Tributyltin-resistant marine bacteria: a summary of recent work

Summary A tributyltin chloride (TBTCl)-resistant bacterium,Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 M TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by whichE. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment fromHindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage andE. coli. TBTCl-tolerant marine bacteria other thanAlteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments fromAlteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.     

Occurrence of tributyltin-tolerant bacteria in tributyltin- or cadmium-containing seawater.

Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.     

Occurrence of tributyltin-tolerant bacteria in tributyltin- or cadmium-containing seawater.

Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.     

溃烂症眼斑拟石首鱼分离的灿烂弧菌的鉴定

摘要：【目的】从患溃烂症的眼斑拟石首鱼分离到优势菌株M-1,并进一步对该菌的系统发育学进行了分析。【方法】采用形态学、生理生化鉴定,结合16S rRNA和HSP60基因序列分析。【结果】16S rRNA基因同源性分析,菌株M-1与灿烂弧菌(AJ874361、AY046955)聚为一群;HSP60基因(groES)序列分析表明M-1与弧菌(EU653883、AY837440)聚为一个分支。【结论】菌株M-1属于灿烂弧菌(Vibrio splendidus)。     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

假单胞菌M-18qscR突变株的构建及其对抗生素合成的调控

在革兰氏阴性菌中,全局性调控因子QscR参与菌群传感调节系统,调节多种毒素因子、次生代谢产物、稳定期基因以及参与生物膜形成的基因的表达,它通过与靶基因DNA启动子的调节元件结合,调节基因转录。假单胞菌株(Pseudomonas sp.)M-18是促进植物生长的根际细菌,能同时分泌藤黄绿菌素(pyoluterion,Plt)和吩嗪-1-羧酸(phenazine-1-carboxylicacid,PCA)。运用同源重组技术,构建了假单胞菌(Pseudomonas sp.)M-18株的qscR突变菌株M-18Q。比较野生株M-18和突变株M-18Q生物合成PCA和Plt的产量,在28℃恒温条件下,在PPM和KMB培养基中M-18Q菌株合成PCA的量分别约为野生型M-18菌株的4～6倍和3～5倍,分别达到480μg/mL和140μg/mL。在PPM培养基中,野生株M-18和突变株M-18Q几乎都没有Plt的合成,而在KMB培养基中,突变菌株和野生型M-18合成Plt的量基本一致。反式互补实验表明,在qscR突变株M-18Q中,PCA生物合成受到抑制而Plt的生物合成却不受影响。phzA基因是吩嗪合成基因簇中第一个基因,phzA‘-’lacZ翻译融合实验表明,qscR基因产物通过抑制PCA合成基因簇的表达,实施负调控作用。结果表明qscR基因是作为一个全局调控基因区别性地调控PCA和Plt的生物合成。     

一株与鳆发光杆菌最相似细菌的研究

对从海鲜食品中分离到的1株少见细菌M1进行系统分类鉴定。采用常规方法[1]进行分离培养,以形态学特征、培养特性、生理生化特征以及分子生物学等方法对其进行分类鉴定。结果可见,该菌株为革兰氏阴性杆状细菌,16SrDNA核甘酸序列测定与鳆发光杆菌最为相似,生理生化特征与发光杆菌属、弧菌属相近,但都不完全相符,而与鳆发光杆菌相似性最高。     

Effect of Temperature on Adhesion of Vibrio Strain AK-1 to Oculina patagonica and on Coral Bleaching

Laboratory aquarium experiments demonstrated that Vibrio strain AK-1 caused rapid and extensive bleaching of the coral Oculina patagonica at 29 degrees C, slower and less-complete bleaching at 23 degrees C, and no bleaching at 16 degrees C. At 29 degrees C, the application of approximately 100 Vibrio strain AK-1 cells directly onto the coral caused 50 and 83% bleaching after 10 and 20 days, respectively. At 16 degrees C, there was no bleaching, even with an initial inoculum of 1.2 x 10 bacteria. To begin to understand the effect of seawater temperature on bleaching of O. patagonica by Vibrio strain AK-1, adhesion of the bacteria to the coral as a function of temperature was studied. Inoculation of 10Vibrio strain AK-1 organisms into flasks containing 20 ml of seawater at 25 degrees C and a fragment of O. patagonica resulted in net levels of bacterial adhesion to the coral of 45, 78, and 84% after 2, 6, and 8 h, respectively. The adhesion was inhibited 65% by 0.001% d-galactose and 94% by 0.001% methyl-beta-d-galactopyranoside (beta-M-Gal). After the incubation of Vibrio strain AK-1 with the coral for 6 h, 42% of the input bacteria were released from the coral with 0.01% beta-M-Gal, compared to less than 0.2% when beta-M-Gal was present during the adhesion step. Adhesion did not occur when Vibrio strain AK-1 was grown at 16 degrees C, regardless of whether the corals were maintained at 16 or 25 degrees C, whereas bacteria grown at 25 degrees C adhered to corals maintained at 16 or 25 degrees C. Bacteria grown at 25 degrees C adhered avidly to Sepharose beads containing covalently bound beta-d-galactopyranoside but failed to bind if grown at 16 degrees C. These data suggest that elevated seawater temperatures may cause coral bleaching by allowing for the expression of adhesin genes of Vibrio strain AK-1.     

