Carbon and hydrogen stable isotope fractionation due to monooxygenation of short-chain alkanes by butane monooxygenase of Thauera butanivorans Bu-B1211

Multi element compound-specific stable isotope analysis (ME-CSIA) is a tool to assess (bio)chemical reactions of molecules in the environment based on their isotopic fingerprints. To that effect, ME-CSIA concepts are initially developed with laboratory model experiments to determine the isotope fractionation factors specific for distinct (bio)chemical reactions. Here, we determined for the first time the carbon and hydrogen isotope fractionation factors for the monooxygenation of the short-chain alkanes ethane, propane, and butane. As model organism we used Thauera butanivorans strain Bu-B1211 which employs a non-haem iron monooxygenase (butane monooxygenase) to activate alkanes. Monooxygenation of alkanes was associated with strong carbon and hydrogen isotope effects: εbulkC = −2.95 ± 0.5 ‰ for ethane, −2.68 ± 0.1 ‰ for propane, −1.19 ± 0.18 ‰ for butane; εbulkH = −56.3 ± 15 ‰ for ethane, −40.5 ± 2.3 ‰ for propane, −14.6 ± 3.6 ‰ for butane. This resulted in lambda (Λ ≈ εHbulk/εCbulk) values of 16.2 ± 3.7 for ethane, 13.2 ± 0.7 for propane, and 11.4 ± 2.8 for butane. The results show that ME-CSIA can be used to track the occurrence and impact of monooxygenase-dependent aerobic processes converting short-chain alkanes in natural settings like marine and terrestrial seeps, gas reservoirs, and other geological formations impacted by natural gas

Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river

Introduction: Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Methods: Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Results: Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. Discussion: We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics

Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chen, S.-C., Ji, J., Popp, D., Jaekel, U., Richnow, H.-H., Sievert, S. M., & Musat, F. Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist. Environmental Microbiology, 24, (2022): 1964-1976, https://doi.org/10.1111/1462-2920.15956.The metabolic potential of the sulfate-reducing bacterium Desulfosarcina sp. strain BuS5, currently the only pure culture able to oxidize the volatile alkanes propane and butane without oxygen, was investigated via genomics, proteomics and physiology assays. Complete genome sequencing revealed that strain BuS5 encodes a single alkyl-succinate synthase, an enzyme which apparently initiates oxidation of both propane and butane. The formed alkyl-succinates are oxidized to CO2 via beta oxidation and the oxidative Wood–Ljungdahl pathways as shown by proteogenomics analyses. Strain BuS5 conserves energy via the canonical sulfate reduction pathway and electron bifurcation. An ability to utilize long-chain fatty acids, mannose and oligopeptides, suggested by automated annotation pipelines, was not supported by physiology assays and in-depth analyses of the corresponding genetic systems. Consistently, comparative genomics revealed a streamlined BuS5 genome with a remarkable paucity of catabolic modules. These results establish strain BuS5 as an exceptional metabolic specialist, able to grow only with propane and butane, for which we propose the name Desulfosarcina aeriophaga BuS5. This highly restrictive lifestyle, most likely the result of habitat-driven evolutionary gene loss, may provide D. aeriophaga BuS5 a competitive edge in sediments impacted by natural gas seeps.This study was financed by the Max Planck Society and by the Helmholtz Association of German Research Centres. The draft genome was sequenced as part of the U.S. Department of Energy Joint Genome Institute (DOE-JGI) Community Science Program project 1078203 awarded to S. M. Sievert and F. Musat. The work conducted by the DOE-JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Lynne Goodwin (Los Alamos National Laboratory) is acknowledged for project management support of the draft genome sequencing. Further support was provided by the U.S. National Science Foundation grant MCB-0702677 (to SMS), and by the Helmholtz Association grant ERC-RA-0020 (to FM). We acknowledge the Centre for Chemical Microscopy (ProVIS) platform at the Helmholtz Centre for Environmental Research – UFZ, for using their analytical facilities. ProVIS is supported by European Regional Development Funds (EFRE – Europe funds Saxony). We acknowledge the Bundesministerium für Bildung und Forschung (BMBF)-funded German Network for Bioinformatics Infrastructure de.NBI (031A537B, 031A533A, 031A538A, 031A533B, 031A535A, 031A537C, 031A534A, 031A532B) for providing computational resources

Intestinal dysbiosis – a new treatment target in the prevention of colorectal cancer

