Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

Abstract

It is still a challenge to link specific metabolic activities to certain species in a microbial community because of methodological limitations. We developed a method to analyze the specific metabolic activity of a single bacterial species within a consortium making use of [ 13 C 7 ]-toluene for metabolic labelling of proteins. Labelled proteins were subsequently analyzed by 2D gel electrophoresis (2-DE) and mass spectrometry (MS) to characterize their identity as well as their 13 C content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of 13 C carbon atoms into proteins of Aromatoleum aromaticum strain EbN1. This strain is capable of metabolizing toluene under nitrate-reducing conditions and was grown in either pure culture or in a mixed consortium with a gluconate-consuming enrichment culture. First, strain EbN1 was grown with non-labelled toluene or labelled [ 13 C 7 ]-toluene as carbon sources, respectively, and their proteins were subjected to 2-DE. In total, 60 unique proteins were identified by MALDI-MS/MS. From 38 proteins, the levels of 13 C incorporation were determined as 92.3 ± 0.8%. Subsequently, we mixed strain EbN1 and the enrichment culture UFZ-1, which does not grow on toluene but on gluconate, and added non-labelled toluene, [ 13 C 7 ]-toluene and/or non-labelled gluconate as carbon sources. The isotope labelling of proteins was analyzed after 2-DE by MS as a quantitative indicator for metabolic transformation of isotopic-labelled toluene by the active species of the consortium. Incorporation of 13 C was exclusively found in proteins from strain EbN1 at a content of 82.6 ± 2.3%, as an average calculated from 19 proteins, demonstrating the suitability of the method used to identify metabolic active species with specific properties within a mixed culture.

Impact of Vitamin B 12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

“…The silver was removed by adding potassium ferricyanide and sodium thiosulfate (9). Subsequently, the sample was proteolytically digested as described earlier (12). Peptides were reconstituted in 0.1% formic acid, injected by an autosampler, and concentrated on a trapping column (nanoAcquity ultrahigh pressure liquid chromatography [UPLC] column, C 18 , 180 m by 2 cm by 5 m; Waters, Eschborn, Germany) with water containing 0.1% formic acid at flow rates of 15 l/min.…”

“…This can be explained by the presence of corrinoid biosynthetic genes in the genome of the organism (25) indicating the de novo formation of the corrinoid cofactor of PceA. In addition, corrinoid salvaging might occur via the functional expression of a genome-encoded vitamin B 12 -specific ATP-binding cassette (ABC) transporter (btuFCD). The structure of the PceA corrinoid cofactor in D. hafniense strain Y51 has not been identified so far.…”

Purification and Characterization of Active-Site Components of the Putative p -Cresol Methylhydroxylase Membrane Complex from Geobacter metallireducens

“…Matrix-assisted laser desorption ionization MS analysis was performed on a dual time-of-flight instrument (Ultraflex III TOF/TOF MS; Bruker Daltonics, Bremen, Germany) as described previously (18). The measurements were performed in positive ion reflector mode and externally calibrated in the peptide range using peptide calibration standard II (Bruker Daltonics).…”

Responses of MethanogenmcrAGenes and Their Transcripts to an Alternate Dry/Wet Cycle of Paddy Field Soil

“…Thus, it might be assumed that analysis at the DNA level may not be fully adequate to link the methanogenic community with the methane production (47). Alternatively, different techniques have been developed in the past decade to link the biogeochemical processes to microbial identity, such as DNA/RNA-SIP (29,33), protein-SIP (15), and NanoSIMS (20). Recently, the transcriptional analysis (mRNA) of functional genes has been applied to detect the metabolically active organisms responsible for biogeochemical processes such as methane oxidation (4,17), ammonium oxidation (30), and nitrate reduction (21).…”