Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c -type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N -oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.     

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.     

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.     

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.     

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.     

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.     

Deciphering the electron transport pathway for graphene oxide reduction by Shewanella oneidensis MR-1

We determined that graphene oxide reduction by Shewanella oneidensis MR-1 requires the Mtr respiratory pathway by analyzing a range of mutants lacking these proteins. Electron shuttling compounds increased the graphene oxide reduction rate 3- to 5-fold. These results may help facilitate the use of bacteria for large-scale graphene production.     

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.     

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.     

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。     

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>MR-1

Background  

EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.     

The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1

We hypothesized that Shewanella oneidensis MR-1, a model dissimilatory metal-reducing bacterium, could utilize environmentally relevant concentrations of tyrosine to produce pyomelanin for enhanced Fe(III) oxide reduction. Because homogentisate is an intermediate of the tyrosine degradation pathway, and a precursor of a redox-cycling metabolite, pyomelanin, we evaluated the process of homogentisate production by S. oneidensis MR-1, in order to identify the key steps involved in pyomelanin production. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. We used genetic analysis and physiological characterization of MR-1 strains either deficient in or displaying substantially increased pyomelanin production. The relative significance imparted by pyomelanin on solid-phase electron transfer was also addressed using electrochemical techniques, which allowed us to extend the genetic and physiological findings to biogeochemical cycling of metals. Based on our findings, environmental production of pyomelanin from available organic precursors could contribute to the survival of S. oneidensis MR-1 when dissolved oxygen concentrations become low, by providing an increased capacity for solid-phase metal reduction. This study demonstrates the role of organic precursors and their concentrations in pyomelanin production, solid phase metal reduction and biogeochemical cycling of iron.     

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.     

MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1

When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO2) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg2+, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.