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First insights into the syntrophic acetate-oxidizing bacteria--a genetic study.

ABSTRACT: Syntrophic acetate-oxidizing bacteria have been identified as key organisms for efficient biogas production from protein-rich materials. They normally grow as lithotrophs or heterotrophs, producing acetate through the Wood-Ljungdahl pathway, but when growing in syntrophy with methanogens, they reportedly reverse this pathway and oxidize acetate to hydrogen and carbon dioxide. However, the biochemical and regulatory mechanisms behind the shift and the way in which the bacteria regain energy remain unknown. In a genome-walking approach, starting with degenerated primers, we identified those gene clusters in Syntrophaceticus schinkii, Clostridium ultunense, and Tepidanaerobacter acetatoxydans that comprise the formyltetrahydrofolate synthetase gene (fhs), encoding a key enzyme of the Wood-Ljungdahl pathway. We also discovered that the latter two harbor two fhs alleles. The fhs genes are phylogenetically separated and in the case of S. schinkii functionally linked to sulfate reducers. The T. acetatoxydans fhs1 cluster combines features of acetogens, sulfate reducers, and carbon monoxide oxidizers and is organized as a putative operon. The T. acetatoxydans fhs2 cluster encodes Wood-Ljungdahl pathway enzymes, which are also known to be involved in C1 carbon metabolism. Isolation of the enzymes illustrated that both formyltetrahydrofolate synthetases of T. acetatoxydans were functionally active. However, only fhs1 was expressed, confirming bidirectional usage of the pathway.

SUBMITTER: Muller B

PROVIDER: S-EPMC3584212 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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First insights into the syntrophic acetate-oxidizing bacteria--a genetic study.

Müller Bettina B Sun Li L Schnürer Anna A

MicrobiologyOpen 20121213 1

Syntrophic acetate-oxidizing bacteria have been identified as key organisms for efficient biogas production from protein-rich materials. They normally grow as lithotrophs or heterotrophs, producing acetate through the Wood-Ljungdahl pathway, but when growing in syntrophy with methanogens, they reportedly reverse this pathway and oxidize acetate to hydrogen and carbon dioxide. However, the biochemical and regulatory mechanisms behind the shift and the way in which the bacteria regain energy remai ...[more]

PMID: 23239474

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Project description:Project description In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane, and is accomplished through obligate mutualistic cross-feeding between SAO bacteria and methanogenic archaea. This pathway is particularly important in anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of the SAO functional guild to the stability and efficiency of the AD process, little is known about their in situ ecophysiologies due to their difficulty of isolation as well as low biomass abundances. Here, we performed a long-term (300 day) continuous enrichment of a thermophilic (55C) SAO community from a municipal AD system. Over 80% of the enriched bioreactor metagenome collectively belonged to a three-member consortium, including an acetate-oxidizing DTU068 bacterium encoding for CO2, H2, and formate production, along with two methanogenic Methanothermobacter_A archaeal species. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. Sample processing After 300 days of chemostat operation, batch microcosms were established in 40 ml glass serum bottles flushed with 80:20 N2:CO2 by anoxically transferring 18 ml of digestate from a single bioreactor (R2) and sealing with butyl rubber septa. Four different incubation conditions were established in triplicate: (1) blank control (e.g. no amendment); (2) 50 mM [12C]-acetate; (3) 50 mM [2 13C]-acetate (e.g. methyl-labeled); (4) 50 mM [1,2 13C]-acetate (universally labeled). Acetate was added to the microcosms (2 ml) as anoxic sterilized basal medium containing [12C], [2 13C], or [1,2 13C] sodium acetate (isotope purity >98%, Cambridge Isotopes). Twelve replicate bottles were established for all universally-labeled acetate-amended microcosms, allowing for three triplicate sets to be sacrificed for protein extraction at 24h, 144 hr, and 408 hr, and a single triplicate set for liquid sampling throughout for VFA analysis. Biomass was pelleted from 10 ml liquid samples via centrifugation (10,000xg) and stored at minus 20C until protein extraction. Protein from cell pellet samples were extracted in an 8M urea solution, then reduced and alkylated. Proteins were then digested with trypsin and subsequently desalted using C18 solid phase extraction. MS analysis was performed using 0.1 ug/ul of peptide solution injected into a Q Exactive HF X mass spectrometer (Thermo Scientific). Data processing Mass spectrometry (MS) data for each biological replicate at all time points (n=18) were analyzed using an implementation of OpenMS implemented in KNIME. Briefly, MS/MS spectra were searched using the MS-GF+ tool against a protein database consisting of all ORFs from the de-replicated set of MAGs, concatenated with reversed (decoy) sequences of all protein entries. Peptide spectra matches (PSMs) were filtered at a 5% false discovery rate (FDR) with Percolator.

Dataset Information

First insights into the syntrophic acetate-oxidizing bacteria--a genetic study.

Publications

First insights into the syntrophic acetate-oxidizing bacteria--a genetic study.

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