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Metabolic regulation of ‘Ca. Methylacidiphilum fumariolicum’ SolV cells grown under different nitrogen and oxygen limitations

ABSTRACT: Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of ‘Ca. Methylacidiphilum fumariolicum’ strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH4 is much more diverse than has previously been assumed in terms of ecology, phylogeny and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of ‘Ca. M. fumariolicum’ SolV cells growing at μmax in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N2 fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.

ORGANISM(S): Candidatus Methylacidiphilum fumarolicum

PROVIDER: GSE40528 | GEO | 2012/09/01

SECONDARY ACCESSION(S): PRJNA174222

REPOSITORIES: GEO

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Project description:Metabolic flexibility in aerobic methane oxidising bacteria (methanotrophs) enhances cell growth and survival in instances where resources are variable or limiting. Examples include the production of intracellular compounds (such as glycogen or polyhydroxyalkanoates) in response to unbalanced growth conditions and the use of some energy substrates, besides methane, when available. Indeed, recent studies show that verrucomicrobial methanotrophs can grow mixotrophically through oxidation of hydrogen and methane gases via respiratory membrane-bound group 1d [NiFe] hydrogenases and methane monooxygenases respectively. Hydrogen metabolism is particularly important for adaptation to methane and oxygen limitation, suggesting this metabolic flexibility may confer growth and survival advantages. In this work, we provide evidence that, in adopting a mixotrophic growth strategy, the thermoacidophilic methanotroph, Methylacidiphilum sp. RTK17.1 changes its growth rate, biomass yields and the production of intracellular glycogen reservoirs. Under nitrogen-fixing conditions, removal of hydrogen from the feed-gas resulted in a 14 % reduction in observed growth rates and a 144% increase in cellular glycogen content. Concomitant with increases in glycogen content, the total protein content of biomass decreased following the removal of hydrogen. Transcriptome analysis of Methylacidiphilum sp. RTK17.1 revealed a 3.5-fold upregulation of the Group 1d [NiFe] hydrogenase in response to oxygen limitation and a 4-fold upregulation of nitrogenase encoding genes (nifHDKENX) in response to nitrogen limitation. Genes associated with glycogen synthesis and degradation were expressed constitutively and did not display evidence of transcriptional regulation. Collectively these data further challenge the belief that hydrogen metabolism in methanotrophic bacteria is primarily associated with energy conservation during nitrogen fixation and suggests its utilisation provides a competitive growth advantage within hypoxic habitats.

Project description:Beller, H. R., T. E. Letain, A. Chakicherla, S. R. Kane, T. C. Legler, and M. A. Coleman. 2006. Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic vs. denitrifying conditions. Journal of Bacteriology 188:7005-7015. Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur-compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately ten percent of the genome) as differentially expressed using Robust Multi-array Average statistical analysis and a 2-fold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated respectively with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur-compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription, quantitative PCR analysis was used to validate these trends. Keywords: bacterial metabolism

Project description:The genome-wide transcriptional responses of the strictly aerobic -proteobacterium Gluconobacter oxydans 621H to oxygen limitation, to the absence of the cytochrome bc1 complex, and to low pH were studied using DNA microarray analyses. Oxygen limitation caused expression changes of 486 genes, representing 20% of the chromosomal genes. Genes with an increased mRNA level included those for terminal oxidases, the cytochrome bc1 complex, transhydrogenase, two alcohol dehydrogenases, heme biosynthesis, PTS proteins, proteins involved in cyclic diGMP synthesis and degradation, two sigma factors, flagella and chemotaxis proteins, several stress proteins, and a putative exporter protein. The downregulated genes comprised those for respiratory dehydrogenases, enzymes of central metabolism, PQQ biosynthesis, outer membrane receptors, Sec proteins, and proteins involved in transcription and translation. A M-NM-^TqrcABC mutant of G. oxydans showed a growth defect during cultivation on mannitol at pH 4 under oxygen saturation. Comparison of the transcriptomes of this mutant versus the wild type under these conditions revealed 51 differentially expressed genes. Interestingly, almost all of the 45 genes with increased expression in the M-NM-^TqrcABC mutant at pH 4 were also upregulated in the wild type grown at pH 6 under oxygen limitation. These results support an active role of the cytochrome bc1 complex in G. oxydans respiration. The transcriptome comparison of G. oxydans wild type at pH 4 versus pH 6 in mannitol medium under oxygen-saturated conditions uncovered only 72 differentially expressed genes. The 35 upregulated genes included those for cytochrome bd oxidase, major polyol dehydrogenase, iron storage and oxidative stress proteins. Among the 37 downregulated genes were some encoding enzymes dealing with carbon dioxide, such as biotin carboxylase, biotin carboxyl carrier protein, and carboanhydrase. These results give first insights into global transcriptional responses of G. oxydans. DNA microarray experiments were repeated independently three times for G. oxydans M-NM-^TqrcABC versus wild type and G. oxydans grown at pH 4 versus pH 6 and four times for G. oxydans grown at oxygen limitation versus pH oxygen saturation in biological replicates.

Project description:The genome-wide transcriptional responses of the strictly aerobic -proteobacterium Gluconobacter oxydans 621H to oxygen limitation, to the absence of the cytochrome bc1 complex, and to low pH were studied using DNA microarray analyses. Oxygen limitation caused expression changes of 486 genes, representing 20% of the chromosomal genes. Genes with an increased mRNA level included those for terminal oxidases, the cytochrome bc1 complex, transhydrogenase, two alcohol dehydrogenases, heme biosynthesis, PTS proteins, proteins involved in cyclic diGMP synthesis and degradation, two sigma factors, flagella and chemotaxis proteins, several stress proteins, and a putative exporter protein. The downregulated genes comprised those for respiratory dehydrogenases, enzymes of central metabolism, PQQ biosynthesis, outer membrane receptors, Sec proteins, and proteins involved in transcription and translation. A ΔqrcABC mutant of G. oxydans showed a growth defect during cultivation on mannitol at pH 4 under oxygen saturation. Comparison of the transcriptomes of this mutant versus the wild type under these conditions revealed 51 differentially expressed genes. Interestingly, almost all of the 45 genes with increased expression in the ΔqrcABC mutant at pH 4 were also upregulated in the wild type grown at pH 6 under oxygen limitation. These results support an active role of the cytochrome bc1 complex in G. oxydans respiration. The transcriptome comparison of G. oxydans wild type at pH 4 versus pH 6 in mannitol medium under oxygen-saturated conditions uncovered only 72 differentially expressed genes. The 35 upregulated genes included those for cytochrome bd oxidase, major polyol dehydrogenase, iron storage and oxidative stress proteins. Among the 37 downregulated genes were some encoding enzymes dealing with carbon dioxide, such as biotin carboxylase, biotin carboxyl carrier protein, and carboanhydrase. These results give first insights into global transcriptional responses of G. oxydans.

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Metabolic regulation of ‘Ca. Methylacidiphilum fumariolicum’ SolV cells grown under different nitrogen and oxygen limitations

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