between some, but not all, of the isolates. Multiple AICEs were shown to be able to transfer during a single mating event. Although we did not obtain experimental evidence for transfer of the sole chromosomal ICE in this population, genotoxic stress mediated its excision from the chromosome, suggesting its functionality. Our results indicate that AICE-mediated HGT in Streptomyces populations is highly dynamic, with likely impact on strain fitness and the ability to adapt to environmental change.

Legume-nodulating betaproteobacteria: diversity, host range, and future prospects.

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.

Mesorhizobium camelthorni sp. nov., isolated from Alhagi sparsifolia.

Nine strains isolated from symbiotic root nodules on Alhagi sparsifolia were previously designated as representing genospecies I. Phylogenetic analyses indicated that genospecies I was related closely to Mesorhizobium alhagi (genospecies II), and clearly formed a new lineage within the genus Mesorhizobium. In this study, we differentiated genospecies I from recognized species of the genus Mesorhizobium based on phylogenetic analyses of additional core genes (recA, glnA), levels of DNA-DNA relatedness (<43.3 %), fatty acid profile (58 % C18:1ω7c, 19 % 11-methyl C18:1ω7c), and biochemical and physiological characteristics. The nine strains are therefore considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium camelthorni sp. nov. is proposed. The type strain is CCNWXJ 40-4(T) (=HAMBI 3020(T) =ACCC 14549(T)).

Elicitation of Antimicrobial Active Compounds by Streptomyces-Fungus Co-Cultures.

The bacteria of the genus Streptomyces and Basidiomycete fungi harbor many biosynthetic gene clusters (BGCs) that are at the origin of many bioactive molecules with medical or industrial interests. Nevertheless, most BGCs do not express in standard lab growth conditions, preventing the full metabolic potential of these organisms from being exploited. Because it generates biotic cues encountered during natural growth conditions, co-culture is a means to elicit such cryptic compounds. In this study, we explored 72 different Streptomyces-fungus interaction zones (SFIZs) generated during the co-culture of eight Streptomyces and nine fungi. Two SFIZs were selected because they showed an elicitation of anti-bacterial activity compared to mono-cultures. The study of these SFIZs showed that co-culture had a strong impact on the metabolic expression of each partner and enabled the expression of specific compounds. These results show that mimicking the biotic interactions present in this ecological niche is a promising avenue of research to explore the metabolic capacities of Streptomyces and fungi.

One-year follow-up of microbial diversity in bioaerosols emitted in a waste sorting plant in France.

Bioaerosols emitted in waste sorting plants (WSP) can induce some adverse health effects on the workers such as rhinitis, asthma and hypersensitivity pneumonitis. The composition of these bioaerosols is scarcely known and most of the time assessed using culture-dependent methods. Due to the well-known limitations of cultural methods, these biodiversity measurements underestimate the actual microbial taxon richness. The aim of the study was to assess the airborne microbial biodiversity by using a sequencing method in a French waste sorting plant (WSP) for one year and to investigate the main factors of variability of this biodiversity. Static sampling was performed in five areas in the plant and compared to an indoor reference (IR), using closed-face cassettes (10 L.min-1) with polycarbonate membranes, every month for one year. Environmental data was measured (temperature, relative humidity). After DNA extraction, microbial biodiversity was assessed by means of sequencing. Bacterial genera Staphylococcus, Streptococcus, Prevotella, Lactococcus, Lactobacillus, Pseudomonas and fungal genera Wallemia, Cladosporium, Debaryomyces, Penicillium, Alternaria were the most predominant airborne microorganisms. Microbial biodiversity was different in the plant compared to the IR and seemed to be influenced by the season.

Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil.

*An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus. *Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815. *Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the delta(15)N ((15)N natural abundance) technique, contributions by biological N(2) fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp. *It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N(2) fixation by beta-rhizobial symbioses in the field.

Burkholderia species are ancient symbionts of legumes.

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia. Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia. The symbiosis-related genes formed deep Burkholderia-specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.

Telomeric and sub-telomeric regions undergo rapid turnover within a Streptomyces population.

Genome dynamics was investigated within natural populations of the soil bacterium Streptomyces. The exploration of a set of closely related strains isolated from micro-habitats of a forest soil exhibited a strong diversity of the terminal structures of the linear chromosome, i.e. terminal inverted repeats (TIRs). Large insertions, deletions and translocations could be observed along with evidence of transfer events between strains. In addition, the telomere and its cognate terminal protein complexes required for terminal replication and chromosome maintenance, were shown to be variable within the population probably reflecting telomere exchanges between the chromosome and other linear replicons (i.e., plasmids). Considering the close genetic relatedness of the strains, these data suggest that the terminal regions are prone to a high turnover due to a high recombination associated with extensive horizontal gene transfer.

Genome Sequences of Five Streptomyces Strains Isolated at Microscale.

