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Lysine-91 of the tetraheme c-type cytochrome CymA is essential for quinone interaction and arsenate respiration in Shewanella sp. strain ANA-3.

ABSTRACT: The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH(2)) substrates. Fluorescence quench titration with the MQH(2) analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a K (d) of 0.1-1 microM. It was also shown that the redox active MQH(2) analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH(2)), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH(2)-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A DeltacymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome.

SUBMITTER: Zargar K

PROVIDER: S-EPMC2762525 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The cymA gene, encoding a tetraheme c-type cytochrome, is required for arsenate respiration in Shewanella species.

Project description:In Shewanella sp. strain ANA-3, utilization of arsenate as a terminal electron acceptor is conferred by a two-gene operon, arrAB, which lacks a gene encoding a membrane-anchoring subunit for the soluble ArrAB protein complex. Analysis of the genome sequence of Shewanella putrefaciens strain CN-32 showed that it also contained the same arrAB operon with 100% nucleotide identity. Here, we report that CN-32 respires arsenate and that this metabolism is dependent on arrA and an additional gene encoding a membrane-associated tetraheme c-type cytochrome, cymA. Deletion of cymA in ANA-3 also eliminated growth on and reduction of arsenate. The DeltacymA strains of CN-32 and ANA-3 negatively affected the reduction of Fe(III) and Mn(IV) but not growth on nitrate. Unlike the CN-32 DeltacymA strain, growth on fumarate was absent in the DeltacymA strain of ANA-3. Both homologous and heterologous complementation of cymA in trans restored growth on arsenate in DeltacymA strains of both CN-32 and ANA-3. Transcription patterns of cymA showed that it was induced under anaerobic conditions in the presence of fumarate and arsenate. Nitrate-grown cells exhibited the greatest level of cymA expression in both wild-type strains. Lastly, site-directed mutagenesis of the first Cys to Ser in each of the four CXXCH c-heme binding motifs of the CN-32 CymA nearly eliminated growth on and reduction of arsenate. Together, these results indicate that the biochemical mechanism of arsenate respiration and reduction requires the interactions of ArrAB with a membrane-associated tetraheme cytochrome, which in the non-arsenate-respiring Shewanella species Shewanella oneidensis strain MR-1, has pleiotropic effects on Fe(III), Mn(IV), dimethyl sulfoxide, nitrate, nitrite, and fumarate respiration.

| S-EPMC1899399 | biostudies-literature

The ArsR repressor mediates arsenite-dependent regulation of arsenate respiration and detoxification operons of Shewanella sp. strain ANA-3.

Project description:Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (arr) and detoxifying (ars) reduction pathways in Shewanella sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking arsR2 grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of arrA and arsC (arsenate reductase-encoding genes) in the DeltaarsR2 mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the arr promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the arr operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the arr promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of arr and ars operons in Shewanella sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP.

| S-EPMC2795299 | biostudies-literature

Characterization of the arsenate respiratory reductase from Shewanella sp. strain ANA-3.

Project description:Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of approximately 131 kDa, composed of one ArrA subunit (approximately 95 kDa) and one ArrB subunit (approximately 27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41 degrees C, a Km of 5 microM, and a Vmax of 11,111 micromol of As(V) reduced min(-1) mg of protein(-1) and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family.

| S-EPMC2223751 | biostudies-literature

Molecular Underpinnings of Nitrite Effect on CymA-Dependent Respiration in Shewanella oneidensis.

Project description:Shewanella exhibit a remarkable versatility of respiration, with a diverse array of electron acceptors (EAs). In environments where these bacteria thrive, multiple EAs are usually present. However, we know little about strategies by which these EAs and their interaction affect ecophysiology of Shewanella. In this study, we demonstrate in the model strain, Shewanella oneidensis MR-1, that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate, and probably many other EAs whose reduction depends on quinol dehydrogenase CymA. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass.

| S-EPMC4954811 | biostudies-literature

Functional roles of arcA, etrA, cyclic AMP (cAMP)-cAMP receptor protein, and cya in the arsenate respiration pathway in Shewanella sp. strain ANA-3.

Project description:Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals--a process that can cause arsenic contamination of water. In Shewanella sp. strain ANA-3, the arsenate respiration genes (arrAB) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator (arcA and etrA [fnr homolog]) and carbon catabolite repression (crp and cya) proteins affect arsenate respiration in ANA-3. Transcription of arcA, etrA, and crp in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking arcA and etrA showed minor to moderate growth defects, respectively, with arsenate. However, crp was essential for growth on arsenate. In contrast to the wild-type strain, arrA was not induced in the crp mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates arr operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the arr promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (cya) genes were identified in the genome. One particular cya-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the cya-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double cya mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in Shewanella sp. strain ANA-3.

| S-EPMC2632105 | biostudies-literature

Expression dynamics of arsenic respiration and detoxification in Shewanella sp. strain ANA-3.

