A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine ...cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA). Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M. extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism. A mutation in mclA, encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, folC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype. Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory mox mutants.
Abstract
Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological ...techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.
Methanogenesis and methane oxidation are the major biological processes affecting the global cycling of the powerful greenhouse gas methane. To carry out the two alternative bioconversions, Nature ...has cleverly recycled key reactions for the C1 transfers between the oxidation levels of formaldehyde and formate, and these involve analogous enzyme systems and common specialized cofactors, methanopterin and methanofuran. Until recently, the distribution of these functions has been limited to methanogenic archaea and methylotrophic proteobacteria, and their evolutionary history remained obscure. Single interdomain lateral transfer of the respective genes has been suggested to play a role. Here we show that genes for C1 transfer reactions linked to methanopterin and methanofuran are also present in diverse representatives of the enigmatic bacterial clade, the Planctomycetes. Phylogenetic analysis places the planctomycete sequences as distantly from their archaeal counterparts as from their proteobacterial counterparts, suggesting novel scenarios for the evolution of the C1 transfer functions in both methanogens and methylotrophs. This finding suggests a possible role for Planctomycetes in the evolution of the methane cycle on Earth.
The numbers of methane‐oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture‐independent methods. Quantitative slot‐blot hybridizations were ...performed with type I and type II methanotroph‐specific probes. These data were compared to data from quantitative hybridizations using a pmoA‐specific probe and a eubacterial probe. From the combined hybridi‐zation data, the methanotroph population in Lake Washington was estimated to be 3.6 × 108–7.4 × 108 cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1ω8) and type II (18:1ω8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 × 108–9.4 × 109 cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 × 108–1.2 × 109 cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two‐ to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.
The results of previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of more than 130 genes, including classical chaperones such as ...the groESL and dnaKJ operons and proteases such as clpB are induced in response to elevated temperature. In addition, previous qualitative whole-cell mass spectrometric studies conducted under heat shock conditions indicated global changes in the D. radiodurans proteome. To enable the discovery of novel heat shock inducible proteins as well as gain greater biological insight into the classical heat shock response at the protein level, we undertook the global whole-cell FTICR mass spectrometric proteomics study reported here. We have greatly increased the power of this approach by conducting a large number of replicate experiments in addition to taking a semiquantitative approach to data analysis, finding good reproducibility between replicates. Through this analysis, we have identified with high confidence a core set of classical heat shock proteins whose expression increases dramatically and reproducibly in response to elevated temperature. In addition, we have found that the heat shock proteome includes a large number of induced proteins that have not been identified previously as heat responsive, and have therefore been designated as candidate responders. Finally, our results are consistent with the hypothesis that elevated temperature stress could lead to cross-protection against other related stresses.
Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that ...degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO2 and water-soluble products. Gas chromatography and 14C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is activity metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products
PDF
