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.G.O. Abu was and R. Weiner and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA. G. O. Abu is now with the Department of Microbiology, University of Port Harcourt, P.M.B. 5323, PH, Nigeria.     

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.     

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

Magnification of tributyl tin toxicity to oyster larvae by bioconcentration in biofilms of Shewanella colwelliana.

The toxic effects of dissolved versus bioconcentrated tributyl tin (TBT) on oyster larvae were compared. Water column TBT levels, which had no effect in solution, inhibited natural attachment and metamorphosis of oyster larvae on bottom surfaces due to bioconcentration by biofilms. This mechanism should be considered when evaluating heavy metal toxicity in the environment.     

Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.     

Molecular cloning of the gene which encodes beta-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.     

Eicosapentaenoic acid facilitates the folding of an outer membrane protein of the psychrotrophic bacterium, Shewanella livingstonensis Ac10

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), are found in various cold-adapted microorganisms. We previously demonstrated that EPA-containing phospholipids (EPA-PLs) synthesized by the psychrotrophic bacterium Shewanella livingstonensis Ac10 support cell division, membrane biogenesis, and the production of membrane proteins at low temperatures. In this article, we demonstrate the effects of EPA-PLs on the folding and conformational transition of Omp74, a major outer membrane cold-inducible protein in this bacterium. Omp74 from an EPA-less mutant migrated differently from that of the parent strain on SDS-polyacrylamide gel, suggesting that EPA-PLs affect the conformation of Omp74 in vivo. To examine the effects of EPA-PLs on Omp74 protein folding, in vitro refolding of recombinant Omp74 was carried out with liposomes composed of 1,2-dipalmitoleoyl-sn-glycero-3-phosphoglycerol and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1:1molar ratio) with or without EPA-PLs as guest lipids. SDS-PAGE analysis of liposome-reconstituted Omp74 revealed more rapid folding in the presence of EPA-PLs. CD spectroscopy of Omp74 folding kinetics at 4°C showed that EPA-PLs accelerated β-sheet formation. These results suggest that EPA-PLs act as chemical chaperones, accelerating membrane insertion and secondary structure formation of Omp74 at low temperatures.     

Manganese removal and product characteristics of a marine manganese-oxidizing bacterium Bacillus sp. FF-1

International Microbiology - Biogenic manganese oxides (BioMnOx) have been found all over the world, and most of them were formed by Mn(II)-oxidizing bacteria (MnOB). In this study, a MnOB...     

Identification of bisphosphatidic acid and its plasmalogen analogues in the phospholipids of a marine bacterium.

A relatively nonpolar unidentified phospholipid (phospholipid X) , isolated from the gram-negative marine bacterium MB 45, was characterized both chromatographically and by chemical analysis. Phospholipid X was shown to be an acidic phospholipid without vicinal hydroxyl, free-amino, or amide groups. The presence of O-alkenyl groups was indicated by a positive reaction for plasmalogen. Mild alkaline methanolysis of phospholipid X yielded only glycerophosphoryglycerol as the derivative. Acetolysis produced only diacyl-glycerol monoacetate. Clevage of O-alkenyl chains by methanolic hydrochloride resulted in the formation of three lyso derivatives. It was estimated that 18.2% of phospholipid X was plasmalogen. From these data, together with chromatographic comparisons with standards, infrared spectra, a molecular weight estimation, and the determination of the glycerol-phosphate-acyl ester ratio, it was concluded that phospholipid X was bisphosphatidic acid mixed with its plasmalogen analogues.     

Purification and characterization of a novel antibacterial protein from the marine bacterium D2.

A biofilm-forming marine bacterium, D2, isolated from the surface of the tunicate Ciona intestinalis, was found to produce a novel, 190-kDa protein with antibacterial activity. The protein contained at least two subunits of 60 and 80 kDa, joined together by noncovalent bonds, and was shown to be released by D2 cells into the surrounding medium during stationary phase. N-terminal sequence analysis revealed no close similarity of this protein to any other proteins within the Swiss Prot database. Bacteriocidal activity against a wide variety of marine and medical bacterial isolates was observed, 77% of the strains tested being sensitive to the protein. Bacterial strains varied in their resistance to the D2 protein, with D2 itself being among the most sensitive with an MBC in liquid suspension of 4 micrograms/ml. An apparent increased resistance of D2 to the protein as the cells progressed further into stationary phase was observed and seen as a possible explanation for its survival despite the production of an autoinhibitory factor. The ability of the D2 bacterium to produce an antibacterial factor in addition to its inhibitory effects on marine invertebrates and algae (S. Egan et al., unpublished data) indicates that D2 has the potential to greatly affect the survival of a range of colonizers of the marine surface environment.     

Transhydrogenase activity in the marine bacterium Beneckea natriegens.

The marine bacterium, Beneckea natriegens, which has previously been reported not to form transhydrogenase, has been shown to synthesize a soluble energy-independent transhydrogenase (NADPH:NADP+ oxidoreductase, EC 1.6.1.1), though no energy-linked activity could be detected. The transhydrogenase is induced maximally in stationary phase cells and its formation is 70-90% repressed by raising the medium phosphate level from 0.33 to 3.3 mM. The enzyme is inhibited by arsenate, inorganic ortho- and pyrophosphate and by a range of organic phosphate-containing compounds, including 2'-AMP, which is an activator of several bacterial transhydrogenases.     

Characterization of carbonic anhydrase isozyme CA2, which is the CAH2 gene product, in Chlamydomonas reinhardtii.

From high-CO2 (5% CO2) grown unicellular green alga, Chlamydomonas reinhardtii, carbonic anhydrase (CA) was isolated by affinity chromatography and characterized. Isolated CA was identified as an isozyme (CA2) which is the product from the second gene CAH2 by peptide sequencing. The CA2 was inactivated by dithiothreitol. This treatment caused dissociation of CA2 into the large (38 kDa) and small subunits (4243 Da). The molecular mass of the CA2 holoenzyme measured by low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC was 87.9 kDa. These results and gene structure indicate that CA2 is a heterotetramer consisting of two large and two small subunits linked by disulfide bonds like CA1, which is the CAH1 gene product. The specific activity of CA2 purified by anion-exchange HPLC was 3300 units per mg protein, which was approximately 1.6 times higher than that of CA1. Therefore, it was concluded that two structurally related isozymes, CA1 and CA2, are present in the wild type cells of C. reinhardtii and differentially regulated by the atmospheric CO2 concentration.     

Genome sequence of the marine bacterium Marinobacter hydrocarbonoclasticus SP17, which forms biofilms on hydrophobic organic compounds

Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.     

Mycoplasma gallisepticum as the first analyzed bacterium in which RNA is not polyadenylated

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. In addition to most eukaryotic mRNAs possessing a stable poly(A)-tail, RNA is polyadenylated as part of a degradation mechanism in prokaryotes, organelles, and the eukaryotic nucleus. To date, only very few systems have been described wherein RNA is metabolized without polyadenylation, including several archaea and yeast mitochondria. The minimal genome of the parasitic bacteria, Mycoplasma, does not encode homologs of any known polyadenylating enzyme. Here, we analyze polyadenylation in Mycoplasma gallisepticum. Our results suggest this organism as being the first described bacterium in which RNA is not polyadenylated.     

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.