The gastrointestinal microbiome contains at least 100 trillion microorganisms (bacteria, viruses, fungi), whose distribution varies from the mouth to the rectum spatially and temporally throughout one\u27s lifetime. The microbiome benefits from advancing research due to its major role in human health. Studies indicate that its functions are immunity, metabolic processes and mucosal barrier. The disturbances of these functions, dysbiosis, influence physiology, lead to diabetes, inflammatory bowel disease, obesity and colon tumorigenesis. The third most common form of cancer, colorectal cancer, is the result of many factors and genes, and although the link between dysbiosis and this type of cancer is poorly characterized, it has been shown that some bacterial species and their metabolites have a critical role in developing colorectal cancer. Also, gut microbiota plays a role in the inflammatory response and immune process perturbations during the progression of colorectal cancer. Some new technologies, such as metagenome sequencing, facilitated the progress by analyzing the metabolic and genetic profile of microbiota, revealing details about the bacterial composition, host interactions, and taxonomic alterations. This review summarizes the studies regarding the link between gut microbiota and colorectal cancer, targeting new therapeutic strategies

Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl) succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O-2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons

Impact of safety-related dose reductions or discontinuations on sustained virologic response in HCV-infected patients: Results from the GUARD-C Cohort

BACKGROUND: Despite the introduction of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, peginterferon alfa/ribavirin remains relevant in many resource-constrained settings. The non-randomized GUARD-C cohort investigated baseline predictors of safety-related dose reductions or discontinuations (sr-RD) and their impact on sustained virologic response (SVR) in patients receiving peginterferon alfa/ribavirin in routine practice. METHODS: A total of 3181 HCV-mono-infected treatment-naive patients were assigned to 24 or 48 weeks of peginterferon alfa/ribavirin by their physician. Patients were categorized by time-to-first sr-RD (Week 4/12). Detailed analyses of the impact of sr-RD on SVR24 (HCV RNA <50 IU/mL) were conducted in 951 Caucasian, noncirrhotic genotype (G)1 patients assigned to peginterferon alfa-2a/ribavirin for 48 weeks. The probability of SVR24 was identified by a baseline scoring system (range: 0-9 points) on which scores of 5 to 9 and <5 represent high and low probability of SVR24, respectively. RESULTS: SVR24 rates were 46.1% (754/1634), 77.1% (279/362), 68.0% (514/756), and 51.3% (203/396), respectively, in G1, 2, 3, and 4 patients. Overall, 16.9% and 21.8% patients experienced 651 sr-RD for peginterferon alfa and ribavirin, respectively. Among Caucasian noncirrhotic G1 patients: female sex, lower body mass index, pre-existing cardiovascular/pulmonary disease, and low hematological indices were prognostic factors of sr-RD; SVR24 was lower in patients with 651 vs. no sr-RD by Week 4 (37.9% vs. 54.4%; P = 0.0046) and Week 12 (41.7% vs. 55.3%; P = 0.0016); sr-RD by Week 4/12 significantly reduced SVR24 in patients with scores <5 but not 655. CONCLUSIONS: In conclusion, sr-RD to peginterferon alfa-2a/ribavirin significantly impacts on SVR24 rates in treatment-naive G1 noncirrhotic Caucasian patients. Baseline characteristics can help select patients with a high probability of SVR24 and a low probability of sr-RD with peginterferon alfa-2a/ribavirin

Microbes trading electricity in consortia of environmental and biotechnological significance

Favorable interspecies associations prevail in natural microbial assemblages. Some of these favorable associations are co-metabolic dependent partnerships in which extracellular electrons are exchanged between species. For such electron exchange to occur, the cells must exhibit electroactive interfaces and get involved in direct cell-to-cell contact (Direct Interspecies Electron Transfer/DIET) or use available conductive mineral grains from their environment (Conductive-particle-mediated Interspecies Electron Transfer/CIET). This review will highlight recent discoveries and knowledge gaps regarding DIET and CIET interspecies associations in artificial co-cultures and consortia from natural and man-made environments and emphasize approaches to validate DIET and CIET. Additionally, we acknowledge the initiation of a movement towards applying electric syntrophies in biotechnology, bioremediation and geoengineering for natural attenuation of toxic compounds. Next, we have highlighted the urgent research needs that must be met to develop such technologies

Microbes trading electricity in consortia of environmental and biotechnological significance.

Favorable interspecies associations prevail in natural microbial assemblages. Some of these favorable associations are co-metabolic dependent partnerships in which extracellular electrons are exchanged between species. For such electron exchange to occur, the cells must exhibit electroactive interfaces and get involved in direct cell-to-cell contact (Direct Interspecies Electron Transfer/DIET) or use available conductive mineral grains from their environment (Conductive-particle-mediated Interspecies Electron Transfer/CIET). This review will highlight recent discoveries and knowledge gaps regarding DIET and CIET interspecies associations in artificial co-cultures and consortia from natural and man-made environments and emphasize approaches to validate DIET and CIET. Additionally, we acknowledge the initiation of a movement towards applying electric syntrophies in biotechnology, bioremediation and geoengineering for natural attenuation of toxic compounds. Next, we have highlighted the urgent research needs that must be met to develop such technologies