Hans-Hermann Richnow

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

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 [13C7]-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 13C content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of 13C 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 [13C7]-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 13C 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, [13C7]-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 13C 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.

6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes.

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin.

ABSTRACT Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.

Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon (13C)- or nitrogen (15N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2–3 d.

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.

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

The identification of metabolically active microbial key players is fundamental for understanding the structure and functions of contaminant-degrading communities. The metabolic activity can be analysed by feeding the microbial culture with stable-isotope-labelled substrates and subsequently tracing their incorporation into the biomass. In this paper we present a method which is able to detect the incorporation of stable isotopes from the substrate into the proteins of a benzene-metabolising microorganism. Pseudomonas putida strain ML2 was grown under aerobic conditions with the substrates (12)C-benzene, (13)C-benzene or (15)N-ammonium and (12)C-benzene. Proteins of these cultures were resolved by two-dimensional gel electrophoresis (2-DE) and corresponding protein spots were subjected to matrix-assisted laser ionization/desorption mass spectrometric (MALDI-MS) analysis. The proteins of the (12)C-sample were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements. The (13)C- or (15)N-content of the peptides from the labelling experiments was determined by MALDI-MS/MS. The incorporation of heavy isotopes into the proteins from cultures grown on (13)C-benzene and (15)N-ammonium was determined based on the mass differences between labelled and non-labelled peptides as well as on the isotopic distribution of the y(1)-ion of arginine. The method we present here principally allows the unravelling of the carbon and nitrogen flow not only in pure cultures, but also in microbial communities consisting of many microbial species.

Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments

BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community.

Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either 13C-labeled benzene or 13C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of 13C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.

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

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 (13C, 15N, 36S) 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.

Carbon and hydrogen isotope fractionation of benzene during biodegradation under sulfate‐reducing conditions: a laboratory to field site approach

The microbial carbon and hydrogen isotope fractionation of benzene under sulfate-reducing conditions was investigated within systems of increasing complexity: (i) batch laboratory microcosms, (ii) a groundwater-percolated column system, and (iii) an aquifer transect. Recent molecular biological studies indicate that, at least in the laboratory microcosms and the column system, benzene is degraded by similar bacterial communities. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) obtained from laboratory microcosms and from the column study varied significantly although experiments were performed under similar redox and temperature conditions. Thus, enrichment factors for only a single element could not be used to distinguish benzene degradation under sulfate-reducing conditions from other redox conditions. In contrast, using correlation of changes of hydrogen vs. carbon isotope ratios (Lambda = Delta delta(2)H/Delta delta(13)C), similar Lambda-values were derived for the benzene biodegradation under sulfate-reducing conditions in all three experimental systems (Lambda(laboratory microcosms) = 23 +/- 5, Lambda(column) = 28 +/- 3, Lambda(aquifer) = 24 +/- 2), showing the robustness of the two-dimensional compound-specific stable isotope analysis (2D-CSIA) for elucidating distinct biodegradation pathways. Comparing carbon and hydrogen isotope fractionation data from recent studies, an overlap in Lambda-values was observed for benzene biodegradation under sulfate-reducing (Lambda = 23 +/- 5 to Lambda = 29 +/- 3) and methanogenic (Lambda = 28 +/- 1 to Lambda = 39 +/- 5) conditions, indicating a similar initial benzene reaction mechanism for both electron-acceptor conditions.