Role of menaquinones in Fe(III) reduction by membrane fractions of Shewanella putrefaciens.
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SUBMITTER: Saffarini DA
PROVIDER: S-EPMC139511 | biostudies-literature | 2002 Feb
REPOSITORIES: biostudies-literature
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Role of menaquinones in Fe(III) reduction by membrane fractions of Shewanella putrefaciens.
Saffarini Daad A DA Blumerman Seth L SL Mansoorabadi Karen J KJ
Journal of bacteriology 20020201 3
Two Tn5-generated mutants of Shewanella putrefaciens with insertions in menD and menB were isolated and analyzed. Both mutants were deficient in the use of several terminal electron acceptors, including Fe(III). This deficiency was overcome by the addition of menaquinone (vitamin K(2)). Isolated membrane fractions from both mutants were unable to reduce Fe(III) in the absence of added menaquinone when formate was used as the electron donor. These results indicate that menaquinones are essential ...[more]
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Project description:Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.
| S-EPMC107715 | biostudies-literature
Project description:Shewanella putrefaciens MR-1 can use a wide variety of terminal electron acceptors for anaerobic respiration, including certain insoluble manganese and iron oxides. To examine whether the outer membrane (OM) cytochromes of MR-1 play a role in Mn(IV) and Fe(III) reduction, mutants lacking the OM cytochrome OmcA or OmcB were isolated by gene replacement. Southern blotting and PCR confirmed replacement of the omcA and omcB genes, respectively, and reverse transcription-PCR analysis demonstrated loss of the respective mRNAs, whereas mRNAs for upstream and downstream genes were retained. The omcA mutant (OMCA1) resembled MR-1 in its growth on trimethylamine N-oxide (TMAO), dimethyl sulfoxide, nitrate, fumarate, thiosulfate, and tetrathionate and its reduction of nitrate, nitrite, ferric citrate, FeOOH, and anthraquinone-2,6-disulfonic acid. Similarly, the omcB mutant (OMCB1) grew on fumarate, nitrate, TMAO, and thiosulfate and reduced ferric citrate and FeOOH. However, OMCA1 and OMCB1 were 45 and 75% slower than MR-1, respectively, at reducing MnO(2). OMCA1 lacked only OmcA. While OMCB1 lacked OmcB, other OM cytochromes were also missing or markedly depressed. The total cytochrome content of the OM of OMCB1 was less than 15% of that of MR-1. Western blots demonstrated that OMCB1 still synthesized OmcA, but most of it was localized in the cytoplasmic membrane and soluble fractions rather than in the OM. OMCB1 had therefore lost the ability to properly localize multiple OM cytochromes to the OM. Together, the results suggest that the OM cytochromes of MR-1 participate in the reduction of Mn(IV) but are not required for the reduction of Fe(III) or other electron acceptors.
| S-EPMC92560 | biostudies-literature
Project description:Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).
| S-EPMC134750 | biostudies-literature
Project description:Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 -a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment.
| S-EPMC4839622 | biostudies-literature
Project description:In the absence of O(2) and other electron acceptors, the Gram-negative bacterium Shewanella oneidensis MR-1 can use ferric [Fe(III)] (oxy)(hydr)oxide minerals as the terminal electron acceptors for anaerobic respiration. At circumneutral pH and in the absence of strong complexing ligands, Fe(III) oxides are relatively insoluble and thus are external to the bacterial cells. S. oneidensis MR-1 and related strains of metal-reducing Shewanella have evolved machinery (i.e., metal-reducing or Mtr pathway) for transferring electrons from the inner-membrane, through the periplasm and across the outer-membrane to the surface of extracellular Fe(III) oxides. The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC, and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt) that belongs to the NapC/NrfH family of quinol dehydrogenases. It is proposed that CymA oxidizes the quinol in the inner-membrane and transfers the released electrons to MtrA either directly or indirectly through other periplasmic proteins. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons through the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. MtrC and OmcA are the outer-membrane decaheme c-Cyts that are translocated across the outer-membrane by the bacterial type II secretion system. Functioning as terminal reductases, MtrC and OmcA can bind the surface of Fe(III) oxides and transfer electrons directly to these minerals via their solvent-exposed hemes. To increase their reaction rates, MtrC and OmcA can use the flavins secreted by S. oneidensis MR-1 cells as diffusible co-factors for reduction of Fe(III) oxides. Because of their extracellular location and broad redox potentials, MtrC and OmcA can also serve as the terminal reductases for soluble forms of Fe(III). In addition to Fe(III) oxides, Mtr pathway is also involved in reduction of manganese oxides and other metals. Although our understanding of the Mtr pathway is still far from complete, it is the best characterized microbial pathway used for extracellular electron exchange. Characterizations of the Mtr pathway have made significant contributions to the molecular understanding of microbial reduction of Fe(III) oxides.
