To understand the energy conversion activities of the anaerobic sulfate-reducing bacteria, it is necessary to identify the components involved in electron flow. The importance of the abundant type I tetraheme cytochrome c 3 (TpIc 3 ) as an electron carrier during sulfate respiration was questioned by the previous isolation of a null mutation in the gene encoding TpIc 3 , cycA, in Desulfovibrio alaskensis G20. Whereas respiratory growth of the CycA mutant with lactate and sulfate was little affected, growth with pyruvate and sulfate was significantly impaired. We have explored the phenotype of the CycA mutant through physiological tests and transcriptomic and proteomic analyses. Data reported here show that electrons from pyruvate oxidation do not reach adenylyl sulfate reductase, the enzyme catalyzing the first redox reaction during sulfate reduction, in the absence of either CycA or the type I cytochrome c 3 :menaquinone oxidoreductase transmembrane complex, QrcABCD. In contrast to the wild type, the CycA and QrcA mutants did not grow with H 2 or formate and sulfate as the electron acceptor. Transcriptomic and proteomic analyses of the CycA mutant showed that transcripts and enzymes for the pathway from pyruvate to succinate were strongly decreased in the CycA mutant regardless of the growth mode. Neither the CycA nor the QrcA mutant grew on fumarate alone, consistent with the omics results and a redox regulation of gene expression. We conclude that TpIc 3 and the Qrc complex are D. alaskensis components essential for the transfer of electrons released in the periplasm to reach the cytoplasmic adenylyl sulfate reductase and present a model that may explain the CycA phenotype through confurcation of electrons. E nvironmental impacts of the sulfate-reducing bacteria (SRB), such as the formation of toxic sulfide and corrosion of metals, stem from SRB sulfate respiration and from vigorous metal metabolism, including both reduction and oxidation. The involvement of these bacteria in microbially influenced metal corrosion (1, 2) and their possible application for the remediation of toxic metal-contaminated environments (3, 4) have driven a systems biology investigation of their metabolism. To predict or control metabolic capabilities for beneficial environmental purposes, the bioenergetic pathways of the SRB need to be elucidated in more detail.Typically, SRB of the genus Desulfovibrio use H 2 , organic acid substrates, formate, or short-chain alcohols as electron donors for sulfate reduction, a process that occurs in the cytoplasm and is mediated by soluble enzymes. With hydrogen as the source of electrons, at an environmentally relevant partial pressure of 10 Pa (reduction potential [E=] ϭ Ϫ300 mV) (5), the first two-electron reduction of sulfate to sulfite (E 0 = ϭ Ϫ516 mV) cannot be accomplished without an additional energy input. Sulfate is activated at the expense of two ATP equivalents through the activity of sulfate adenylyltransferase (equation 1), producing adenosine phosphosulfate (APS), which increases th...
The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically,Desulfovibrio vulgarisHildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
The role of tetraheme cytochrome c 3 (CycA) in the metabolism of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was investigated by deletion of the cycA gene using a marker-exchange deletion strategy. A highly abundant periplasmic cytochrome, CycA has the important function of transferring electrons from periplasmic hydrogenases (Hyd, Hyn, Hys) to transmembrane complexes which transport the electrons to the cytoplasm where sulfate is reduced. Previous studies have indicated that during its interaction with periplasmic hydrogenases, CycA is also involved in the reduction of toxic metals.Growth of the cycA mutant strain on lactate as the electron donor and sulfate as the terminal electron acceptor showed that, despite its abundance, CycA is not essential for DvH growth. However, the rate of growth of the mutant strain was significantly lower, and the extent of growth less, than rates and extents of growth of the wild type and complement strains on lactate/sulfate medium. This indicates that a portion of the electrons generated from cytoplasmic lactate oxidation are transported by CycA for energy production, possibly in a hydrogen cycling mechanism employed to generate ATP. Failure of the mutant strain to grow on either formate or H 2 , with sulfate or sulfite as electron acceptors, further indicated that CycA may be the only redox partner of periplasmic hydrogenases.The cycA mutant strain also did not grow as well as either the wild type or complement strains on medium supplemented with pyruvate/sulfate. Final growth on pyruvate/sulfate was comparable, but the mutant grew more slowly than the wild type and complement strains. Interestingly, the mutant grew better than the wild type or complement strains on pyruvate alone, possibly due to the release of H 2 and/or CO 2 in concentrations which may be somewhat inhibitory to wild type growth.
This study evaluates the efficiency of a full‐scale, 81 m‐wide permeable reactive barrier (PRB) configured by injection of dairy whey in the downgradient region of a contaminant source zone to enhance the in situ biodegradation of high concentrations (102 to 103μg/L) of chlorinated ethenes (CEs). Ten biannual whey injections were completed in a 3.5‐year pilot phase and 1.5‐year operational phase. Improved and sustained dechlorination was observed at extraction/injection and downgradient wells in the fully‐operational phase, when dried whey masses were increased from 13.6 kg to 230–360 kg, whey slurry volumes were increased from 2300 L to 307,000–480,000 L, and extraction/injection well loops were employed for the application of whey. At extraction/injection wells, CEs decreased to low (≤10 μg/L) or undetectable levels. At downgradient wells, average trichloroethene concentrations decreased, by as much as 100% (from ≤384.2 during the pilot phase to ≤102.6 μg/L during the operational phase), while average cis‐dichloroethene concentrations decreased by as much as 57.5% (from ≤6466.1 to ≤4912.2 μg/L). Downgradient vinyl chloride averages either increased by as much as 63.8% (from ≤859.6 to ≤1407.9 μg/L) or decreased by 64.0% (from 1375.4 to 880 μg/L). Downgradient ethene + ethane averages increased by as much as 73.2% (from ≤1145.3 to ≤1347.1 μg/L). On the basis of the 2008 average market price, the estimated material cost of whey is $1.96/kg organic carbon or, for the configuration of an 81 m PRB by biannual application of 300 kg whey, $325/year. Carbon substrate cost comparisons and implications for efficient in situ treatment design are discussed.
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