Shewanella Colwelliana Research Articles

Enhanced biofilm formation and melanin synthesis by the oyster settlement-promoting Shewanella colwelliana is related to hydrophobic surface and simulated intertidal environment

A direct relationship between biofilm formation and melanogenesis in Shewanella colwelliana with increased oyster recruitment is already established. Previously, S. colwelliana was grown in a newly patented biofilm-cultivation device, the conico-cylindrical flask (CCF), offering interchangeable hydrophobic/hydrophilic surfaces. Melanization was enhanced when S. colwelliana was cultivated in a hydrophobic vessel compared with a hydrophilic vessel. In the present study, melanogenesis in the CCF was positively correlated with increased architectural parameters of the biofilm (mean thickness and biovolume obtained by confocal laser scanning microscopy) and melanin gene (melA) expression observed by densitometry. Niche intertidal conditions were mimicked in a process operated in an ultra-low-speed rotating disk bioreactor, which demonstrated enhanced biofilm formation, melanogenesis, exopolysaccharide synthesis and melA gene expression compared with a process where 12-h periodic immersion and emersion was prevented. The wettability properties of the settling plane as well as intermittent wetting and drying, which influenced biofilm formation and melA expression, may affect oyster settlement in nature.

Effects of dietary supplementation of probiotic Shewanella colwelliana WA64, Shewanella olleyana WA65 on the innate immunity and disease resistance of abalone, Haliotis discus hannai Ino

The effects of dietary administration of two probiotics, Shewanella colwelliana WA64 and Shewanella olleyana WA65, on the innate immunity of abalone (Haliotis discus hannai Ino), and survival of juvenile abalone challenged with Vibrio harveyi have been studied. Two groups of abalone were fed with three different diets: one control, and two diets supplemented with 109 cell g−1 of probiotic WA64 (WA64 diet) and WA65 (WA65 diet) for up to four weeks. Results showed that abalone fed diets containing S. colwelliana WA64 and S. olleyana WA65 had led to an enhanced cellular and humoral immune response, notably higher haemocytes, respiratory burst activity, serum lysozyme activity and total protein levels were recorded after one week of probiotic administration. On the other hand, mortality after the challenges with V. harveyi in the group fed with control diet ranged from 77 to 80%, while mortality rates observed in the groups fed with diets supplemented with WA64 and WA65 ranged from 27 to 50% and 30–43%, respectively. The results demonstrated potential for S. colwelliana WA64 and S. olleyana WA65 to improve innate immunity and disease resistance in H. discus hannai.

A novel conico-cylindrical flask aids easy identification of critical process parameters for cultivation of marine bacteria

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment and ports to allow air supply was employed for melanin production by Shewanella colwelliana and antibiotic production by Pseudoalteromonas rubra. The design allowed comparison of production between (1) CCF with hydrophobic surface (PMMA-CCF), (2) CCF with hydrophilic glass surface (GS-CCF), and (3) standard unbaffled Erlenmeyer flask (EF). Melanin production in the PMMA-CCF was higher by at most 33.5% and growth of S. colwelliana by at most 309.2% compared to the other vessels. Melanin synthesis was positively correlated with reactor surface area and hydrophobicity, suspended cell growth, and biofilm formation. Antibiotic production in the EF was higher by at most 83.3%, but growth of P. rubra was higher in the PMMA-CCF by at most 54.5% compared to the other vessels. A hydrophilic vessel surface, abundant air supply, but low shear stress enhanced antibiotic production. The CCF together with the EF allowed identification of the crucial parameters (vessel surface characteristics, growth, biofilm formation, and aeration) influencing productivity, knowledge of which in the initial stages of process development will facilitate informed decisions at the later phases.

A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.

Gammaproteobacteria as a Possible Source of Eicosapentaenoic Acid in Anoxic Intertidal Sediments

Eicosapentaenoic acid (EPA; n-20:5omega3) was found to be a constituent of phospholipids in three mesophilic strains of Gammaproteobacteria, which were isolated from anoxic most probable number series prepared with sediments from an intertidal flat of the German North Sea coast. Their partial 16S rRNA gene sequences identified the isolates as close relatives of Shewanella colwelliana, Vibrio splendidus, and Photobacterium lipolyticum. So far, eicosapentaenoic acid has mainly been reported to occur in eukaryotes and some piezophilic or psychrophilic bacteria. With decreasing temperature, relative contents of EPA (up to 14% of total fatty acids) increased in all strains. Additionally, Shewanella and Vibrio spp. showed a significant increase in monounsaturated fatty acids with lower growth temperature. Analysis of the phospholipid compositions revealed that EPA was present in all three major phospholipid types, namely, phosphatidyl glycerol (PG), cardiolipin and phosphatidyl ethanolamine (PE). However, EPA was enriched in PG and cardiolipin relative to PE. In the tidal flat sediments from which the isolates were obtained, substantial amounts of EPA-containing PG were detected, whereas other typical microeukaryotic phospholipids-being also a possible source of EPA-were abundant at the sediment surface but were present in clearly lower amounts in the anoxic layers beneath 5 cm depth. Therefore, the EPA-containing PG species in the deeper layers in these sediments may indicate the presence of Gammaproteobacteria closely related to the isolates. These bacteria appear to be an important source of EPA in buried, anoxic sediments beneath the layers harboring significant populations of benthic eukaryotes.