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Project description:BackgroundShewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.ResultsOmp35 was purified from the OM of anaerobically grown cells, the gene encoding Omp35 was identified, and an omp35 null mutant (OMP35-1) was isolated and characterized. Although OMP35-1 grew on all electron acceptors tested, a significant lag was seen when grown on fumarate, nitrate, and Fe(III). Complementation studies confirmed that the phenotype of OMP35-1 was due to the loss of Omp35. Despite its requirement for wild-type rates of electron acceptor use, analysis of Omp35 protein and predicted sequence did not identify any electron transport moieties or predicted motifs. OMP35-1 had normal levels and distribution of known electron transport components including quinones, cytochromes, and fumarate reductase. Omp35 is related to putative porins from MR-1 and S. frigidimarina as well as to the PorA porin from Neisseria meningitidis. Subcellular fraction analysis confirmed that Omp35 is an OM protein. The seven-fold anaerobic upregulation of Omp35 is mediated post-transcriptionally.ConclusionOmp35 is a putative porin in the OM of MR-1 that is markedly upregulated anaerobically by a post-transcriptional mechanism. Omp35 is required for normal rates of growth on Fe(III), fumarate, and nitrate, but its absence has no effect on the use of other electron acceptors. Omp35 does not contain obvious electron transport moieties, and its absence does not alter the amounts or distribution of other known electron transport components including quinones and cytochromes. The effects of Omp35 on anaerobic electron acceptor use are therefore likely indirect. The results demonstrate the ability of non-electron transport proteins to influence anaerobic respiratory phenotypes.
| S-EPMC459216 | biostudies-literature
Project description:Microbes play a critical role in the global arsenic biogeocycle. Most studies have focused on redox cycling of inorganic arsenic in bacteria and archaea. The parallel cycles of organoarsenical biotransformations are less well characterized. Here we describe organoarsenical biotransformations in the environmental microbe Shewanella putrefaciens. Under aerobic growth conditions, S. putrefaciens reduced the herbicide MSMA (methylarsenate or MAs(V)) to methylarsenite (MAs(III)). Even though it does not contain an arsI gene, which encodes the ArsI C-As lyase, S. putrefaciens demethylated MAs(III) to As(III). It cleaved the C-As bond in aromatic arsenicals such as the trivalent forms of the antimicrobial agents roxarsone (Rox(III)), nitarsone (Nit(III)) and phenylarsenite (PhAs(III)), which have been used as growth promoters for poultry and swine. S. putrefaciens thiolated methylated arsenicals, converting MAs(V) into the more toxic metabolite monomethyl monothioarsenate (MMMTAs(V)), and transformed dimethylarsenate (DMAs(V)) into dimethylmonothioarsenate (DMMTAs(V)). It also reduced the nitro groups of Nit(V), forming p-aminophenyl arsenate (p-arsanilic acid or p-AsA(V)), and Rox(III), forming 3-amino-4-hydroxybenzylarsonate (3A4HBzAs(V)). Elucidation of organoarsenical biotransformations by S. putrefaciens provides a holistic appreciation of how these environmental pollutants are degraded.
| S-EPMC4984541 | biostudies-literature
Project description:BackgroundThe completion of genome sequencing in a number of Shewanella species, which are most renowned for their metal reduction capacity, offers a basis for comparative studies. Previous work in Shewanella oneidensis MR-1 has indicated that some genes within a cluster (mtrBAC-omcA-mtrFED) were involved in iron reduction. To explore new features of iron reduction pathways, we experimentally analyzed Shewanella putrefaciens W3-18-1 since its gene cluster is considerably different from that of MR-1 in that the gene cluster encodes only four ORFs.ResultsAmong the gene cluster, two genes (mtrC and undA) were shown to encode c-type cytochromes. The ?mtrC deletion mutant revealed significant deficiencies in reducing metals of Fe2O3, ?-FeO(OH), ?-FeO(OH), ferric citrate, Mn(IV) and Co(III), but not organic compounds. In contrast, no deficiency of metal reduction was observed in the ?undA deletion mutant. Nonetheless, undA deletion resulted in progressively slower iron reduction in the absence of mtrC and fitness loss under the iron-using condition, which was indicative of a functional role of UndA in iron reduction.ConclusionsThese results provide physiological and biochemical evidences that UndA and MtrC of Shewanella putrefaciens W3-18-1 are involved in iron reduction.
| S-EPMC4222724 | biostudies-literature
Project description:A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37 degrees C, with an optimum growth temperature of 18 degrees C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37 degrees C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation.
| S-EPMC1472395 | biostudies-literature
Project description:Pelobacter carbinolicus is phylogenetically intertwined with the Geobacteraceae, a family of deltaproteobacteria which couple oxidation of organic compounds to Fe(III) reduction. Whereas Geobacter species completely oxidize organic compounds to CO2, Pelobacter species either ferment or oxidize short chain alcohols to acetate. Pelobacter species also contain far fewer c-type cytochromes, proteins which play a role in electron transfer during Fe(III) respiration, compared to their Geobacter counterparts. Keywords: two-condition comparison
2008-05-22 | GSE8338 | GEO