Tetsuro Koga

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.

Bactericidal activities of essential oils of basil and sage against a range of bacteria and the effect of these essential oils on Vibrio parahaemolyticus

Basil and sage essential oils were examined for bactericidal activity against a range of Gram-positive and Gram-negative bacteria by viable count determinations. Generally, Gram-positive bacteria showed higher resistance to basil and sage essential oils than Gram-negative bacteria. Vibrio species showed a high sensitivity to both essential oils. Stationary growth phase cells of selected bacteria showed higher resistance to these essential oils than exponential growth phase cells. Basil-resistant (b21) and sage-resistant (s20) strains of Vibrio parahaemolyticus were isolated. Both strains showed higher resistance to heat and H2O2 than parent strain. Conversely, heat-adapted V. parahaemolyticus also showed a higher resistance to these essential oils than nonadapted cells.

A Broad-Host-Range Vibriophage, KVP40, Isolated from Sea Water

A broad‐host‐range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host. The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species. KVP40 was a large tailed phage containing double‐stranded DNA and belonged to Ackermanns morphotype A2. KVP40 DNA was cleaved by 11 different type II restriction endo‐nucleases including EcoRl and Hindlll, but not by 17 other enzymes including BamHI, KpnI and SalI.1

Nutrient Starvation Induces Cross Protection against Heat, Osmotic, or H2O2 Challenge in Vibrio parahaemolyticus

Glucose‐starved cells of Vibrio parahaemolyticus were compared with non‐starved counterparts with respects to heat, osmotic, and oxidative challenges. The starved cells demonstrated greater thermal and oxidative resistance than did the non‐starved cells. The starved cells also showed greater resistance against low osmotic challenge than did the non‐starved cells although both cells showed a comparable resistance against high osmotic challenge.

Classification of Vibrio bacteriophages

85 Vibrio phages, 84 of them tailed and 1 filamentous, were surveyed. The tailed phages belonged to six basic morphotypes and to the Myoviridae, Siphoviridae, or Podoviridae families. 63 phages were classified into 18 species. The filamentous phage is a member of the Inovirus genus of the Inoviridae family. Vibrio phages are very heterogenous and include some morphologically interesting viruses. Several Vibrio phages closely resemble phages of other gram-negative bacteria, possibly indicating phylogenetic relationships between their hosts.

Antibacterial activity of Lactobacillus species against Vibrio species

Forty-one Lactobacillus strains were tested for antagonistic activity against nine strains of Vibrio. L. plantarum and L. casei were the most effective, and L. brevis was the least effective in inhibiting the growth of Vibrio species. L. gasseri and L. helveticus strains showed higher activity, while L. reuteri and L. fermentum showed lower inhibitory activity against Vibrio species. L. acidophilus strains exhibited various degrees of antagonistic activities against Vibrio species. However, none of the Lactobacillus species were able to inhibit the growth of Salmonella enteritidis, S. typhimurium, Escherichia coli, and Staphylococcus aureus. Inhibition of the Vibrio species was probably due to the production of organic acids by the Lactobacillus species.

Alkaline adaptation induces cross-protection against some environmental stresses and morphological change in Vibrio parahaemolyticus

The relationship between alkaline adaptation and the resistance against environmental stresses was examined in Vibrio parahaemolyticus. Alkali-adapted cells were found to have increased resistance against various stresses, including heat, crystal violet, deoxycholic acid, and hydrogen peroxide. However, alkali-adapted cells showed no increased resistance against acid stress and heat-adapted cells did not show increased resistance against alkaline stress. Furthermore, alkaline treatment induced cell elongation with heterogenous size of the bacterium.

Isolation and characterization of the outer membrane from Vibrio parahaemolyticus

The outer membrane of Vibrio parahaemolyticus strain 3283-61 (serotype O2:K3) was isolated from blebs released upon spheroplast formation, in the presence of lysozyme and EDTA, by isopycnic sucrose density gradient centrifugation. SDS-PAGE of the outer membrane fraction prepared from cells grown in nutrient broth containing 3% (w/v) NaCl revealed five major proteins, designated a to e, with apparent approximate molecular weights: a, 44 000; b, 36 000; c, 33 500; d, 26 500; e, 22 000. An increase in NaCl concentration in the growth medium resulted in an increase of proteins b and c, whereas a decrease to 0.5% (w/v) induced two additional major proteins with respective molecular weights of about 35 000 and 32 000. Proteins a and b appeared to be loosely associated with the peptidoglycan layer since they were largely retained after extraction with 2% (w/v) SDS at 50 degrees C for 30 min. Proteins c and/or e may play a role in phage VP1-receptors since phage-resistant mutants derived from strain 3283-61 had significantly diminished amounts of both proteins. The major outer membrane proteins varied in number and molecular weight in strains of V. parahaemolyticus belonging to different K-serotypes.

Structure of a Novel Bacteriophage VP3 for Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine bacterium which causes gastroenteritis after consumption of contaminated sea food (7). Nakanishi et al (8) isolated three bacteriophages specific for this bacterium from sea water, human feces and a lysogenic strain of the bacterium, but they did not reveal the structure of these phages. Although many phages of V. parahaemolyticus were later also isolated from marine samples (2-4), their structural characteristics are still unknown, except for one phage that is morphologically similar to the T phages of Escherichia coli (10). We have attempted to isolate V. parahaemolyticus phages from sea water in order to investigate the morphology of these phages. Among the phages isolated, one, designated VP3, exhibits knob-like projections (probably capsomeres) around its head. Here we describe the ultrastructure of this novel phage. Strain Y-75-l (08:K20) of V. parahaemolyticus was used as the host stratin for propagation of phage VP3. Seventeen K-serotype pilot strains and three other strains of V. parahaemolyticus were also used for testing the host range of phage VP3. All the strains, except strain 3283-61 which has been maintained in our laboratory for many years, were obtained from the Research Institute for Microbial Diseases, Osaka University, Osaka. Bacteria were grown in 3%-NaCl broth (8) at 30 C with shaking. Solid medium contained 3%-NaCl broth supplemented with 1.5 and 0.6% agar for bottom and top agar layers, respectively. For isolation of phages, sea water samples collected near Tokushima were mixed with equal volumes of double-strength 3%-NaCl broth. The enrichment mixtures were inoculated with log-phase cultures of strain Y-75-l and incubated at 30 C for 5 hr. Single-plaque isolation of phage VP3 from the enrichment culture was carried out repeatedly as described by Adams (1). Phage titers were determined by the agar layer method (1). After incubation overnight at 30 C the efficiency of plating was estimated relative to that on strain Y-75-1. For preparation of phage stock, phage VP3 was added to a log-phase culture of host strain Y-75-l in 3%-NaCl broth and incubated at 30 C with shaking. After incubation for 5 hr phages were precipitated by 10% (w/v) polyethylene glycol 6000 and 1 M NaCl, collected by centrifugation at 8,000 Xg for 10 min, and resuspended in AAM solution containing 1% ammonium acetate and 0.01 M MgC1 2 (11). The phage suspension was placed on the top of stepwise CsCl gradients

Characterization of a Novel Vibrio parahaemolyticus Phage, KVP241, and Its Relatives Frequently Isolated from Seawater

A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA‐DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4‐type phages having a more elongated head.