Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction

We would like to thank C. Dilßner and M. Neytschev (UFZ) for excellent technical assistance with CoA thioester synthesis, strain cultivation and HPLC analyses. In addition, we thank Birgit Würz (UFZ) for invaluable analytical advice and help with GC mass spectrometry. We are also indebted to L. von Wintzingerode, A. Grunwald, and J. Grabengießer (UFZ) for assistance in the cultivation and enzyme assay experiments. Many thanks to K. Eismann (UFZ) as well, for help with the proteome analysis and fruitful discussions regarding different protein extraction methods.The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.00691/full#supplementary-materialThe tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B12-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO2 by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.Program Topic "Chemicals in the Environment" within the Research Program "Terrestrial Environment" of the Helmholtz Association European Union (EU) 62485

Insights into Autotrophic Activities and Carbon Flow in Iron-Rich Pelagic Aggregates (Iron Snow)

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes

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

Benzylsuccinate Synthase is Post-Transcriptionally Regulated in the Toluene-Degrading Denitrifier Magnetospirillum sp. Strain 15-1

The facultative denitrifying alphaproteobacterium Magnetospirillum sp. strain 15-1 had been isolated from the hypoxic rhizosphere of a constructed wetland model fed with toluene. This bacterium can catabolize toluene anaerobically but not aerobically. Here, we used strain 15-1 to investigate regulation of expression of the highly oxygen-sensitive glycyl radical enzyme benzylsuccinate synthase, which catalyzes the first step in anaerobic toluene degradation. In cells growing aerobically with benzoate, the addition of toluene resulted in a ~20-fold increased transcription of bssA, encoding for the catalytically active subunit of the enzyme. Under anoxic conditions, bssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate. Proteomics showed that abundance of benzylsuccinate synthase increased in cells growing anaerobically with toluene. In contrast, peptides of this enzyme were never detected in oxic conditions. These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes

Quantification of glyphosate and aminomethylphosphonic acid from microbiome reactor fluids

Rationale: Glyphosate is one of the most widely used herbicides and it is suspected to affect the intestinal microbiota through inhibition of aromatic amino acid synthesis via the shikimate pathway.In vitromicrobiome bioreactors are increasingly used as model systems to investigate effects on intestinal microbiota and consequently methods for the quantitation of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in microbiome model systems are required. Methods: An optimized protocol enables the analysis of both glyphosate and AMPA by simple extraction with methanol:acetonitrile:water (2:3:1) without further enrichment steps. Glyphosate and AMPA are separated by liquid chromatography on an amide column and identified and quantified with a targeted tandem mass spectrometry method using a QTRAP 5500 system (AB Sciex). Results: Our method has a limit of detection (LOD) in extracted water samples of <2 ng/mL for both glyphosate and AMPA. In complex intestinal medium, the LOD is 2 and 5 ng/mL for glyphosate and AMPA, respectively. These LODs allow for measurement at exposure-relevant concentrations. Glyphosate levels in a bioreactor model of porcine colon were determined and consequently it was verified whether AMPA was produced by porcine gut microbiota. Conclusions: The method presented here allows quantitation of glyphosate and AMPA in complex bioreactor fluids and thus enables studies of the impact of glyphosate and its metabolism on intestinal microbiota. In addition, the extraction protocol is compatible with an untargeted metabolomics analysis, thus allowing one to look for other perturbations caused by glyphosate in the same sample

Calculation of partial isotope incorporation into peptides measured by mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Stable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. However in order to track carbon fluxes in microbial communities new methods are required that allow the calculation of partial incorporation into biomolecules.</p> <p>Results</p> <p>In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial¹³C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.</p> <p>We used a freely available peptide mass dataset from <it>Mycobacterium tuberculosis </it>consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from <it>Pseudomonas putida </it>ML2 grown on labeled substrate of various known concentrations were used and¹³C isotopic incorporation was successfully predicted. An easy-to-use script <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> was further developed to guide users through the calculation procedure for their own data series.</p> <p>Conclusion</p> <p>Our method is valuable for estimating¹³C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.</p

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand.We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota

Genome and secretome of Chondrostereum purpureum correspond to saprotrophic and phytopathogenic life styles

