A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Self Cite

bRecently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H 2 . Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H 2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H 2 . The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 mol of H 2 per min and mg of protein. However, an unexpectedly high Michaelis constant (K m ) for H 2 of 3.6 ؎ 0.5 M was determined, which is in contrast to the previously proposed low K m of group 5 hydrogenases and makes atmospheric H 2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H 2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O 2 .

Section: Resultsmentioning

confidence: 74%

“…For quantification of AH transcripts, cells of R. eutropha strains were grown in FGN medium to an OD 436 of 8. Total RNA isolation and reverse transcription to cDNA was done as described previously (24). Quantitative PCR was performed on the obtained cDNA using SYBR green fluorescent dye and a 7500 Fast PCR Cycler (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

Schäfer

Friedrich

Lenz

2013

Self Cite

“…The Hydrogenophaga-associated metagenomic data included genes involved in carbon fixation via RuBisCO and in the aerobic oxidation of hydrogen and carbon monoxide. Characterized Hydrogenophaga species are known to be facultatively autotrophic, i.e., they oxidize hydrogen to power carbon fixation only when organic carbon is unavailable (44,45). They are also aerobes or facultative anaerobes (45).…”

Section: Discussionmentioning

confidence: 99%

Bacterial Communities Associated with Subsurface Geochemical Processes in Continental Serpentinite Springs

Brazelton

Morrill

Szponar

et al. 2013

102

125

Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.

“…One example from R. eutropha is the chaperone GroEL, which was present in substantial amounts in the PHB granule fraction of a ⌬phaP1 mutant (13). On the other hand, some phasin proteins were identified in a membrane fraction from R. eutropha by proteome analysis (31). However, PHB granules were not the subject of that study, and PHB granuleassociated proteins might have contaminated the membrane fraction.…”

mentioning

confidence: 92%

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Sznajder

Pfeiffer

Jendrossek

2015

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative ␣/␤-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. P olyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHA) are storage compounds for carbon and energy and are widespread in prokaryotic species (1). PHB is deposited in the form of 200-to 500-nm particles (granules) when PHB-accumulating bacteria are cultivated in the presence of a surplus of a suitable carbon source. Ralstonia eutropha H16 (alternative designation, Cupriavidus necator) has become the model organism of PHB research, and the species is also used in industrial processes to produce PHB as a biodegradable polymer with plastic-like properties (2-4). The intensive research of the last few decades has led to a good understanding of the biochemical routes leading to PHB/PHA and of the biochemical properties of proteins with established functions in PHB/PHA metabolism. For example, the key enzymes of PHB synthesis, the PHB synthases (PhaCs), of many PHB-accumulating species have been purified; the coding genes were cloned; and the polymerization reaction has been studied (for overviews and recent results, see references 5 to 11). Meanwhile, much evidence has accumulated showing that PHB granules are not simply storage molecules but represent well-defined subcellular organelles that consist of a polymer core and a surface layer to which many proteins with specific functions are attached (12). Besides the aforementioned PHB synthase, small amphiphilic polypeptides, so-called phasin proteins (PhaP...