The genomes of five Streptomyces strains belonging to the same soil community were sequenced and assembled. The strains, which were isolated at microscale, belonged to different Streptomyces species. This sample provides access to understand the functioning of a Streptomyces community in an ecological context.

Mining the Biosynthetic Potential for Specialized Metabolism of a Streptomyces Soil Community.

The diversity and distribution of specialized metabolite gene clusters within a community of bacteria living in the same soil habitat are poorly documented. Here we analyzed the genomes of 8 Streptomyces isolated at micro-scale from a forest soil that belong to the same species or to different species. The results reveal high levels of diversity, with a total of 261 biosynthesis gene clusters (BGCs) encoding metabolites such as terpenes, polyketides (PKs), non-ribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs) with potential bioactivities. A significant part of these BGCs (n = 53) were unique to only one strain when only 5 were common to all strains. The metabolites belong to very diverse chemical families and revealed that a large diversity of metabolites can potentially be produced in the community. Although that analysis of the global metabolome using GC-MS revealed that most of the metabolites were shared between the strains, they exhibited a specific metabolic pattern. We also observed that the presence of these accessory pathways might result from frequent loss and gain of genes (horizontal transfer), showing that the potential of metabolite production is a dynamic phenomenon in the community. Sampling Streptomyces at the community level constitutes a good frame to discover new biosynthetic pathways and it appears as a promising reservoir for the discovery of new bioactive compounds.

Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions.

Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.

A new clade of Mesorhizobium nodulating Alhagi sparsifolia.

We isolated 33 nodule bacteria from the legume Alhagi sparsifolia growing in the desert of northwest China. They fell into three groups by restriction analysis of their rrs (small subunit ribosomal RNA) genes, and these, together with dnaK and dnaJ genes, were sequenced from representative isolates to assess their taxonomic position by phylogenetic analysis. The bacteria in each group belonged to different lineages that might represent three different new Mesorhizobium species, two of which form a novel clade very distinct from other species in the genus. Most A. sparsifolia symbionts harboured closely related nodA and nodC genes forming new lineages. The presence of these closely related symbiosis genes in various genomic backgrounds and the incongruence observed between the different gene phylogenies indicate a history of horizontal gene transfer of symbiosis genes between the A. sparsifolia symbionts.

Genome Sequences of 11 Conspecific Streptomyces sp. Strains.

The genomes of 11 conspecific Streptomyces strains, i.e., from the same species and inhabiting the same ecological niche, were sequenced and assembled. This data set offers an ideal framework to assess the genome evolution of Streptomyces species in their ecological context.

Massive Gene Flux Drives Genome Diversity between Sympatric Streptomyces Conspecifics.

In this work, by comparing genomes of closely related individuals of Streptomyces isolated at a spatial microscale (millimeters or centimeters), we investigated the extent and impact of horizontal gene transfer in the diversification of a natural Streptomyces population. We show that despite these conspecific strains sharing a recent common ancestor, all harbored significantly different gene contents, implying massive and rapid gene flux. The accessory genome of the strains was distributed across insertion/deletion events (indels) ranging from one to several hundreds of genes. Indels were preferentially located in the arms of the linear chromosomes (ca. 12 Mb) and appeared to form recombination hot spots. Some of them harbored biosynthetic gene clusters (BGCs) whose products confer an inhibitory capacity and may constitute public goods that can favor the cohesiveness of the bacterial population. Moreover, a significant proportion of these variable genes were either plasmid borne or harbored signatures of actinomycete integrative and conjugative elements (AICEs). We propose that conjugation is the main driver for the indel flux and diversity in Streptomyces populations.IMPORTANCE Horizontal gene transfer is a rapid and efficient way to diversify bacterial gene pools. Currently, little is known about this gene flux within natural soil populations. Using comparative genomics of Streptomyces strains belonging to the same species and isolated at microscale, we reveal frequent transfer of a significant fraction of the pangenome. We show that it occurs at a time scale enabling the population to diversify and to cope with its changing environment, notably, through the production of public goods.

Diversity and antimicrobial activities of Streptomyces isolates from Fetzara Lake, north eastern Algeria.

A total of 125 Streptomyces strains were isolated from an Algerian wetland (Fetzara Lake) and characterized by growth on different culture media. Phylogenetic analyses were carried out by 16S rRNA sequence comparison after PCR amplification using universal primers. Antibacterial bioassays performed by the agar diffusion method enabled us to retain 33 Streptomyces isolates for their activity against two Gram-positive bacteria (Bacillus subtilis and Micrococcus luteus) and one Gram-negative bacteria (Escherichia coli). Among them, six isolates inhibited all three indicator strains. Antibacterial compounds were then extracted from the solid culture media with ethanol and ethyl acetate as organic solvents. The minimal inhibitory concentration (% v/v) of the extracts was evaluated by a standardized broth dilution method against different clinical-resistant bacterial isolates and Candida albicans. The most active crude extracts were selected for further characterization by chromatographic analysis (RP-HPLC).