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The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dôme (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

The complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy.

International audience; Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation suggests a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as a sole source of carbon and energy for growth, Methylorubrum extorquens DM4 is a longstanding reference strain. Here, the primary 5′-ends of transcripts were obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM or methanol. In total, 7231 putative TSSs were annotated and classified with respect to their localization to coding sequences (CDSs). TSSs on the opposite strand of CDS (antisense TSS) account for 31% of all identified TSSs. One-third of the detected TSSs were located at a distance to the start codon inferior to 250 nt (average of 84 nt) with 7% of leaderless mRNA. Taken together, the global TSS map for bacterial growth using DCM or methanol will facilitate future studies in which transcriptional regulation is crucial, and efficient DCM removal at polluted sites is limited by regulatory processes.

Background: Chloromethane (CH3Cl) is the most abundant chlorinated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. Results and Conclusions: We investigated chloromethane-degrading taxa associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13C-labelled CH3Cl combined with metagenomic DNA sequencing. Metagenome assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterised methylotrophic cmuA-independent pathway likely drives CH3Cl degradation in the investigated tree ferns.

International audience; Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Halogenated volatile organic compounds (VOCs) emitted by terrestrial ecosystems, such as chloromethane (CH3Cl), have pronounced effects on troposphere and stratosphere chemistry and climate. The magnitude of the global CH3Cl sink is uncertain since it involves a largely uncharacterized microbial sink. CH3Cl represents a growth substrate for some specialized methylotrophs, while methanol (CH3OH), formed in much larger amounts in terrestrial environments, may be more widely used by such microorganisms. Direct measurements of CH3Cl degradation rates in two field campaigns and in microcosms allowed the identification of top soil horizons (i.e., organic plus mineral A horizon) as the major biotic sink in a deciduous forest. Metabolically active members of Alphaproteobacteria and Actinobacteria were identified by taxonomic and functional gene biomarkers following stable isotope labeling (SIP) of microcosms with CH3Cl and CH3OH, added alone or together as the [C-13]-isotopologue. Well-studied reference CH3Cl degraders, such as Methylobacterium extorquens CM4, were not involved in the sink activity of the studied soil. Nonetheless, only sequences of the cmuA chloromethane dehalogenase gene highly similar to those of known strains were detected, suggesting the relevance of horizontal gene transfer for CH3Cl degradation in forest soil. Further, CH3Cl consumption rate increased in the presence of CH3OH. Members of Alphaproteobacteria and Actinobacteria were also C-13-labeled upon [C-13]-CH3OH amendment. These findings suggest that key bacterial CH3Cl degraders in forest soil benefit from CH3OH as an alternative substrate. For soil CH3Cl-utilizing methylotrophs, utilization of several one-carbon compounds may represent a competitive advantage over heterotrophs that cannot utilize one-carbon compounds.

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

Engineering organisms for biotechnology applications requires knowledge of their essential genes and associated regulatory networks. A new study of methylotrophic metabolism in Methylobacterium reveals essentiality of the unregulated, off-pathway phosphoribulokinase gene and an unexpected key regulatory role for its product ribulose-1,5-bisphosphate.

The genome sequence of Hyphomicrobium sp. strain GJ21, isolated in the Netherlands from samples of environments contaminated with halogenated pollutants and capable of using dichloromethane as its sole carbon and energy source, was determined.

The genomes of the aerobic methanotrophs “ Methyloterricola oryzae ” strain 73a T and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73a T represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum .

International audience; Chloromethane (CH3 Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of chloromethane characterized so far. Hydrogen isotope fractionation for degradation of chloromethane was determined for the first time, and yielded enrichment factors (ε) of -29‰ and -27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in (13) C of untransformed CH3 Cl was also observed, and similar isotope enrichment factors (ε) of -41‰ and -38‰ were obtained for degradation of chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of chloromethane will contribute to refine models of the global atmospheric budget of chloromethane.

