Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post–transfer refinement

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.04279.00

Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance

<p>Abstract</p> <p>Background</p> <p>Lactic acid bacteria are commonly marketed as probiotics based on their putative or proven health-promoting effects. These effects are known to be strain specific but the underlying molecular mechanisms remain poorly understood. Therefore, unravelling the determinants behind probiotic features is of particular interest since it would help select strains that stand the best chance of success in clinical trials. Bile tolerance is one of the most crucial properties as it determines the ability of bacteria to survive in the small intestine, and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study was to investigate the natural protein diversity within the <it>Lactobacillus plantarum </it>species with relation to bile tolerance, using comparative proteomics.</p> <p>Results</p> <p>Bile tolerance properties of nine <it>L. plantarum </it>strains were studied <it>in vitro</it>. Three of them presenting different bile tolerance levels were selected for comparative proteomic analysis: <it>L. plantarum </it>299 V (resistant), <it>L. plantarum </it>LC 804 (intermediate) and <it>L. plantarum </it>LC 56 (sensitive). Qualitative and quantitative differences in proteomes were analyzed using two-dimensional electrophoresis (2-DE), tryptic digestion, liquid chromatography-mass spectrometry analysis and database search for protein identification. Among the proteins correlated with differences in the 2-DE patterns of the bacterial strains, 15 have previously been reported to be involved in bile tolerance processes. The effect of a bile exposure on these patterns was investigated, which led to the identification of six proteins that may be key in the bile salt response and adaptation in <it>L. plantarum</it>: two glutathione reductases involved in protection against oxidative injury caused by bile salts, a cyclopropane-fatty-acyl-phospholipid synthase implicated in maintenance of cell envelope integrity, a bile salt hydrolase, an ABC transporter and a F0F1-ATP synthase which participate in the active removal of bile-related stress factors.</p> <p>Conclusions</p> <p>These results showed that comparative proteomic analysis can help understand the differential bacterial properties of lactobacilli. In the field of probiotic studies, characteristic proteomic profiles can be identified for individual properties that may serve as bacterial biomarkers for the preliminary selection of strains with the best probiotic potential.</p

Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere

Chloromethane (CH3Cl) is a toxic gas mainly produced naturally, in particular by plants, and its emissions contribute to ozone destruction in the stratosphere. Conversely, CH3Cl can be degraded and used as the sole carbon and energy source by specialised methylotrophic bacteria, isolated from a variety of environments including the phyllosphere, i.e. the aerial parts of vegetation. The potential role of phyllospheric CH3Cl-degrading bacteria as a filter for plant emissions of CH3Cl was investigated using variants of Arabidopsis thaliana with low, wild-type and high expression of HOL1 methyltransferase previously shown to be responsible for most of CH3Cl emissions by A. thaliana. Presence and expression of the bacterial chloromethane dehalogenase cmuA gene in the A. thaliana phyllosphere correlated with HOL1 genotype, as shown by qPCR and RT-qPCR. Production of CH3Cl by A. thaliana paralleled HOL1 expression, as assessed by a fluorescence-based bioreporter. The relation between plant production of CH3Cl and relative abundance of CH3Cl-degrading bacteria in the phyllosphere suggests that CH3Cl-degrading bacteria co-determine the extent of plant emissions of CH3Cl to the atmosphere

Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post-transfer refinement. eLife 2014

Abstract 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

Hydrogen and carbon isotope fractionation during degradation of chloromethane by methylotrophic bacteria

Chloromethane (CH3Cl) 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 (epsilon) of -29 parts per thousand and -27 parts per thousand for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in C-13 of untransformed CH3Cl was also observed, and similar isotope enrichment factors (e) of -41 parts per thousand and -38 parts per thousand 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

Draft genome sequences of gammaproteobacterial methanotrophs isolated from marine ecosystems

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems

Hopanoid-free Methylobacterium extorquens DM4 overproduces carotenoids and has widespread growth impairment

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

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems

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

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.Organismic and Evolutionary Biolog

Draft genomes of gammaproteobacterial methanotrophs isolated from terrestrial ecosystems

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