We thank R. Ullrich for his mycological expertise and K. Eismann for useful technical assistance.The basidiomycete Chondrostereum purpureum (Silverleaf fungus) is a saprotroph and plant pathogen commercially used for combatting forest “weed” trees in vegetation management. However, little is known about its lignocellulose-degrading capabilities and the enzymatic machinery that is responsible for the degradative potential, and it is not yet clear to which group of wood-rot fungi it actually belongs. Here, we sequenced and analyzed the draft genome of C. purpureum (41.2 Mbp) and performed a quantitative proteomic approach during growth in submerged and solid-state cultures based on soybean meal suspension or containing beech wood supplemented with phenol-rich olive mill residues, respectively. The fungus harbors characteristic lignocellulolytic hydrolases (GH6 and GH7) and oxidoreductases (e.g. laccase, heme peroxidases). High abundance of some of these genes (e.g. 45 laccases, nine GH7) can be explained by gene expansion, e.g. identified for the laccase orthogroup ORTHOMCL11 that exhibits a total of 18 lineage-specific duplications. Other expanded genes families encode for proteins more related to a pathogenic lifestyle (e.g. protease and cytochrome P450s). The fungus responds to the presence of complex growth substrates (lignocellulose, phenolic residues) by the secretion of most of these lignocellulolytic and lignin-modifying enzymes (e.g. alcohol and aryl alcohol oxidases, laccases, GH6, GH7). Based on the genetic and enzymatic constitution, we consider the ‘marasmioid’ fungus C. purpureum as a ‘phytopathogenic’ white-rot fungus (WRF) that possesses a complex extracellular enzyme machinery to accomplish efficient lignocellulose degradation during both saprotrophic and phytopathogenic life phases.The work was financially and scientifically supported by the European Union [integrated projects INDOX – KBBE-7-2013-613549; ENZOX2 – 720297], by the DFG project PeroxiDiv HO 1961/8-1 and the AiF project PeroxyMEER IGF 19636 BG/3. The work has been partly funded by the DFG Priority Program 1374 "Infrastructure-Biodiversity-Exploratories" with the projects HO 1961/6-1, KE 1742/2-1 and JE 724/7-4 (AOBJ: 635952) and the Spanish Ministry of Economy and Competitiveness [project AGL2012-32873]. RR thanks the JAE-Program of the Spanish National Research Council [CSIC] and EA thanks MINECO and FEDER Co-Funds [RyC-2013-12481]

The Simplified Human Intestinal Microbiota (SIHUMIx) Shows High Structural and Functional Resistance against Changing Transit Times in In Vitro Bioreactors

Many functions in host–microbiota interactions are potentially influenced by intestinal transit times, but little is known about the effects of altered transition times on the composition and functionality of gut microbiota. To analyze these effects, we cultivated the model community SIHUMIx in bioreactors in order to determine the effects of varying transit times (TT) on the community structure and function. After five days of continuous cultivation, we investigated the influence of different medium TT of 12 h, 24 h, and 48 h. For profiling the microbial community, we applied flow cytometric fingerprinting and revealed changes in the community structure of SIHUMIx during the change of TT, which were not associated with changes in species abundances. For pinpointing metabolic alterations, we applied metaproteomics and metabolomics and found, along with shortening the TT, a slight decrease in glycan biosynthesis, carbohydrate, and amino acid metabolism and, furthermore, a reduction in butyrate, methyl butyrate, isobutyrate, valerate, and isovalerate concentrations. Specifically, B. thetaiotaomicron was identified to be affected in terms of butyrate metabolism. However, communities could recover to the original state afterward. This study shows that SIHUMIx showed high structural stability when TT changed—even four-fold. Resistance values remained high, which suggests that TTs did not interfere with the structure of the community to a certain degree

Environmentally Relevant Concentration of Bisphenol S Shows Slight Effects on SIHUMIx

Bisphenol S (BPS) is an industrial chemical used in the process of polymerization of polycarbonate plastics and epoxy resins and thus can be found in various plastic products and thermal papers. The microbiota disrupting effect of BPS on the community structure of the microbiome has already been reported, but little is known on how BPS affects bacterial activity and function. To analyze these effects, we cultivated the simplified human intestinal microbiota (SIHUMIx) in bioreactors at a concentration of 45 µM BPS. By determining biomass, growth of SIHUMIx was followed but no differences during BPS exposure were observed. To validate if the membrane composition was affected, fatty acid methyl esters (FAMEs) profiles were compared. Changes in the individual membrane fatty acid composition could not been described; however, the saturation level of the membranes slightly increased during BPS exposure. By applying targeted metabolomics to quantify short-chain fatty acids (SCFA), it was shown that the activity of SIHUMIx was unaffected. Metaproteomics revealed temporal effect on the community structure and function, showing that BPS has minor effects on the structure or functionality of SIHUMIx