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PubMed2001

Isolation and characterization of a Shewanella putrefaciens MR-1 electron transport regulator etrA mutant: reassessment of the role of EtrA.

Shewanella putrefaciens MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O(2). It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from Escherichia coli, may regulate gene expression for anaerobic electron transport. An etrA knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy. Reverse transcription-PCR analysis of total RNA demonstrated the loss of the etrA mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine N-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O(2), as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO. The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript. ETRA-153 was also deficient in one type of ubiquinone. These results are in contrast to those previously reported for the putative etrA mutant METR-1. Molecular analysis of METR-1 indicated that its etrA gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions.

Aerobiosis↗
PubMed2006

Multiscale dynamics of the cell envelope of Shewanella putrefaciens as a response to pH change.

The bacterial surface properties of gram-negative Shewanella putrefaciens were characterized by microbial adhesion to hydrocarbons (MATH), adhesion to polystyrene dishes, and electrophoresis at different values of pH and ionic strength. The bacterial adhesion to these two apolar substrates shows significant variations according to pH and ionic strength. Such behavior could be partly explained by electrostatic repulsions between bacteria and the solid or liquid interface. However, a similar trend was also observed at rather high ionic strength where electrostatic interactions are supposed to be screened. The nanomechanical properties at pH 4 and 10 and at high ionic strength were investigated by using atomic force microscopy (AFM). The indentation curves revealed the presence of a polymeric external layer that swells and softens up with increasing pH. This suggests a concomitant increase of the water permeability and so did of the hydrophilicity of the bacterial surface. Such evolution of the bacterial envelope in response to changes in pH brings new insight to the pH dependence in the bacterial adhesion tests. It especially demonstrates the necessity to consider the hydrophobic/hydrophilic surface properties of bacteria as not univocal for the various experimental conditions investigated.

Bacterial Adhesion↗
PubMed2001

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces.

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.

Bacterial Adhesion↗
PubMed1993

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens.

Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria (Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.

Animals↗
PubMed1994

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.

Anaerobiosis↗
PubMed1994

Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens.

The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.

Journal Article↗
PubMed2002

Genetic complementation of an outer membrane cytochrome omcB mutant of Shewanella putrefaciens MR-1 requires omcB plus downstream DNA.

Anaerobically grown cells of the metal-reducing bacterium Shewanella putrefaciens MR-1 contain multiple outer membrane (OM) cytochromes. A gene replacement mutant (strain OMCB1) lacking the OM cytochrome OmcB is markedly deficient in the reduction of MnO2 and exhibits reduced rates of Fe(III) reduction. The levels of other OM cytochromes are also decreased in OMCB1. Complementation of OMCB1 with wild-type omcB did not restore any of these defects. However, a 21-kb genomic fragment from MR-1, which included omcB and 19 kb of downstream DNA, fully restored MnO2 and Fe(III) reduction and the full complement of OM cytochromes to OMCB1. A 14.7-kb DNA fragment, including omcB and 12 kb of downstream DNA, provided only a modest increase in MnO2 reduction and OM cytochrome content, but it fully restored Fe(III) citrate reduction and partially restored FeOOH reduction. While omcB mRNA was readily detected in this complement, the OmcB protein was not detected in any cellular compartment. The restoration of Fe(III) reduction despite the absence of OmcB suggests that OmcB itself is not required for Fe(III) reduction. Another OM cytochrome, OmcA, was mislocalized to the cytoplasmic membrane of OMCB1. Only the 21-kb genomic fragment was able to restore proper localization of OmcA to the OM. This 21-kb fragment does not contain omcA, but it does contain several open reading frames (ORFs) downstream from omcB. The most downstream of these ORFs (altA) encodes a putative AraC-like transcriptional regulator. However, a gene replacement mutant of altA resembled the wild type with respect to MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM. Since OMCB1 continues to express genes immediately downstream from omcB, the lack of expression of this downstream DNA does not explain its phenotype or the need for the large complementing fragment. The results suggest that the DNA downstream of omcB must be present in cis in order to restore Fe(III) reduction, MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM.

Bacterial Outer Membrane Proteins↗
PubMed1994

Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens.

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.

Amino Acids↗
PubMed1994

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

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.

Biodegradation, Environmental↗
PubMed1997

Physiology and enzymology involved in denitrification by Shewanella putrefaciens.

Nitrate reduction to N2O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7. All three strains reduced nitrate to nitrite to N2O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 micromol liter-1). All S. putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three- to sixfold higher than those found under oxic conditions. Nitrate reductase activities (31 to 60 micromol of nitrite min-1 mg of protein-1) detected in intact cells of S. putrefaciens were equal to or higher than those seen in Escherichia coli LE 392. Km values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor. Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen. Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound. When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels. The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification.

Aerobiosis↗
PubMed1999

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.

Amino Acid Oxidoreductases↗
PubMed2001

Growth and metabolic activity of Shewanella putrefaciens maintained under different CO2 and O2 concentrations.

