Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.     

Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.     

Ferric iron reduction-linked growth yields of Shewanella putrefaciens MR-1

The anaerobic reduction of ferric citrate by Shewanella putrefaciens MR-1 cells was inhibited markedly by p -chloromercuriphenylsulphonate, moderately by potassium cyanide, and to a small extent by 2-heptyl-4-hydroxyquinolone- N -oxide. Iron reduction was accompanied by increases in total cellular protein, with values of 0.33-7.54 g cell protein produced per mol Fe(III) reduced. The growth yields were dependent upon the growth conditions of the inoculum and the initial concentration of Fe(III) citrate in the medium. Specifically, maximum growth yields were obtained when the inoculum was pregrown anaerobically and when the initial Fe(III) citrate concentrations were 5–10 mmol l^-1. Lower growth yields were obtained with initial Fe(III) citrate concentrations of 20–30 mmol l^-1, suggesting that cell growth was partially inhibited by higher concentrations of Fe(III) or Fe(II). Maximal growth yields were also observed early (6–24 h), after which continued increases in cell protein were minimal.     

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor. Received: 26 September 1997 / Accepted: 8 December     

Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1

C.R. MYERS, B.P. CARSTENS, W.E. ANTHOLINE and J.M. MYERS.2000. Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas l -lactate or NADPH did not support any activity. The addition of 10 μmol l⁻¹ FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p -chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.     

Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.     

Replication of plasmids with the p15A origin in Shewanella putrefaciens MR-1

The plasmid pACYC184 was introduced into Shewanella putrefaciens MR-1 by electroporation. In 100% of the transformants examined, the plasmid was maintained as a free replicon outside the chromosome. This was the case whether or not the plasmid contained a 224-bp DNA insert derived from an open-reading frame of MR-1 genomic DNA. Therefore, in contrast to a report in the literature, plasmids containing the p15A origin of replication can replicate freely in S. putrefaciens MR-1, and do not make convenient vectors for gene replacement in this bacterium. However, we found that plasmids with the pMB1 origin of replication (e.g. pBR322) cannot replicate in MR-1 and could therefore have potential as vectors for gene replacement.     

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S. putrefaciens etrA is able to complement an fnr mutant of E. coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etrA mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S. putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.     

Effects of electron acceptors and donors on transformation of tetrachloromethane by Shewanella putrefaciens MR-1

Abstract Transformation of chlorinated aliphatic compounds was examined in Shewanella putrefaciens strain MR-1, an obligately respiring facultative anaerobe. Under anaerobic conditions, MR-1 has been shown to transform tetrachloromethane to trichloromethane (24%), CO₂ (7%), cell-bound material (50%) and unidentified nonvolatile products (4%). The highest rate and extent of transformation were observed with MR-1 cells grown under iron(III)-respiring conditions. Lactate, formate and hydrogen were the most effective electron donors. Tetrachloromethane was not degraded in the presence of oxygen. Transformation of other chlorinated methanes and ethenes was not observed.     

Role of menaquinone in the reduction of fumarate, nitrate, iron(III) and manganese(IV) by Shewanella putrefaciens MR-1

Abstract Mutants of Shewanella putrefaciens MR-1 deficient in menaquinone and methylmenaquinone, but which have wild-type levels of ubiquinone, retain the ability to use trimethylamine N -oxide as an electron acceptor, but they lose the ability to use nitrate, iron(III), and fumarate as electron acceptors. These mutants also show a reduced rate of manganese(IV) reduction. One of these mutants could be restored to essentially wild-type phenotype by supplementing the medium with 1,4-dihydroxy-2-naphthoic acid. A requirement for naphthoquinones in iron(III) reduction and a preference for naphthoquinones in manganese(IV) reduction provide further support that the metal reducing systems in MR-1 are linked to anaerobic respiration.     

一株奥奈达希瓦氏菌烈性噬菌体的分离、纯化及鉴定

【目的】从环境中分离获得希瓦氏菌烈性噬菌体,并对其性质进行研究。【方法】以4株希瓦氏菌为宿主菌,采用双层平板法从污水样品中分离得到奥奈达希瓦氏菌MR-1烈性噬菌体M1;观察噬菌斑特征;利用超速离心法浓缩M1颗粒,进一步用氯化铯密度梯度离心纯化;采用透射电子显微镜观察纯化的M1颗粒;提取M1核酸,通过核酸酶处理分析其核酸类型及结构;绘制一步生长曲线。【结果】噬菌体M1在双层平板上形成圆形的噬菌斑,清晰透明,边缘光滑,直径为2.3 mm-2.5 mm;经电镜观察,噬菌体M1头部呈二十面体,直径约为55 nm,尾长约为170 nm,尾部可收缩,属于肌尾噬菌体科(Myoviridae);通过酶切分析表明噬菌体M1核酸为线形双链DNA;一步生长曲线显示该噬菌体感染后完成一个复制循环所需要的时间约为15-20 min。【结论】噬菌体M1属肌尾噬菌体科,研究结果为后续研究病毒在地球微生物成岩过程中所起的作用提供了实验材料。     

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens AU63 deficient in antifungal activity against Pythium ultimum

Tn5 transposon mutagenesis via electroporation of Pseudomonas fluorescens AU63 was used to generate mutants deficient in antifungal activity against the phytopathogenic fungi Pythium ultimum and Thielaviopsis basicola. Mutant C-45 was obtained by an initial screen for the loss of antibacterial activity against Bacillus subtilis and a subsequent screen of mutants obtained for the loss of antifungal activity against pathogenic fungi. A single chromosomal insertion of Tn5 in the chromosome of Ps. fluorescens C-45 was confirmed by Southern blot hybridization. A metabolite responsible for the observed antibacterial and antifungal activities was identified using thin layer chromatography. The antimicrobial activities of the partially purified substance present in the parental strain and missing in the C-45 mutant were not affected by protease, high temperature, acid or alkali treatment. These results provide the basis for a structural analysis of this new antimicrobial substance and the genetic elucidation of its biosynthesis.     

Localization of cytochromes to the outer membrane of anaerobically grown Shewanella putrefaciens MR-1.

In gram-negative bacteria, numerous cell functions, including respiration-linked electron transport, have been ascribed to the cytoplasmic membrane. Gram-negative bacteria which use solid substrates (e.g., oxidized manganese or iron) as terminal electron acceptors for anaerobic respiration are presented with a unique problem: they must somehow establish an electron transport link across the outer membrane between large particulate metal oxides and the electron transport chain in the cytoplasmic membrane. When the metal-reducing bacterium Shewanella putrefaciens MR-1 is grown under anaerobic conditions and membrane fractions are purified from cells lysed by an EDTA-lysozyme-polyoxyethylene cetyl ether (Brij 58) protocol, approximately 80% of its membrane-bound cytochromes are localized in its outer membrane. These outer membrane cytochromes could not be dislodged by treatment with chaotropic agents or by increased concentrations of the nonionic detergent Brij 58, suggesting that they are integral membrane proteins. Cytochrome distribution in cells lysed by a French press protocol confirm the localization of cytochromes to the outer membrane of anaerobically grown cells. This novel cytochrome distribution could play a key role in the anaerobic respiratory capabilities of this bacterium, especially in its ability to mediate manganese and iron reduction.     

MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c-type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c-type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.