Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porincytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.This work was supported by Wageningen University & Research through its investment theme Resilience, the Technology Foundation (STW), the Applied Science Division of the Dutch Research Council (NWO; project 08053), NWO grant 016.Vidi.189.050, and a Gravitation grant of the Netherlands Ministry of Education, Culture and Science and NWO (project 024.002.002 SIAM). B.K. was supported by the Villum foundation, Denmark (VYI Grant 25491).info:eu-repo/semantics/publishedVersio

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a -hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to -hydroxybutyrate that may be converted to the energy and carbon storage compound, poly--hydroxybutyrate. Accordingly, we confirmed the formation of poly-?-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.This work was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO) [project 08053]. Additional funding was provided by BE-BASIC [grant F08.004.01 to SA], an ERC grant [project 323009 to AJMS] and the Gravitation grant [project 024.002.002 to AJMS] of the Netherlands Ministry of Education, Culture and Science and NWO

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Plant-wide systems microbiology for the wastewater industry

The wastewater treatment sector embraces mixed-culture biotechnologies for sanitation, environmental protection, and resource recovery. Bioprocess design, monitoring and control thrive on microbial processes selected in complex microbial communities. Microbial ecology and systems microbiology help access microbiomes and characterize microorganisms, metabolisms and interactions at increased resolution and throughput. Big datasets are generated from the sequencing of informational molecules extracted from biomasses sampled across process schemes. However, they mostly remain on science benches and computing clusters, without reaching the industry in a clear engineering objective function. A bilateral bridge should actionize this information. As systems microbiologists, we miss that engineering designs and operations rely on stoichiometry and kinetics. The added-value provided by microbial ecology and systems microbiology to improve capital (CAPEX) and operating expenditures (OPEX) needs to be addressed. As engineers, we miss that microbiology can be provide powerful microbial information on top of physical-chemical measurements for quantitative process design (e.g., nutrient removal systems) with detailed scientific description of phenomena inside microbiomes. In this perspective article, we allied academia and industry to address the state of shared knowledge, successes and failures, and to establish joint investigation platforms. Our roadmap involves three milestones to (i) elaborate an essential list of microbiological information needed to implement methods at the process line; (ii) characterize microbiomes from microorganisms to metabolisms, and shape conceptual ecosystem models as primer for process ecology understanding; (iii) bridge engineering and mathematical models with an analytical toolbox for fast- vs. high-throughput analyses to discover new microbial processes and engineer assemblies. We praise for a harmonized "language of love"(incorporating common vocabulary, units, protocols) across the water and environmental biotechnology sector to team up mindsets for a sewer- and plant-wide integration of systems microbiology and engineering.BT/Environmental Biotechnolog

Plant-Wide Systems Microbiology for the Wastewater Industry

The wastewater treatment sector embraces mixed-culture biotechnologies for sanitation, environmental protection, and resource recovery. Bioprocess design, monitoring and control thrive on microbial processes selected in complex microbial communities. Microbial ecology and systems microbiology help access microbiomes and characterize microorganisms, metabolisms and interactions at increased resolution and throughput. Big datasets are generated from the sequencing of informational molecules extracted from biomasses sampled across process schemes. However, they mostly remain on science benches and computing clusters, without reaching the industry in a clear engineering objective function. A bilateral bridge should actionize this information. As systems microbiologists, we miss that engineering designs and operations rely on stoichiometry and kinetics. The added-value provided by microbial ecology and systems microbiology to improve capital (CAPEX) and operating expenditures (OPEX) needs to be addressed. As engineers, we miss that microbiology can be more powerful than physical-chemical measurements for quantitative process design (e.g., nutrient removal systems) with detailed scientific description of phenomena inside microbiomes. In this perspective article, we allied academia and industry to address the state of shared knowledge, successes and failures, and to establish joint investigation platforms. Our roadmap involves three milestones to (i) elaborate an essential list of microbiological information needed to implement methods at process line; (ii) characterize microbiomes from microorganisms to metabolisms, and shape conceptual ecosystem models as primer for process ecology understanding; (iii) bridge engineering and mathematical models with an analytical toolbox for fast- vs. high-throughput analyses to discover new microbial processes and engineer assemblies. We praise for a harmonized “language of love” (incorporating common vocabulary, units, protocols) across the water and environmental biotechnology sector to team up mindsets for a sewer- and plant-wide integration of systems microbiology and engineering

Genome Sequences of Alicycliphilus denitrificans Strains BC and K601T▿

Alicycliphilus denitrificans strain BC and A. denitrificans strain K601T degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601T but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of A. denitrificans strains BC and K601T were sequenced. Here, we report the complete genome sequences of A. denitrificans strains BC and K601T

Degradation of benzene (1), toluene (2) and acetate (3) with chlorate (a), nitrate (b) or oxygen (c) as electron acceptor by <i>A. denitrificans</i> strain BC.

<p>Benzene, toluene and acetate degradation is indicated with diamonds. Benzene and toluene concentrations are outlined on a secondary y-axis while acetate and electron acceptor concentrations are indicated on the primary y-axis. Chlorate, nitrate and oxygen consumption is depicted with squares. Chloride production when chlorate is used as electron acceptor is indicated with triangles and no electron acceptor consumption is shown when no significant difference could be observed because of presence of the electron acceptor in abundance.</p

Main metabolic pathways of <i>A. denitrificans</i>.

<p>Pathways are indicated using arrows. Black arrows indicate pathways of both strain BC and K601^T, red arrows indicate pathways of strain BC, and blue arrows pathways of strain K601^T. Red gene numbers indicate genes of strain BC (geneID is Alide_red gene number) and blue gene numbers genes of strain K601^T (geneID is Alide2_blue gene number).</p

General features of the genomes of <i>A. denitrificans</i> strains BC and K601^T.

<p>General features of the genomes of <i>A. denitrificans</i> strains BC and K601^T.</p