Description of Shewanella glacialipiscicola sp. nov. and Shewanella algidipiscicola sp. nov., isolated from marine fish of the Danish Baltic Sea, and proposal that Shewanella affinis is a later heterotypic synonym of Shewanella colwelliana

Two novel species belonging to the genus Shewanella are described on the basis of a polyphasic taxonomic approach. A total of 40 strains of Gram-negative, psychrotolerant, H2S-producing bacteria were isolated from marine fish (cod and plaice) caught in the Baltic Sea off Denmark. Strains belonging to group 1 (seven strains) were a lactate-assimilating variant of Shewanella morhuae with a G+C content of 44 mol%. The strains of group 2 (33 strains) utilized lactate, N-acetylglucosamine and malate but did not produce DNase or ornithine decarboxylase. Their G+C content was 47 mol%. Phylogenetic analysis of the 16S rRNA gene sequence data placed the two novel species within the genus Shewanella. Group 1 showed greatest sequence similarity with S. morhuae ATCC BAA-1205T (99.9 %). However, gyrB gene sequence analysis and DNA-DNA hybridization differentiated these isolates from S. morhuae, with 95.6 % sequence similarity and less than 57 % DNA relatedness, respectively. Group 2 strains shared more than 99 % 16S rRNA gene sequence similarity with the type strains of Shewanella colwelliana and Shewanella affinis, but gyrB sequence similarity ( approximately 85 %) and the results of DNA hybridization ( approximately 28 %) indicated that the new isolates represented a novel species. Furthermore, when compared to each other, the type strains of S. colwelliana and S. affinis had almost identical gyrB sequences and significantly high DNA reassociation values (76-83 %), indicating that they belonged to the same species. Based on the conclusions of this study, we propose the novel species Shewanella glacialipiscicola sp. nov. (type strain T147T=LMG 23744T=NBRC 102030T) for group 1 strains and Shewanella algidipiscicola sp. nov. (type strain S13T=LMG 23746T=NBRC 102032T) for group 2 strains, and we propose that Shewanella affinis as a later heterotypic synonym of Shewanella colwelliana.

Shewanella affinis sp. nov., isolated from marine invertebrates.

Four marine bacterial strains, designated KMM 3587T, KMM 3586, KMM 3821 and KMM 3822, were isolated from the sipuncula Phascolosoma japonicum, a common inhabitant of Troitza Bay in the Gulf of Peter the Great (Sea of Japan region), and from an unidentified hydrocoral species collected in Makarov Bay (Iturup Islands), Kuril Islands, North-West Pacific Ocean. The strains were characterized to clarify their taxonomic position. 16S rRNA gene sequences of KMM 3587T and KMM 3586 indicated 99% similarity to Shewanella colwelliana. Despite such a high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization experiments demonstrated only 45-52% binding with DNA of S. colwelliana ATCC 39565T. The DNA G+C contents of the novel strains were 45 mol% and the shared level of DNA hybridization was conspecific (81-97%), indicating that they represent a single genospecies. The novel strains were mesophilic (able to grow at 10-34 degrees C), neutrophilic and haemolytic, and able to degrade gelatin, casein and Tween 20, 40 and 80, but not starch, agar, elastin, alginate or chitin. The major fatty acids were i13 : 0, i15 : 0, 16 : 0, 16 : 1omega7 and 17 : 1omega8 (68.9% of total). The major isoprenoid quinones were Q7 (47-62%) and Q8 (26-47%). Eicosapentaenoic acid was produced in minor amounts. Based on these data, the strains are assigned to a novel species, Shewanella affinis sp. nov. (type strain KMM 3587T=CIP 107703T=ATCC BAA-642T).

Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana

The enzyme p-hydroxyphenylpyruvate hydroxylase (HPPH) is involved in pigmentation (pyomelanin) via homogentisic acid (HGA). Pyomelanin formation is correlated with HGA production and expression of HPPH in three disparate marine species: Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Induction of pigmentation in V. cholerae 569B by nutrient limitation also correlated with production of HGA.

Cloning and expression of a gene encoding a T-cell reactive protein from coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen

The gene which encodes a previously described T-cell reactive protein (TCRP) of the human fungal pathogen Coccidioides immitis (Ci) was cloned and sequenced. Both the genomic and cDNA sequences were determined. The transcription start point was confirmed. The tcrP gene has three introns and a 1197-bp ORF which translates to a 399-amino-acid (aa) protein (45.2 kDa). The predicted protein has approx. 50% aa sequence identity and 70% similarity to mammalian 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins and mammalian F-antigens. Expression of the Ci tcrP in Escherichia coli resulted in production of a deep brown pigment, consistent with E. coli expression of the bacterial HPPD homolog from Shewanella colwelliana. The TCRP is likely the Ci form of HPPD.

