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Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

ABSTRACT: The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectroscopy to determine the ability of donating electrons to chlorate reductase. Our results show that one of the c cytochromes (6 kDa) is able to donate electrons, both to chlorate reductase and to the membrane-bound cytochrome c oxidase, whereas the roles of the remaining c cytochromes still remain to be elucidated. Peptide extracts of the c cytochromes were obtained by tryptic in-gel digestion for matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Peptide sequences obtained indicate that the 6-kDa cytochrome c protein is similar to c cytochromes from the chlorate-reducing bacterium Dechloromonas aromatica.

SUBMITTER: Backlund AS

PROVIDER: S-EPMC2675203 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

Bäcklund Anna Smedja AS Bohlin Jan J Gustavsson Niklas N Nilsson Thomas T

Applied and environmental microbiology 20090220 8

The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectr ...[more]

PMID: 19233956

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Project description:The genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains (Ideonella dechloratans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobilized at least once from a perchlorate reducer to build chlorate respiration. Genome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction.

Project description:Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1T, a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI, encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle.IMPORTANCE Halogenated organic and inorganic compounds are important environmental pollutants that have carcinogenic and genotoxic effects on both animals and humans. Previous research studied the degradation of organic and inorganic halogenated compounds separately but not concurrently. This study shows concurrent degradation of halogenated alkanoates and chlorate as an electron donor and acceptor, respectively, coupled to growth in a single bacterium, Pseudomonas chloritidismutans AW-1T Hence, besides biogenesis of molecular oxygen from chlorate reduction enabling a distinctive placement of strain AW-1T between aerobic and anaerobic microorganisms, we can now add another unique metabolic potential of this bacterium to the roster. The degradation of different halogenated compounds under anoxic conditions by a single bacterium is also of interest for the natural halogen cycle in different aquatic and terrestrial ecosystems where ample natural production of halogenated compounds has been documented.

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Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

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Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

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