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.     

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.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Reduction of ferric green rust by Shewanella putrefaciens

AIMS: To reduce carbonated ferric green rust (GR*) using an iron respiring bacterium and obtain its reduced homologue, the mixed Fe(II)-Fe(III) carbonated green rust (GR). METHODS AND RESULTS: The GR* was chemically synthesized by oxidation of the GR and was incubated with Shewanella putrefaciens cells at a defined [Fe(III)]/[cell] ratio. Sodium methanoate served as the sole electron donor. The GR* was quickly transformed in GR (iron reducing rate = 8.7 mmol l(-1) h(-1)). CONCLUSIONS: Ferric green rust is available for S. putrefaciens respiration as an electron acceptor. The reversibility of the GR redox state can be driven by bacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work suggests that GRs would act as an electronic balance in presence of bacteria. It provides also new perspectives for using iron reducing bacterial activity to regenerate the reactive form of GR during soil or water decontamination processes.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Structure and properties of novel inclusions in Shewanella putrefaciens

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.     

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.     

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.     

Corrosion-enhancing potential of Shewanella putrefaciens isolated from industrial cooling waters

Microbially influenced corrosion (MIC) is catalysed by a series of metabolic activities of selected micro-organisms, notably by oxidation of cathodic hydrogen by hydrogenase, by hydrogen sulphide and by reduction of ferric iron. The sulphate-reducing bacteria are considered to be the most common catalyst of MIC, whereas the role of other bacteria has been neglected. This study examined the corrosive potential of the facultative sulphide producer, Shewanella putrefaciens , isolated from an industrial cooling water system. Shewanella putrefaciens was shown to reduce ferric iron and sulphite under anaerobic conditions and with ferric iron being the preferred electron acceptor. The isolate could utilize cathodic hydrogen as an energy source, especially when using sulphite as a terminal electron acceptor. In pure culture corrosion experiments, the highest mass loss of mild steel was observed in the presence of sulphite as sole electron acceptor, although mass loss was also detected where ferric iron was the sole electron acceptor. Our data indicate that S. putefaciens plays a role in MIC as it was able to catalyse a variety of corrosion-promoting reactions and to corrode mild steel under pure culture conditions.     

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.     

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°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10⁸ CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10² CFU/cm²) than in a batch system (reaching 10⁷ CFU/cm²), 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.     

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.     

Influence of respiratory substrate on the cytochrome content of Shewanella putrefaciens

Shewanella putrefaciens can use trimethylamine oxide (TMAO) as electron acceptor under anoxic conditions. The associated cytochromes induced during growth under various respiratory conditions have been separated by liquid chromatography (DEAE Sepharose CL6b) and SDS-PAGE and characterized spectrophotometrically and by redox potentiometry. Two major low potential cytochromes and at least three minor low potential cytochromes, likely to be involved in TMAO reduction, were found. No cytochrome specific for TMAO reductase was found.     

Anaerobic respiration of Shewanella putrefaciens requires both chromosomal and plasmid-borne genes

Abstract Pleiotropic respiratory mutants, incapable of growth on any electron acceptor other than oxygen, were isolated from two strains of Shewanella putrefaciens (MR-1 and sp200). All anaerobic respiratory functions were restored by complementation of the mutants with specific cloned DNA fragments. Southern hybridization experiments revealed that the fragment that complements the MR-1 mutant was localized on the megaplasmids of both strains, while the fragment that complements the sp200 mutant was chromosomal. Neither of these fragments hybridized with the anaerobic regulatory genes of S. putrefaciens ( etrA ) or E. coli ( fnr ).     

Structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6

The structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6 has been elucidated. Chemical modifications of the polymer in conjunction with 1H and 13C NMR spectroscopy, including 2D techniques, were employed in the analysis. It is concluded that the repeating unit is composed of two nine-carbon sugars as follows: -->4)-alpha-NonpA-(2-->3)-beta-Sugp-(1--> where alpha-NonpA is 5-acetamido-7-acetamidino-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-non-2-ulosonic acid (8eLeg) and beta-Sugp is 2-acetamido-2,6-dideoxy-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-beta-D-galactopyranose, with the proposed name Shewanellose (She).     

Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3′-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.     

Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction

The growth of bacteria by dissimilatory elemental sulfur reduction is generally associated with obligate anaerobes and thermophiles in particular. Here we describe the sulfur-dependent growth of the facultatively anaerobic mesophile Shewanella putrefaciens. Six of nine representative S. putrefaciens isolates from a variety of environments proved able to grow by sulfur reduction, and strain MR-1 was chosen for further study. Growth was monitored in a minimal medium (usually with 0.05% Casamino Acids added as a growth stimulant) containing 30 mM lactate and limiting concentrations of elemental sulfur. When mechanisms were provided for the removal of the metabolic end product, H2S, measurable growth was obtained at sulfur concentrations of from 2 to 30 mM. Initial doubling times were ca. 1.5 h and substrate independent over the range of sulfur concentrations tested. In the cultures with the highest sulfur concentrations, cell numbers increased by greater than 400-fold after 48 h, reaching a maximum density of 6.8 x 10(8) cells ml-1. Yields were determined as total cell carbon and ranged from 1.7 to 5.9 g of C mol of S(0) consumed-1 in the presence of the amino acid supplement and from 0.9 to 3.4 g of C mol of S(0-1) in its absence. Several lines of evidence indicate that cell-to-sulfur contact is not required for growth. Approaches for the culture of sulfur-metabolizing bacteria and potential ecological implications of sulfur reduction in Shewanella-like heterotrophs are discussed.     

Ornithine decarboxylase activities of a Shewanella putrefaciens which produces only diamines

Ornithine decarboxylase (ODC) activity in cell extracts of Shewanella putrefaciens was surveyed. The pH dependency of the ODC activity revealed that the bacterium has two different ODC having optimum pH at 8·25 and 6·50. They were considered to be biosynthetic and biodegradative enzymes, respectively. Their activity ratio varied when the bacterium was cultured at pH 7·0 and 6·0. Both ODC activities were inhibited by α-difluoromethylornithine but cell growth was not affected.     

Anaerobic production of polyunsaturated fatty acids by Shewanella putrefaciens strain ACAM 342

Abstract The fatty acid composition of cultures of Shewanella putrefaciens strain ACAM 342 grown aero-bically and anaerobically at 15°C and 25°C were analysed by capillary gas chromatography. The bacterium was found to produce the polyunsaturated fatty acids (PUFA) 18:2ω3, 18:3ω3 and 20:5ω3 under aerobic and anaerobic conditions at both growth temperatures. This result suggests that the bacterium possesses both the aerobic and anaerobic pathways for unsaturated fatty acid synthesis, where an alternate terminal electron acceptor(s) is utilised in the absence of oxygen.