The protein encoded by the Shewanella colwelliana melA gene is a p-hydroxyphenylpyruvate dioxygenase

The identity of the product of the melA gene from Shewanella colelliana with the enzyme p-hydroxyphenylpyruvic dioxygenase is shown. Cloning of the melA gene endowed Escherichia coli with the capacity to synthesize melanin-like pigments from L-tyrosine. E. coli contained transaminases that transforms L-tyrosine into p-hydroxyphenylpyruvate. This keto acid was detected in the cultures. On the other hand, E. coli containing melA was able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously polymerize giving the pigment. Furthermore, p-hydroxyphenylpyruvate dioxygenase activity was detected in this system. Analysis of the deduced amino acid sequence revealed a high homology with the p-hydroxyphenylpyruvate deoxygenase enzyme from different organisms.

Glucose metabolism and polysaccharide accumulation in the marine bacterium, Shewanella colwelliana

Shewanella colwelliana, a marine bacterium isolated in association with the oyster Crassostrea virginica, produces an abundant exopolysaccharide with potential commercial value as an adhesive under aqueous conditions. Its utilization of glucose was modulated by stoichiometric concentrations of yeast extract. In Brain Heart Infusion medium containing glucose, growth was diauxic with delayed glucose utilization and incorporation into exopolysaccharide. Data from radio-respirometry protocols indicate that glucose is catabolized through a combination of the hexose monophosphate and Entner-Doudoroff pathways. Exopolysaccharide production could be significantly enhanced by adjusting glucose concentrations of the growth medium.

Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D

Shewanella colwelliana D is a marine procaryote which produces a diffusible brown pigment that correlates with melA gene expression. Previously, melA had been cloned, sequenced, and expressed in Escherichia coli; however, the reaction product of MelA had not been identified. This report identifies that product as homogentisic acid, provides evidence that the pigment is homogentisic acid-melanin (pyomelanin), and suggests that MelA is p-hydroxyphenylpyruvate hydroxylase. This is the first report of pyomelanin in an obligate marine bacterium.

Sequence determination and mutational analysis of the lly locus of Legionella pneumophila

The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.

Human 4-hydroxyphenylpyruvate dioxygenase. Primary structure and chromosomal localization of the gene.

We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence. These peptide sequences covered 86% of the cDNA-derived amino-acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa. There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C-terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases. The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e. amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.

Characterization of melA: a gene encoding melanin biosynthesis from the marine bacterium Shewanella colwelliana

A recombinant plasmid with the ability to impart melanin synthesis to an Escherichia coli host was isolated from a Shewanella colwelliana genomic library. The genetic determinant of the Mel + phenotype is carried on a 1.3-kb DNA fragment and sequence analysis of this revealed a single intact open reading frame that was sufficient for melanin synthesis ( mel). This gene is expressed as a monocistronic transcript and a putative transcription start point is located 115 nucleotides upstream from the translational start codon. The mel gene encoded a protein of 39.5 kDa [346 amino acids (aa)] that showed no aa sequence homology with other proteins known to mediate melanin synthesis (e.g., tyrosinases).

Properties of an extracellular adhesive polymer from the marine bacterium,Shewanella Colwelliana

Shewanella colwelliana, a marine bacterium which grows in association with oysters, has been found to produce an unusual extracellular polymer which mediates irreversible adhesion of the bacterium to surfaces. Production of the polymer was enhanced when the bacterium was grown on marine agar, overlaid with a dialysis membrane, and seeded with a liquid suspension of the cells. Purified exopolymer was shown to contain Ca, S, P and Si (40–45%), lipid (10%), carbohydrate (15–35%) and protein (<5%). Glucose was the predominant component of the carbohydrate moiety. Uronic acids were not detected. The exopolymer forms a loose capsule that surrounds the bacterium, demonstrated by light and electron microscopy.

Interactions between Shewanella colwelliana , Oyster Larvae, and Hydrophobic Organophosphate Pesticides

Shewanella colwelliana (strain D) is a periphytic estuarine bacterium that forms biofilms beneficial to oyster set. Our study examined whether these and other films concentrated two hydrophobic, organophosphate pesticides, Abate and malathion, that are detected in Chesapeake Bay oyster waters. Both biofilms and purified exopolysaccharide of S. colwelliana did not adsorb more of the Abate or malathion than could be accounted for by adsorption to control surfaces. Similar results were obtained by using Deleya marina, Hyphomonas MHS3, and autochthonous biofilms. Conversely, decapsulated S. colwelliana D cells, prepared in the laboratory, bioconcentrated Abate. Significantly, the S. colwelliana D biofilms exposed to Abate did not inhibit the settlement and metamorphosis of Crassostrea gigas larvae.