International audience; Dichloromethane (DCM) is a volatile toxic halogenated solvent mainly produced and used industrially. DCM-degrading bacteria have long been models of choice for studying bacterial dehalogenation metabolism at the physiological, biochemical and genetic levels, and have also been used in bioremediation processes. DCM-degrading strains isolated in recent years will be discussed in the context of enzymes known to catalyze dehalogenation of DCM. Insights into the modes of adaptation of bacteria to DCM gained by comparative genomic analysis, highlight the importance of horizontal gene transfer in the dissemination of genes for DCM metabolism in the environment.

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.

Toxic man-made compounds released into the environment represent potential nutrients for bacteria, and microorganisms growing with such compounds as carbon and energy sources can be used to clean up polluted sites. However, in some instances, microorganisms contribute to contaminant degradation without any apparent benefit for themselves. Such cometabolism plays an important part in bioremediation, but is often difficult to control. Microbial degradation of tetrachloromethane (carbon tetrachloride, CCl(4)), a toxic ozone-depleting organic solvent mainly of anthropogenic origin, is only known to occur by cometabolic reduction under anoxic conditions. Yet no microbial system capable of using CCl(4) as the sole carbon source has been described. Microbial growth based on CCl(4) as a terminal electron acceptor has not been reported, although corresponding degradation pathways would yield sufficient energy. Known modes for the biodegradation of CCl(4) involve several microbial metabolites, mainly metal-bound coenzymes and siderophores, which are produced by facultative or strictly anaerobic bacteria and methanogenic Archaea. Recent reports have demonstrated that CCl(4) dechlorination rates are enhanced by redox-active organic compounds such as humic acids and quinones, which act as shuttles between electron-providing microorganisms and CCl(4) as a strong electron acceptor. The key factors underlying dechlorination of CCl(4), the practical aspects and specific requirements for microorganism-associated degradation of CCl(4) at contaminated sites and perspectives for future developments are discussed.

Genome sequences of Methylobacter luteus , Methylobacter whittenburyi , Methylosarcina fibrata , Methylomicrobium agile , and Methylovulum miyakonense were generated. The strains represent aerobic methanotrophs typically isolated from various terrestrial ecosystems.

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum . At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO 2 -requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR 1 BCAa 1 Ab 1 DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. PyrR 1 is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR 2 was investigated by allelic gene replacement. Unlike the pyrR 1 mutant, the ΔpyrR 2 strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO 2 was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR 1 and pyrR 2 mutants suggested that only PyrR 2 positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR 1 and PyrR 2 in the pyr regulon expression.

