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Staphylococcus aureus releases a large number of exoproteins such as toxins and enzymes, as extracellular and surface-associated forms, many of which have been shown to contribute to pathogenic activity or to enhance virulence (17,35,48,57). These exoproteins include (i) factors involved in invasion/tissue penetration, (ii) factors involved in evasion of host defenses and (iii) factors involved in attachment. The first group of exoproteins are alpha-toxin (14), beta-hemolysin (47), gammahemolysin (46), delta-hemolysin (20), and phospholipase C (9). The second group are toxic shock syndrome toxin-1 (TSST-1) (29), enterotoxins (3,4,8,15,22,52), protein A (34), and others (6,31,32,50,53), and the third group contains clumping factor (37) and others (7,12,23,45). More than 30 of these exoproteins that are significantly linked to pathogenesis have been purified to homogeneity, and the genes encoding these exoproteins have been cloned and sequenced (5,48 Abstract: We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC3, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.

Strains of lactic acid bacteria (LAB) were isolated from several different sources and evaluated in vitro for potential probiotic effects in abalones. Two isolates (Lactobacillus sp. strain a3 and Enterococcus sp. strain s6) were highly resistant to bile salt and/or gastric juice and inhibited the growth of three abalone pathogens (Listonella anguillarum, Vibrio harveyi, and V. carchariae). Each of the LAB isolates was used to supplement diet of the abalone Haliotis gigantea for a period of 3 weeks. One group of animals received Lactobacillus sp. strain a3 added to commercial dry feed, one group received Enterococcus sp. strain s6 added to the feed, and a control group received only standard commercial feed. Culturable LAB counts of gut homogenates indicate the a3 colonized in the gut of abalones. Digestive enzyme activities and the concentrations of a number of volatile short-chain fatty acids (VSCFA) were elevated in the gut of abalones receiving feed supplemented with the two LAB strains. These results indicate that dietary supplementation can enable LAB colonization or persistence in the gut of abalone species and can potentially enhance probiotic effects.

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The hydrocarbon-degrading bacterium Dietzia maris WR-3 was isolated from a consortium comprising ammonia-oxidizing and denitrifying bacteria derived from marine sediments. Here, we examined biosurfactant production by strain WR-3 when cultured using several different carbon (D-glucose, n -decane, n -hexadecane, motor oil, olive oil, and rapeseed oil) and nitrogen (NH(4) )(2) SO(4) , NaNO(3) , yeast extract, and polypeptone) sources as growth substrates. Strain WR-3 was able to grow and reduce the surface tension of culture broth to 31±1.0 mN m(-1) when cultured using n -hexadecane and nitrate ions. The surface-active compounds produced by strain WR-3 were extracted and analyzed by thin layer chromatography. Moreover, the main components in the extract were further purified and subjected to gas chromatography/mass spectrometry (GC/MS). From the analysis, the surface-active compounds were tentatively identified as wax ester-like compounds, which were synthesized from the degradation process of n -alkane. The production of surface-active compounds by strain WR-3 promoted attachment of cells to hydrocarbon droplets via increased cell hydrophobicity, thus allowing enhanced degradation of water immiscible substrates. As Dietzia spp. can grow and produce wax esters from the degradation process of hydrocarbons, these marine bacteria are potentially useful for the bioremediation of hydrocarbon-contaminated environments.

Marinobacter comprises Gram-negative, aerobic, motile, and rod-shaped bacteria within the gamma-subclass of the Proteobacteria and is known to be halophilic or halotolerant, heterotrophic neutrophile. Two strains classified as belonging to Marinobacter, named PAD-2 and SeT-1, were isolated from marine sediment. The most closely related species of PAD-2 and SeT-1 are M. alkaliphilus and M. guinea, respectively. The strain PAD-2 exhibited remarkably higher denitrification at concentrations of 0.5 to 1 M NaCl (3-6% w/w) than at other salinities (2 and 3 M NaCl, 12-18% w/w), and optimal denitrification was observed in media with 0.5 M NaCl. The effect of pH on denitrification by strain PAD-2 was also examined, and the optimum denitrification occurred at neutral pH rather than under alkaline conditions. Overall, strain PAD-2 appears to be a novel halotolerant species belonging to the genus Marinobacter that shares many characteristics, such as substrate utilization profile and optimum NaCl concentration for growth with M. alkaliphilus.

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerollipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their Nterminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins. Key words: Staphylococcal exoproteins, Extracellular proteases, Two-dimensional polyacrylamide-gel electrophoresisStaphylococcus aureus is an important human pathogen implicated in a wide range of diseases including septicemia, meningitis, endocarditis, osteomyelitis, toxic-shock syndrome (TSS) and food poisoning (21,28,34). Incidences of both community-and hospitalacquired staphylococcal infections are increasing because of its rapid development of antibiotic resistance (29).S. aureus secretes a large number of extracellular proteins (exoproteins), which have been shown to contribute to the pathogenic activities or to enhance the virulence of this organism (15,21,25,33). Recently, renewed interest has focused on proteases secreted from S. aureus under the control of agr (accessory gene regulator) and sar (staphylococcal accessory regulator), the main regulators of S. aureus virulence determinant genes (6,7,12,24). The expression of a specific serine glutamyl endopeptidase, V8 protease, was found to be *Address correspondence to Dr. Michio Ohta, Department of Molecular Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. Fax: + 81-52-744-2107. E-mail: mohta @med.nagoya-u.ac.jp 285 important for virulence in animal models of staphylococcal infections (8). In addition to V8 protease, S. au reus secretes at least two other proteases, staphopain (papain-like thiol protease) (2) and aureolysin (typical metalloproteinase) (I). These proteases also encompass enzymes including Pseudomonas aeruginosa elastase (23), Legionella pneumophila (18) and Listeria monocytogenes metalloproteinases (9), Vibrio cholerae hemagglutinin protease (5), Staphylococcus epidermidis elastase (32), and the ...

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