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Gene regulatory and metabolic adaptation processes of Dinoroseobacter shibae DFL12T during oxygen depletion.

ABSTRACT: Metabolic flexibility is the key to the ecological success of the marine Roseobacter clade bacteria. We investigated the metabolic adaptation and the underlying changes in gene expression of Dinoroseobacter shibae DFL12(T) to anoxic life by a combination of metabolome, proteome, and transcriptome analyses. Time-resolved studies during continuous oxygen depletion were performed in a chemostat using nitrate as the terminal electron acceptor. Formation of the denitrification machinery was found enhanced on the transcriptional and proteome level, indicating that D. shibae DFL12(T) established nitrate respiration to compensate for the depletion of the electron acceptor oxygen. In parallel, arginine fermentation was induced. During the transition state, growth and ATP concentration were found to be reduced, as reflected by a decrease of A578 values and viable cell counts. In parallel, the central metabolism, including gluconeogenesis, protein biosynthesis, and purine/pyrimidine synthesis was found transiently reduced in agreement with the decreased demand for cellular building blocks. Surprisingly, an accumulation of poly-3-hydroxybutanoate was observed during prolonged incubation under anoxic conditions. One possible explanation is the storage of accumulated metabolites and the regeneration of NADP(+) from NADPH during poly-3-hydroxybutanoate synthesis (NADPH sink). Although D. shibae DFL12(T) was cultivated in the dark, biosynthesis of bacteriochlorophyll was increased, possibly to prepare for additional energy generation via aerobic anoxygenic photophosphorylation. Overall, oxygen depletion led to a metabolic crisis with partly blocked pathways and the accumulation of metabolites. In response, major energy-consuming processes were reduced until the alternative respiratory denitrification machinery was operative.

SUBMITTER: Laass S

PROVIDER: S-EPMC4036333 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Gene regulatory and metabolic adaptation processes of Dinoroseobacter shibae DFL12T during oxygen depletion.

Laass Sebastian S Kleist Sarah S Bill Nelli N Drüppel Katharina K Kossmehl Sebastian S Wöhlbrand Lars L Rabus Ralf R Klein Johannes J Rohde Manfred M Bartsch Annekathrin A Wittmann Christoph C Schmidt-Hohagen Kerstin K Tielen Petra P Jahn Dieter D Schomburg Dietmar D

The Journal of biological chemistry 20140319 19

Metabolic flexibility is the key to the ecological success of the marine Roseobacter clade bacteria. We investigated the metabolic adaptation and the underlying changes in gene expression of Dinoroseobacter shibae DFL12(T) to anoxic life by a combination of metabolome, proteome, and transcriptome analyses. Time-resolved studies during continuous oxygen depletion were performed in a chemostat using nitrate as the terminal electron acceptor. Formation of the denitrification machinery was found enh ...[more]

PMID: 24648520

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Project description:The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

Project description:Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na(+)/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies.

Dataset Information

Gene regulatory and metabolic adaptation processes of Dinoroseobacter shibae DFL12T during oxygen depletion.

Publications

Gene regulatory and metabolic adaptation processes of Dinoroseobacter shibae DFL12T during oxygen depletion.

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