Project description:Because arsenate [As(V)] reduction by bacteria can significantly enhance arsenic mobility in the environment, it is important to be able to predict when this activity will occur. Currently, two bacterial systems are known that specifically reduce As(V), namely, a respiratory system (encoded by the arr genes) and a detoxification system (encoded by the ars genes). Here we analyze the conditions under which these two systems are expressed in Shewanella sp. strain ANA-3. The ars system is expressed under both aerobic and anaerobic conditions, whereas the arr system is only expressed anaerobically and is repressed by oxygen and nitrate. When cells are grown on As(V), the arr system is maximally induced during exponential growth, with peak expression of the ars system occurring at the beginning of stationary phase. Both the arr and ars systems are specifically induced by arsenite [As(III)], but the arr system is activated by a concentration of As(III) that is 1,000 times lower than that required for the arsC system (< or =100 nM versus < or =100 microM, respectively). A double mutant was constructed that does not reduce As(V) under any growth conditions. In this strain background, As(V) is capable of inducing the arr system at low micromolar concentrations, but it does not induce the ars system. Collectively, these results demonstrate that the two As(V) reductase systems in ANA-3 respond to different amounts and types of inorganic arsenic.

| S-EPMC1272973 | biostudies-literature

Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1.

Project description:The cymA gene, which encodes a tetraheme cytochrome c, was cloned from Shewanella putrefaciens MR-1. This gene complemented a mutant which had a TnphoA insertion in cymA and which was deficient in the respiratory reduction of iron(III), nitrate, fumarate, and manganese(IV). The 561-bp nucleotide sequence of cymA encodes a protein of 187 amino acids with a predicted molecular mass of 20.8 kDa. No N-terminal signal sequence was readily apparent; consistent with this, a cytochrome with a size of 21 kDa was detected in the wild type but was absent in the insertional mutant. The cymA gene is transcribed into an mRNA; the major transcript was approximately 790 bases, suggesting that it is not part of a multicistronic operon. This RNA transcript was not detected in the cymA mutant. The CymA protein was found in the cytoplasmic membrane and soluble fraction of MR-1, and it shares partial amino acid sequence homology with multiheme c-type cytochromes from other bacteria. These cytochromes are ostensibly involved in the transfer of electrons from the cytoplasmic membrane to acceptors in the periplasm. The localization of the fumarate and iron(III) reductases to the periplasm and outer membrane of MR-1, respectively, suggests the possibility of a similar electron transfer role for CymA.

| S-EPMC178810 | biostudies-other

Mutational and gene expression analysis of mtrDEF, omcA and mtrCAB during arsenate and iron reduction in Shewanella sp. ANA-3.

Project description:Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA-3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA-3) compared with wild-type ANA-3. However, the presence of arsenate adsorbed onto ferrihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild-type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA-3), arsenate was reduced similarly to wild-type ANA-3 indicating the Fe(III) reduction pathway is not required for ferrihydrite-associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA-3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre-adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsenate can co-induce operons (arr and mtr) implicated in arsenic mobilization.

| S-EPMC4559585 | biostudies-literature

Partial functional replacement of CymA by SirCD in Shewanella oneidensis MR-1.

Project description:The gammaproteobacterium Shewanella oneidensis MR-1 utilizes a complex electron transfer network composed primarily of c-type cytochromes to respire under anoxic conditions a variety of compounds, including fumarate, nitrate, and dimethyl sulfoxide (DMSO), in addition to the minerals Fe(III) and Mn(IV). Central to several respiratory pathways is CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome that functions as the major hydroquinone dehydrogenase. To investigate functional redundancy and plasticity in S. oneidensis MR-1 electron transport, we isolated ?cymA suppressor mutants and characterized one biochemically and genetically. Interestingly, in the characterized ?cymA suppressor mutant, respiration of fumarate, ferric citrate, and DMSO was restored but that of nitrate was not. The suppression was found to be due to transcriptional activation of sirC and sirD, encoding a periplasmic iron sulfur protein and an integral membrane hydroquinone dehydrogenase, respectively. Biochemical in vitro reconstitution experiments confirmed electron transport between formate and fumarate via fumarate reductase by suppressor membrane fractions. The suppression was found to be caused by insertion of an ISSod1 element upstream of the sirCD transcriptional start site, generating a novel, constitutively active hybrid promoter. This work revealed that adaptation of an alternative electron transfer pathway from quinol to terminal oxidoreductases independent of CymA occurs rapidly in S. oneidensis MR-1.

| S-EPMC3133100 | biostudies-literature

The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3.

Project description:Arsenate [As(V); HAsO(4)(2-)] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO(2)]. The generation time and lactate molar growth yield (Y(lactate)) are 2.8 h and 10.0 g of cells mol of lactate(-1), respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y(lactate) value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.

| S-EPMC154534 | biostudies-literature

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