Incorporation of carbon and nitrogen atoms into proteins measured by protein‐based stable isotope probing (Protein‐SIP)

Jehmlich, Nico; Schmidt, Frank; Hartwich, Mathias; Bergen, Martin von; Richnow, Hans H.; Vogt, Carsten

doi:10.1002/rcm.3684

Cited by 76 publications

(65 citation statements)

References 32 publications

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“…After phenol extraction and acetate precipitation of the organic and aqueous phase, extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970). Five sections of each lane were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and treated according to Jehmlich et al (2008). Eluted peptides from the aqueous-phase gel slices were combined.…”

Section: Masd Clone Librarymentioning

confidence: 99%

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

et al. 2014

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Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with 13 C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA-and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short-and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent b-oxidation and the reverse Wood-Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.

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Section: Masd Clone Librarymentioning

confidence: 99%

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

et al. 2014

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“…SDS gel slices were destained, dehydrated and proteolytically cleaved overnight at 37 uC using trypsin (Promega) (Jehmlich et al, 2008). Extracted peptides were desalted using ZipTip-mC18 material (Merck Millipore).…”

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confidence: 99%

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family

Hartwig¹,

Thomas²,

Krumova³

et al. 2015

Microbiology

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Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN) 2 CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1.In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O 2 -sensitive H 2 -uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H 2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries. INTRODUCTION[NiFe]-hydrogenases are widespread amongst archaeal and bacterial species (Vignais & Billoud, 2007). These enzymes can either oxidize H 2 to generate a chemiosmotic proton gradient via a membrane-based electron transfer chain, as well as provide a source of reducing power, or dissipate accumulated intracellular reductant by reducing protons to produce H 2 ; some perform both functions under certain physiological conditions (Lubitz et al., 2014;Pinske et al., 2015;Vignais & Billoud, 2007 (Ogata et al., 2015). The biosynthesis of this cofactor is complicated, requiring the combined activities of six Hyp accessory proteins, whose functions include synthesis and insertion of an Fe(CN) 2 CO moiety into the apo-catalytic subunit followed by introduction of the nickel ion (Böck et al., 2006;Forzi & Sawers, 2007). Subsequent to successful cofactor insertion, a C-terminal peptide present on the large subunit of most [NiFe]-hydrogenases is cleaved by a hydrogenase-specific protease and further assembly of the enzyme can then be completed (Böck et al., 2006;Pinske & Sawers, 2014).The Hyp proteins include HypA and the GTPase HypB, which, together with the peptidyl-prolyl cis/trans isomerase SlyD (Zhang et al., 2005), deliver the nickel ion; HypC, which is a small iron-and CO 2 -binding protein (Soboh et al., 2013); the FeS cluster protein HypD, which acts as a scaffold for assembl...

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“…Protein-SIP is based on the metabolic incorporation of isotopically labeled substrates, where 13 C, 15 N or 36 S can be used (Jehmlich et al, 2008a(Jehmlich et al, , b, 2012. The metabolism of the labeled substrates leads to assimilation of heavy isotopes and subsequent incorporation into the various classes of biomolecules, including proteins and can be detected by mass spectrometry of peptides as reviewed in Seifert et al (2012).…”

Section: Features Of Protein-sip To Simultaneously Detect Metabolic Amentioning

confidence: 99%

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

et al. 2013

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The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ( 13 C, 15 N, 36 S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

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Incorporation of carbon and nitrogen atoms into proteins measured by protein‐based stable isotope probing (Protein‐SIP)

Cited by 76 publications

References 32 publications

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

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