Project description:(from abstract): Iron oxidation is a desirable trait of biomining microorganisms, although the mechanism is not well-understood in extreme thermoacidophiles. The complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up-regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. Keywords: substrate response
Project description:(from abstract): Iron oxidation is a desirable trait of biomining microorganisms, although the mechanism is not well-understood in extreme thermoacidophiles. The complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up-regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. Dye flip of Mse cells includes two samples, yeast exact (YE) and yeast extract + ferrous sulfate (YEF). The first slide, Y3F5, has YE RNA labeled with cy3 and YEF RNA labeled with cy5, while the second slide, F3Y5, has YEF RNA labeled with cy3 and YE RNA labeled with cy5. YE serves as the reference condition, with the expectation that ORFs involved with Fe2+ oxidation (and SO4 metabolism) will be upregulated on YEF (log2 fold change of YE-YEF < -1).
Project description:Dimethylsufoniopropionate (DMSP) is an important and abundant organic sulfur compound and an important substrate for marine bacterioplankton. The Roseobacter clade of marine alpha-proteobacteria, including Silicibacter pomeroyi strain DSS3, are known to be a key phylogenetic group involved in DMSP degradaton. The fate of DMSP has important implications for the global sulfur cycle, but the genes involved in this process and their regulation are largely unknown. S. pomeroyi is capable of performing two major pathways of DMSP degradation, making it an interesting model organism. Based on the full genome sequence of this strain we designed an oligonucleotide-based microarray for the detection of transcripts of nearly all genes. The array was used to study the transcriptional response of S. pomeroyi cultures to additions of DMSP or Acetate in a time series experiment. We identified a number of DMSP-upregulated genes that could be assigned to potential roles in the metabolization of DMSP. DMSP also affected the transcription of other groups of genes, including genes for transport and metabolization of peptides, amino-acids and polyamines. High DMSP concentrations may be a chemical signal indicating phytoplankton abundance and elicit a regulatory response aimed at making maximum use of the available nutrients under these conditions. Keywords: Microarray, marine bacterium, messenger RNA, transcription, sulfur metabolism The array design is based on the complete genome sequence of S. pomeroyi strain DSS 3 and available from Genbank (Accession numbers CP000031 and CP000032). Probes for all identified potential genes were designed by Combimatrix using proprietary software. A total of 4161 genes out of the 4348 identified potential genes on the S. pomeroyi genome are represented on the array. When possible, two probes per gene were designed.
Project description:The non-methane-utilizing methylotroph, Methylophilus sp. TWE2, was isolated from tap-water during the enrichment of methanotrophs with methane. The complete genome sequence of strain TWE2 showed that this bacterium may convert methanol to formaldehyde via catalysis of methanol dehydrogenase (MDH), after which formaldehyde would be assimilated to biomass through the ribulose monophosphate (RuMP) pathway or dissimilated via the tetrahydromethanopterin (H4MPT) pathway. The deficiency of glycolysis and the TCA cycle indicate that strain TWE2 may be an obligate methylotroph. This is the first complete genome sequence of the genus Methylophilus.
Project description:Natural and anthropogenic wetlands are main sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic microorganisms and by processes at the oxic-anoxic interface, such as sulfur cycling, that reduce the activity of methanogens. In this study, we obtained a pure culture (strain HY1) of a versatile wetland methanotroph that oxidizes various organic and inorganic compounds. This strain represents (i) the first isolate that can aerobically oxidize both methane and reduced sulfur compounds and (ii) a new alphapoteobacterial species, named Candidatus Methylovirgula thiovorans. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are the only enzymes for methane and methanol oxidation, respectively. Unexpectedly, strain HY1 harbors various pathways for respiratory sulfur oxidation and oxidized reduced sulfur compounds to sulfate using the Sox-rDsr pathway (without SoxCD) and the S4I system. It employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on the reduced sulfur compounds. Methane and thiosulfate were independently and simultaneously oxidized by strain HY1 for growth. Proteomic and microrespiratory analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of their substrates. The discovery of this versatile methanotroph demonstrates that methanotrophy and thiotrophy is compatible in a single bacterium and adds a new aspect to interactions of methane and sulfur cycles in oxic-anoxic interface environments.