When microbes acquire new abilities through horizontal gene transfer, the genes and pathways must function under conditions with which they did not coevolve. If newly-acquired genes burden the host, their utility will depend on further evolutionary refinement of the recombinant strain. We used laboratory evolution to recapitulate this process of transfer and refinement, demonstrating that effective use of an introduced dichloromethane degradation pathway required one of several mutations to the bacterial host that are predicted to increase chloride efflux. We then used this knowledge to identify parallel, beneficial mutations that independently evolved in two natural dichloromethane-degrading strains. Finally, we constructed a synthetic mobile genetic element carrying both the degradation pathway and a chloride exporter, which preempted the adaptive process and directly enabled effective dichloromethane degradation across diverse Methylobacterium environmental isolates. Our results demonstrate the importance of post–transfer refinement in horizontal gene transfer, with potential applications in bioremediation and synthetic biology. DOI: http://dx.doi.org/10.7554/eLife.04279.001, eLife digest Many microbes can rapidly evolve to adapt to new or extreme habitats. Most often the characteristics that develop via evolution result from individuals inheriting new combinations of genes from their parents. However, many species can also acquire new genes through a process called horizontal gene transfer, where organisms that share an environment exchange potentially useful genes. The ability of bacteria to acquire genes by horizontal gene transfer is thought to be the main reason for the rapid evolution of antibiotic-resistant bacteria such as MRSA. A horizontally transferred gene rarely works efficiently when first transferred, and may even cause problems for its new host. The host organism must therefore evolve further after receiving a new gene, but it is difficult to trace how this occurs. A toxic chemical called dichloromethane (or DCM) has been used in many industries since World War II, and has caused widespread contamination to the environment. Strains from several microbial genera have been identified that have adapted to break down DCM and use it as their sole energy source. Moreover, a gene responsible for breaking down DCM appears to have been shared between different species by horizontal gene transfer. In work that was presented earlier in 2014, researchers introduced this gene into several bacterial strains from the genus Methylobacterium that had not been previously exposed to DCM. However, the resulting new strains of bacteria still had difficulties growing on DCM. There were diverse ways that the bacteria could have been prevented from growing—for example, multiple by-products generated when DCM is broken down are highly toxic. Now Michener et al.—including the researchers that performed the 2014 work—have experimentally evolved five of the modified strains of Methylobacterium to discover how they adapt over time to living on DCM. Examining the DNA sequences of these bacteria showed several mutations that both improved the ability of the bacteria to survive on DCM and helped the bacteria to more efficiently remove chloride ions from their systems. Chloride ions accumulate as DCM is broken down; therefore, Michener et al. suggest that being exposed to too much chloride prevented the bacteria in previous experiments from growing well. This idea is further supported by mutations found in previously isolated bacteria that live on DCM. These mutations increase the bacteria's ability to excrete chloride ions from their cells, and when Michener et al. removed these mutations, the bacteria no longer grew well on DCM. Understanding how bacteria adapt once they have acquired the DCM-degrading gene could make it easier to help bacteria to mop up DCM and other contaminants in the environment. DOI: http://dx.doi.org/10.7554/eLife.04279.002

Capnophiles are bacteria stimulated by bicarbonate and CO2, the two major forms of inorganic carbon (IC) in physiological neutral liquids. Capnophiles are often pathogenic heterotrophs found in IC-rich ecological niches such as human cavities. Like capnophiles, the growth of lactic acid bacteria (LAB) such as Lactobacillus plantarum and Enterococcus faecalis is stimulated by IC. CO2 or HC are substrates in carbamoyl phosphate (CP) synthesis and other carboxylation reactions in amino acid and nucleotide biosynthesis. When media were supplemented with nucleotides and all the amino acids, potassium bicarbonate still stimulated L. plantarum growth. This suggests that IC may be involved in other aspects of L. plantarum physiology besides its implication as a substrate in carboxylation reactions. Carbonic anhydrase (CA) catalyses the hydration of CO2 into bicarbonate. Since inorganic carbon stimulated L. plantarum growth, we searched for CA encoding genes in LAB genomes. CA can be classified into three classes according to their protein relatedness: α, β and γ . A class α CA was found in the L. plantarum, Leuconostoc mesenteroides, Streptococcus thermophilus, Oenococcus oeni, Enterococcus faecalis and Enterococcus faecium. These enterococci harboured a second CA encoding gene belonging to the γ class. No CA encoding gene was found in the Lactococcus lactis genome. These observations are discussed with regard to LAB evolution and ecological niches, which are often rich in IC. Lactic acid bacteria / Lactobacillus plantarum / carbon dioxide / bicarbonate / carbonic anhydrase

