Characterization of a flavocytochrome that is induced during the anaerobic respiration of Fe3+ by Shewanella frigidimarina NCIMB400. (1/494)

A 63.9 kDa periplasmic tetrahaem flavocytochrome c(3), designated Ifc(3), was found to be expressed in Shewanella frigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates. Ifc(3) was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c(3)-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV. A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc(3), ifcA, did not lead to a significant change in the rate of Fe(3+) reduction in batch culture. However, during such growth the Ifc(3)-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane beta-barrel protein of 324 amino acid residues.  (+info)

Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide. (2/494)

The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules. S. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.  (+info)

Shewanella pealeana sp. nov., a member of the microbial community associated with the accessory nidamental gland of the squid Loligo pealei. (3/494)

A new, mesophillic, facultatively anaerobic, psychrotolerant bacterium, strain ANG-SQ1T (T = type strain), was isolated from a microbial community colonizing the accessory nidamental gland of the squid Loligo pealei. It was selected from the community on the basis of its ability to reduce elemental sulfur. The cells are motile, Gram-negative rods (2.0-3.0 microns long, 0.4-0.6 micron wide). ANG-SQ1T grows optimally over the temperature range of 25-30 degrees C and a pH range of 6.5-7.5 degrees C in media containing 0.5 M NaCl. 16S rRNA sequence analysis revealed that this organism belongs to the gamma-3 subclass of the Proteobacteria. The closest relative of ANG-SQ1T is Shewanella gelidimarina, with a 16S rRNA sequence similarity of 97.0%. Growth occurs with glucose, lactate, acetate, pyruvate, glutamate, citrate, succinate, Casamino acids, yeast extract or peptone as sole energy source under aerobic conditions. The isolate grows anaerobically by the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate or elemental sulfur as terminal electron acceptor with lactate. Growth of ANG-SQ1T was enhanced by the addition of choline chloride to growth media lacking Casamino acids. The addition of leucine or valine also enhanced growth in minimal growth media supplemented with choline. The results of both phenotypic and genetic characterization indicate that ANG-SQ1T is a Shewanella species. Thus it is proposed that this new isolate be assigned to the genus Shewanella and that it should be named Shewanella pealeana sp. nov., in recognition of its association with L. pealei.  (+info)

A study of deep-sea natural microbial populations and barophilic pure cultures using a high-pressure chemostat. (4/494)

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.  (+info)

Homogeneity of Danish environmental and clinical isolates of Shewanella algae. (5/494)

Danish isolates of Shewanella algae constituted by whole-cell protein profiling a very homogeneous group, and no clear distinction was seen between strains from the marine environment and strains of clinical origin. Although variation between all strains was observed by ribotyping and random amplified polymorphic DNA analysis, no clonal relationship between infective strains was found. From several patients, clonally identical strains of S. algae were reisolated up to 8 months after the primary isolation, indicating that the same strain may be able to maintain the infection.  (+info)

Voltammetry of a flavocytochrome c(3): the lowest potential heme modulates fumarate reduction rates. (6/494)

Iron-induced flavocytochrome c(3), Ifc(3), from Shewanella frigidimarina NCIMB400, derivatized with a 2-pyridyl disulfide label, self-assembles on gold electrodes as a functional array whose fumarate reductase activity as viewed by direct electrochemistry is indistinguishable from that of Ifc(3) adsorbed on gold or graphite electrodes. The enhanced stability of the labeled protein's array permits analysis at a rotating electrode and limiting catalytic currents fit well to a Michaelis-Menten description of enzyme kinetics with K(M) = 56 +/- 20 microM, pH 7.5, comparable to that obtained in solution assays. At fumarate concentrations above 145 microM cyclic voltammetry shows the catalytic response to contain two features. The position and width of the lower potential component centered on -290 mV and corresponding to a one-electron wave implicates the oxidation state of the lowest potential heme of Ifc(3) as a defining feature in the mechanism of fumarate reduction at high turnover rates. We propose the operation of dual pathways for electron transfer to the active site of Ifc(3) with the lowest potential heme acting as an electron relay on one of these pathways.  (+info)

Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400. (7/494)

Two membranous c-type cytochromes from the Fe(III)-respiring bacterium Shewanella frigidimarina NCIMB400, CymA and OmcA, have been purified and characterized by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. The 20-kDa CymA is a member of the NapC/NirT family of multiheme cytochromes, which are invariably anchored to the cytoplasmic membrane of Gram-negative bacteria, and are postulated to mediate electron flow between quinols and periplasmic redox proteins. CymA was found to contain four low-spin c-hemes, each with bis-His axial ligation, and midpoint reduction potentials of +10, -108, -136, and -229 mV. The 85-kDa OmcA is located at the outer membrane of S. frigidimarina NCIMB400, and as such might function as a terminal reductase via interaction with insoluble Fe(III) substrates. This putative role is supported by the finding that the protein was released into solution upon incubation of harvested intact cells at 25 degrees C, suggesting an attachment to the exterior face of the outer membrane. OmcA was revealed by magneto-optical spectrocopies to contain 10 low-spin bis-His ligated c-hemes, with the redox titer indicating two sets of near iso-potential components centered at -243 and -324 mV.  (+info)

Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme. (8/494)

Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.  (+info)