Project description:Dimethylsulfide is a volatile organic sulfur compound that provides the largest input of biogenic sulfur from the oceans to the atmosphere, and thence back to land, constituting an important link in the global sulfur cycle. Microorganisms degrading DMS affect fluxes of DMS in the environment, but the underlying metabolic pathways are still poorly understood. Methylophaga thiooxydans is a marine methylotrophic bacterium capable of growth on DMS as sole source of carbon and energy. Using proteomics and transcriptomics we identified genes expressed during growth on dimethylsulfide and methanol to refine our knowledge of the metabolic pathways that are involved in DMS and methanol degradation in this strain. Amongst the most highly expressed genes on DMS were the two methanethiol oxidases driving the oxidation of this reactive and toxic intermediate of DMS metabolism. Growth on DMS also increased expression of the enzymes of the tetrahydrofolate linked pathway of formaldehyde oxidation, in addition to the tetrahydromethanopterin linked pathway. Key enzymes of the inorganic sulfur oxidation pathway included flavocytochrome c sulfide dehydrogenase, sulfide quinone oxidoreductase, and persulfide dioxygenases. A sulP permease was also expressed during growth on DMS. Other enzymes of organic and inorganic sulfur metabolism previously detected in cell extracts of Methylophaga have not been characterised at the genetic level yet; their expression level and regulation could not be analysed. A pan-genome analysis of six available Methylophaga genomes suggests that only two of the six investigated bacteria have the metabolic potential to utilize methanethiol, the degradation product of DMS. These results mirror phenotypic analyses and demonstrate that DMS-utilization and subsequent C1 and sulfur oxidation are not conserved across the entire genus.
Project description:The purple sulfur bacterium Allochromatium vinosum DSM 180T is one of the best studied sulfur-oxidizing anoxygenic phototrophic bacteria and has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism’s high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur or sulfite as compared to photoorganoheterotrophic growth on malate. Differential expression (at least twofold) of 1149 genes was observed, corresponding to 30% of the A. vinosum genome. A total of 549 genes were identified for which relative transcription increased at least twofold during growth on one of the different sulfur sources while relative transcription of 599 genes decreased. A significant number of genes that were strongly induced have documented sulfur-metabolism-related functions. Among these are the dsr genes including dsrAB for dissimilatory sulfite reductase and the sgp genes for the proteins of the sulfur globule envelope thus confirming former results. In addition we were able to identify new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Two of these were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for the oxidation of sulfide and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria.
Project description:The purple sulfur bacterium Allochromatium vinosum DSM 180T is one of the best studied sulfur-oxidizing anoxygenic phototrophic bacteria and has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism’s high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur or sulfite as compared to photoorganoheterotrophic growth on malate. Differential expression (at least twofold) of 1149 genes was observed, corresponding to 30% of the A. vinosum genome. A total of 549 genes were identified for which relative transcription increased at least twofold during growth on one of the different sulfur sources while relative transcription of 599 genes decreased. A significant number of genes that were strongly induced have documented sulfur-metabolism-related functions. Among these are the dsr genes including dsrAB for dissimilatory sulfite reductase and the sgp genes for the proteins of the sulfur globule envelope thus confirming former results. In addition we were able to identify new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Two of these were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for the oxidation of sulfide and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria.
Project description:Dimethylsufoniopropionate (DMSP) is an important and abundant organic sulfur compound and an important substrate for marine bacterioplankton. The Roseobacter clade of marine alpha-proteobacteria, including Silicibacter pomeroyi strain DSS3, are known to be a key phylogenetic group involved in DMSP degradaton. The fate of DMSP has important implications for the global sulfur cycle, but the genes involved in this process and their regulation are largely unknown. S. pomeroyi is capable of performing two major pathways of DMSP degradation, making it an interesting model organism. Based on the full genome sequence of this strain we designed an oligonucleotide-based microarray for the detection of transcripts of nearly all genes. The array was used to study the transcriptional response of S. pomeroyi cultures to additions of DMSP or Acetate in a time series experiment. We identified a number of DMSP-upregulated genes that could be assigned to potential roles in the metabolization of DMSP. DMSP also affected the transcription of other groups of genes, including genes for transport and metabolization of peptides, amino-acids and polyamines. High DMSP concentrations may be a chemical signal indicating phytoplankton abundance and elicit a regulatory response aimed at making maximum use of the available nutrients under these conditions. Keywords: Microarray, marine bacterium, messenger RNA, transcription, sulfur metabolism