Les bacteries lactiques necessitent des milieux riches pour leur croissance car des mutations dans des genes impliques dans la biosynthese de nombreux acides amines, de vitamines ou de nucleobases ont invalide leurs capacites a synthetiser ces composes. La nature et la frequence des lesions genetiques responsables de ces auxotrophies naturelles ont ete recherchees dans les genes codant pour la synthese de deux voies de biosynthese, celle de l'arginine et celle des pyrimidines. Ces deux voies possedent un intermediaire de biosynthese commun, le carbamyl phosphate (CP). Une collection de 150 souches de Lactobacillus plantarum isolees de produits fermentes et de niches ecologiques diverses a ete testee. Aucune souche n'etait auxotrophe vis-a-vis des pyrimidines et 7 souches seulement presentaient une auxotrophie vis-a-vis de la citrulline, un precurseur de l'arginine. Les auxotrophes Arg - etaient plus frequemment rencontres parmi les isolats laitiers que parmi des isolats vegetaux ou d'origine humaine. Nous faisons l'hypothese que l'association des lactobacilles aux produits laitiers favoriserait la selection d'auxotrophes vis-a-vis de l'arginine. Les genes impliques dans la biosynthese de la citrulline sont regroupes dans l'operon argCJBDF et sont fonctionnels dans la plupart des souches etudiees. En cas d'inactivation, seul un gene est altere dans plus de la moitie des auxotrophes Arg- examines. Dans trois cas, l'inactivation de gene resulte d'evenements de mutation ponctuelle de type transversion. Dans les quatre autres auxotrophes, des alterations non reversibles de type deletion ou insertion ont ete mis en evidence. L'absence de cible privilegiee pour les mutations suggere qu'un processus de mutagenese aleatoire est a l'origine de l'apparition de ces auxotrophes naturels. Une analyse in silico du genome sequence de L. plantarum a permis la mise en evidence de 6 groupes de copies de genes impliques dans la biosynthese de l'arginine et des pyrimidines. Les genes codant l'enzyme impliquee dans la biosynthese du CP, le precurseur des deux voies metaboliques, sont presents en trois exemplaires. Parmi les copies du gene codant la grande sous-unite de cette enzyme, le gene pyrAb2 revet les caracteristiques d'un pseudogene, porteur de plusieurs mutations qui changent le cadre de lecture et ecourtent la proteine. Ainsi, ces resultats appuient l'hypothese selon laquelle les bacteries lactiques et plus particulierement L. plantarum, evoluent en s'adaptant a leurs habitats. Le genome de L. plantarum semble degenerer par accumulation de lesions genetiques. La diversite metabolique rencontree au sein des bacteries lactiques peut s'expliquer par ces modifications intra-genomiques. Cependant, l'apport d'information par des mecanismes de transferts horizontaux joue egalement un role crucial dans l'evolution des genomes de ces bacteries.

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO 2 . We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum , 32 strains of L. pentosus , 15 strains of L. paraplantarum , and 10 strains of L. casei . The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO 2 requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.

Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of Methylobacterium extorquens DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids-the other major isoprenoid product of this strain-and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in M. extorquens DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for M. extorquens DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane.

Background: The use of fossil carbon sources for fuels and petrochemicals has serious impacts on our environment and is unable to meet the demand in the future. A promising and sustainable alternative is to substitute fossil carbon sources with microbial cell factories converting lignocellulosic biomass into desirable value added products. However, such bioprocesses require availability of suitable and efficient microbial biocatalysts, capable of utilizing C5 sugars and tolerant to inhibitory compounds generated during pretreatment of biomass. In this study, the performance of a collection of lactic acid bacteria was evaluated regarding their properties with respect to the conversion of lignocellulosic feedstocks. The strains were examined for their ability to utilize xylose and arabinose as well as their resistance towards common inhibitors from pretreated lignocellulosic biomass (furan derivatives, phenolic compounds, weak acids). Results: Among 296 tested Lactobacillus and Pediococcus strains, 3 L. pentosus, 1 P. acidilactici and 1 P. pentosaceus isolates were found to be both capable of utilizing xylose and arabinose and highly resistant to the key inhibitors from chemically pretreated lignocellulosic biomass. When tested in broth with commonly found combinations of inhibitors, the selected strains showed merely 4%, 1% and 37% drop in growth rates for sugarcane bagasse, wheat straw and soft wood representatives, respectively, as compared to Escherichia coli MG1655 showing decreased growth rates by 36%, 21% and 90%, respectively, under the same conditions. Conclusion: The study showed that some strains of Lactobacilli and Pediococci have the potential to be used as production platforms for value-added products from pretreated lignocellulosic biomass. Selected Lactobacilli and Pediococci strains were able to tolerate the key inhibitors in higher concentrations compared to E. coli; in addition, as these isolates were also capable of fermenting xylose and arabinose, they constitute good candidates for efficient lignocellulosic feedstock bioconversions.