Growth, trimethylamine (TMA), off-odour and biogenic amine production by a strain of Shewanella putrefaciens isolated from spoiled hake (Merlucius merluccius L.) and cultured in a model system, were tested under four different gas compositions (60% CO2/40% O2, 60% CO2/15% O2/25% N2, 40% CO2/60% O2, 40% CO2/40% O2/20% N2) and under air. After 3 weeks of incubation, the control (air) batch showed the highest microbial counts (> 9 log cfu/ml) and TMA concentrations (45 mg N-TMA/100 ml), and strong putrid off-odours were detected from day 15. High amounts of putrescine and cadaverine were produced in this batch, but histamine increased only slightly. Batches under controlled atmospheres showed reduced growth, TMA, off-odour and biogenic amine production. The 40% CO2/60% O2 mixture had the strongest inhibitory effect on bacterial growth, while the 60% CO2/15% O2/25% N2 mixture was less effective. Putrescine and histamine production was lowest in S. putrefaciens under the 40% CO2/60% O2 mixture. However, the level of histamine in S. putrefaciens was higher under 40% CO2/40% O2/20% N2 than when the bacteria was incubated in air. Under the gas mixtures, there was a similar decrease in the production of cadaverine and agmatine by S. putrefaciens, irrespective of the gas concentrations. The production of 2-phenylethylamine appeared to be inhibited under any atmospheric condition.

Animals↗
PubMed2005

Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy.

The nanomechanical properties of gram-negative bacteria (Shewanella putrefaciens) were investigated in situ in aqueous solutions at two pH values, specifically, 4 and 10, by atomic force microscopy (AFM). For both pH values, the approach force curves exhibited subsequent nonlinear and linear regimens that were related to the progressive indentation of the AFM tip in the bacterial cell wall, including a priori polymeric fringe (nonlinear part), while the linear part was ascribed to compression of the plasma membrane. These results indicate the dynamic of surface ultrastructure in response to changes in pH, leading to variations in nanomechanical properties, such as the Young's modulus and the bacterial spring constant.

Bacterial Outer Membrane Proteins↗
PubMed2000

Design and application of a rapid screening technique for isolation of selenite reduction-deficient mutants of Shewanella putrefaciens.

A rapid screening technique for isolation of selenite (Se(IV)) reduction-deficient (Ser) mutants was developed and used to identify four Ser mutants of Shewanella putrefaciens. Two Ser mutants were unable to grow anaerobically on fumarate, nitrate or nitrite. Two other Ser mutants were unable to grow anaerobically on all compounds tested as sole terminal electron acceptor. Previously isolated Mn(IV) reduction-deficient mutants displayed Ser-positive phenotypes and reduced Se(IV) at wild-type rates, while two of nine Fe(III) reduction-deficient mutants displayed Ser-negative phenotypes and reduced Se(IV) at low rates. This study provides the first reported method for isolation of Ser mutants and demonstrates that Se(IV) reduction by S. putrefaciens is respiratory chain-linked.

Anaerobiosis↗
PubMed2001

Cell surface electrochemical heterogeneity of the Fe(III)-reducing bacteria Shewanella putrefaciens.

Acid-base titration experiments and electrostatic force microscopy (EFM) were used to investigate the cell surface electrochemical heterogeneity of the Fe(III)-reducing bacteria, Shewanella putrefaciens. The acid-base titrations extended from pH 4 to 10, and the titration data were fit using a linear programming pKa spectrum approach. Overall, a five-site model accounted for the observed titration behavior with the most acidic sites corresponding to carboxylic groups and phosphodiester groups, intermediate sites phosphoryl groups, and two basic sites equivalent to amine or hydroxyl groups. The pH for the point of zero charge on the bacteria was 5.4. In EFM images of cells rinsed in solutions at pH 4.0, 7.0, and 8.0, a pronounced increase in small (< or = 100 nm diameter) high contrast patches was observed on the cells with increasing pH. The pH dependence of EFM image contrast paralleled the pattern of cell surface charge development inferred from the titration experiments; however, quantitative analysis of high contrast regions in the EFM images yielded lower surface charge values than those anticipated from the titration data. For example at pH 7, the calculated surface charge of high contrast regions in EFM images of the bacterial cells was -0.23 microC/cm2 versus -20.0 microC/cm2 based on the titration curve. The differences in surface charge estimates between the EFM images and titration data are consistent not only with charge development throughout the entire volume of the bacterial cell wall (i.e., in association with functional groups that are not directly exposed at the cell surface) but also with the presence of a thin structural layer of water containing charge-compensating counterions. In combination, the pKa spectra and EFM data demonstrate that a particularly high degree of electrochemical heterogeneity exists within the cell wall and at the cell surface of S. putrefaciens.

Cell Membrane↗
PubMed2000

Isolation of U(VI) reduction-deficient mutants of Shewanella putrefaciens.

A U(VI) reduction-deficient mutant (Urr) screening technique was developed and combined with chemical mutagenesis procedures to identify a Urr mutant of Shewanella putrefaciens strain 200. The Urr mutant lacked the ability to grow anaerobically on U(VI) and NO(2)(-), yet retained the ability to grow anaerobically on eight other compounds as terminal electron acceptor. All 11 members of previously isolated sets of Fe(III) and Mn(IV) reduction-deficient mutants of S. putrefaciens 200 displayed Urr-positive phenotypes with the Urr screen and were capable of anaerobic growth on U(VI). This is the first reported isolation of a respiratory mutant that is unable to grow anaerobically on U(VI) as terminal electron acceptor.

Anaerobiosis↗