Monomethylamine (MMA, CH 3 NH 2 ) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N -methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS , mgsABC , and mgdABCD for the three enzymes of the pathway, γ-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4 gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4 gmaS and DM4 gmaS Δmetdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4 gmaS and DM4 gmaS Δmetdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source.

Horizontal gene transfer plays a crucial role in microbial evolution. While much is known about the mechanisms that determine whether physical DNA can be transferred into a new host, the factors determining the utility of the transferred genes are less clear. We have explored this issue using dichloromethane consumption in Methylobacterium strains. Methylobacterium extorquens DM4 expresses a dichloromethane dehalogenase (DcmA) that has been acquired through horizontal gene transfer and allows the strain to grow on dichloromethane as the sole carbon and energy source. We transferred the dcmA gene into six Methylobacterium strains that include both close and distant evolutionary relatives. The transconjugants varied in their ability to grow on dichloromethane, but their fitness on dichloromethane did not correlate with the phylogeny of the parental strains or with any single tested physiological factor. This work highlights an important limiting factor in horizontal gene transfer, namely, the capacity of the recipient strain to accommodate the stress and metabolic disruption resulting from the acquisition of a new enzyme or pathway. Understanding these limitations may help to rationalize historical examples of horizontal transfer and aid deliberate genetic transfers in biotechnology for metabolic engineering.

Carbamoyl-phosphate synthetases (CPS) catalyze carbamoyl phosphate (CP) biosynthesis from glutamine, bicarbonate and ATP. CPS are formed of two subunits, a small glutaminase subunit and a large synthetase subunit. CP is a common intermediate of arginine and pyrimidine biosynthe- sis. CPS in prokaryotes are either arginine-regulated (CPS-A), pyrimidine-regulated (CPS-P) or reg- ulated by both components. Two to zero CPS are present in the four lactic acid bacteria studied (Lactobacillus plantarum, Enterococcus faecalis, Lactococcus lactisand Lactobacillus delbrueckii ssp. lactis). Only L. plantarum harbours two CPS with a CPS-P providing CP for both metabolic path- ways. CPS-A can only supplement CP for arginine biosynthesis in higher concentrations of CO 2 or bicarbonate. The CPS-P present in L. plantarum and E. faecalis is encoded by genes within the pyr operon, and genes dispersed within the chromosome in Lc. lactis. CPS is absent in L. delbrueckii ssp. lactis and the catabolism of arginine via the arginine deiminase pathway (ADI) provides the CP for pyrimidine biosynthesis. In addition to their functional CPS-P, E. faecalis and Lc. lactis also harbour an ADI pathway so that arginine catabolism may regulate CP biosynthesis in these species. Lactic acid bacteria CPS were compared to CPS of 13 Gram-positive bacteria with sequenced or partially sequenced genomes. Most organisms harbour a CPS-P. CPS-P is also found in the few organisms (L. plantarum, B. subtilisand B. stearothermophilus) which harbour a CPS-A. The number of CPS and the organization of their genes is variable in Gram-positive bacteria. arginine / pyrimidine biosynthesis / lactic acid bacteria / Gram-positive bacteria / carbamoyl- phosphate synthetase

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

Les bacteries lactiques requierent des milieux de culture tres riches, du fait de leurs multiples auxotrophies. L'etude des besoins nutritionnels en arginine et en pyrimidines de 113 souches de lactobacilles homofermentaires, principalement des Lactobacillus plantarum et des L. pentosus, a montre que seulement 64 % des souches etaient prototrophes pour ces composes ; 33 % des souches necessitent la presence d'arginine et d'uracile dans le milieu de culture en aerobiose (mais non en atmosphere enrichie en gaz carbonique) suggerant une deficience au niveau de l'intermediaire commun entre ces deux metabolismes : le carbamyl phosphate (CP). Aucun auxotrophe pour les pyrimidines seules n'a ete detecte et parmi les quatre souches auxotrophes pour l'arginine seul, trois souches sont depourvues d'activite ornithine transcarbamylase (EC 2. 1. 3. 3) qui est une enzyme utilisant le CP comme substrat. Ainsi, le metabolisme du CP semble etre un bon modele d'etude de l'evolution et de l'adaptation des lactobacilles homofermentaires a leurs niches ecologiques. Dans cet article nous faisons le point de l'etat des connaissances et de la variabilite du metabolisme du CP chez les bacteries lactiques. Selon les especes, le CP est soit synthetise par la carbamyl phosphate synthetase (CPSase ; EC 6.3.5.5) a partir de gaz carbonique, d'ATP et de glutamine (L. plantarum), soit provient du catabolisme de l'arginine par la voie de l'arginine desiminase (ADI) (Lactobacillus leichmannii). Au sein des bacteries lactiques, la voie de l'ADI peut etre absente, partiellement ou totalement presente avec differentes regulations de son expression.

A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation. The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster. Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented. Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found. Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase. A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L. plantarum and one with ArgF of B. subtilis, which are paralogous. This divergence was not observed in vivo because an L. plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L. plantarum and B. subtilis. No OTCase activity was detectable when L. plantarum was grown in the presence of saturating amounts of arginine or citrulline. Arginine may repress the citrulline biosynthetic genes in L. plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn. The carA and argCJBDF genes are divergently transcribed. Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation.

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH 3 Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.

The complete genome sequence of Methylomicrobium album strain BG8, a methane-oxidizing gammaproteobacterium isolated from freshwater, is reported. Aside from a conserved inventory of genes for growth on single-carbon compounds, M. album BG8 carries a range of gene inventories for additional carbon and nitrogen transformations but no genes for growth on multicarbon substrates or for N fixation.

Robust growth of the gammaproteobacterium Methylomicrobium buryatense strain 5G on methane makes it an attractive system for CH 4 -based biocatalysis. Here we present a draft genome sequence of the strain that will provide a valuable framework for metabolic engineering of the core pathways for the production of valuable chemicals from methane.

International audience; Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.

Four strains of facultatively heterofermentative lactobacilli isolated from beer and human feces have physiological characteristics similar to those of Lactobacillus plantarum. Unlike 66% of the L. plantarum strains tested (F. Bringel, M.-C. Curk, and J.-C. Hubert, Int. J. Syst. Bacteriol. 46:588-594, 1996), these strains do not catabolize alpha-methyl-D-mannoside. However, because they exhibit little DNA relatedness to L. plantarum and Lactobacillus pentosus, these four strains were classified as members of a new species, Lactobacillus paraplantarum; strain CNRZ 1885 (= CIP 104668) is the type strain.

International audience; The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Methylomicrobium strains are widespread in saline environments. Here, we report the complete genome sequence of Methylomicrobium alcaliphilum 20Z, a haloalkaliphilic methanotrophic bacterium, which will provide the basis for detailed characterization of the core pathways of both single-carbon metabolism and responses to osmotic and high-pH stresses. Final assembly of the genome sequence revealed that this bacterium contains a 128-kb plasmid, making M. alcaliphilum 20Z the first methanotrophic bacterium of known genome sequence for which a plasmid has been reported.

Summary Transposition of conjugative transposons proceeds by excision and formation of a covalently closed circular Intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double-stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.

Hyphomicrobium sp. strain MC1 is an aerobic methylotroph originally isolated from industrial sewage. This prosthecate bacterium was the first strain reported to grow with chloromethane as the sole carbon and energy source. Its genome, consisting of a single 4.76-Mb chromosome, is the first for a chloromethane-degrading bacterium to be formally reported.