Enhancement of virulence of Entamoeba histolytica by histamine in vitro

Three isolates of E. histolytica isolated and maintained in modified Boeck and Drbohlav's medium and the axenic nonpathogenic strain NIH-200 maintained in Diamond's TPS-1 medium were used to assess the effect of histamine added to the cultures on their pathogenicity in just weaned rats of Charles Foster strain. Initially when the three polyxenic isolates were examined for their Pathogenicity after growing them with graded concentrations of histamine viz. M-2 through M-6 (log dilutions of the molar concentration of histamine dihydrochloride) the caecal scores were significantly enhanced in cultures grown with M-2 and M-3 concentrations. Subsequently NIH-200 strain was examined similarly after growing it with 20 micrograms/ml of histamine dihydrochloride through three generations. The Neal's caecal score of NIH-200 increased significantly stepwise up to second subculture and became stabilised at third generation with histamine.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.     

Purification and characterization of Vibrio cholerae O139 fimbriae

Abstract A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V. cholerae non-O1 S7-like pili. The former was purified and characterized. The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V. cholerae non-O1 S7 pili. This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested. The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.     

Improved β-glucan yield using an Aureobasidium pullulans M-2 mutant strain in a 200-L pilot scale fermentor targeting industrial mass production

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches are known to be immune activation agents and are incorporated in anti-cancer drugs and health-promoting supplements. β-Glucan concentration was 9.2 g/L in a 200-L pilot scale fermentor using mutant strain Aureobasidium pullulans M-2 from an imperfect fungal strain belonging to A. pullulans M-1. The culture broth of A. pullulans M-2 had a faint yellow color, whereas that of the wild-type had an intense dark green color caused by the accumulation of melanin-like pigments. β-Glucan produced by A. pullulans M-2 was identified as a polysaccharide of D-glucose monomers linked by β-(1→3, 1→6)-glycosidic bonds through GC/MS and NMR analysis. When a conventional medium was used in the culture of A. pullulans M-2 in a 3-L jar fermentor, β-glucan concentration was 1.4-fold that produced by the wild-type. However, when a medium optimized by statistical experimental design was used with dissolved oxygen at 10%, the β-glucan concentration was 9.9 g/L with a yield of 0.52 (g β-glucan/g consumed sucrose), 2.9-fold that of the wild-type. This level of productivity was reproduced when the fermentation was scaled up 200-L. The industrial production of high β-glucan without melanin-like pigments is highly expected, as a health-promoting supplement or functional food.     

Inhibition of Vibrio parahaemolyticus by a bacteriocin-like inhibitory substance (BLIS) produced by Vibrio mediterranei 1

AIMS: The aim of this research was to identify and partially purify new bacteriocin-like substances from strains of halophilic 'non-cholera' vibrios isolated from food sources. METHODS AND RESULTS: Forty-five halophilic Vibrio spp. strains were screened for antimicrobial production. Vibrio mediterranei 1, a nonpathogenic strain, showed antimicrobial activity towards Vibrio parahaemolyticus spp. and related species. The bacteriocin-like inhibitory substance (BLIS), released by the bacteria into growth media, was concentrated by ultrafiltration and characterized. BLIS was sensitive to proteinase K, was stable in the pH range 5-9, was resistant to organic solvents and was heat stable up to 75 degrees C. Initial purification of BLIS by size exclusion chromatography showed an apparent molecular mass of 63-65 kDa. CONCLUSIONS: This study reports the ability of V. mediterranei 1 to produce a bacteriocin-like substance inhibiting growth of V. parahaemolyticus spp. and other closely related bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The strong activity of BLIS towards the human and fish pathogen V. parahaemolyticus and the persistence of antimicrobial properties under a variety of different conditions suggest its potential application in food microbiology.     

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.     

Isolation and properties of extracellular lipase of native (B-10) and mutant (M-1) Serratia marcescens strains

The cultivation conditions of wild-type strain V-10 and mutant strain M-1 (overproducer of endonuclease and chitinase) of Serratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximal lipase yield (840 AU/ml) after 10-12 h of cultivation; the strain M-1 (33 AU/ml), after 25-30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acid medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed a higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and inactive in the presence of cetyltrimethylammonium bromide.     

Ethanol fermentation of beet molasses by a yeast resistant to distillery waste water and 2-deoxyglucose

A flocculent killer yeast, Saccharomyces cerevisiae strain H-1, which was selected for ethanol fermentation of beet molasses, has a tendency to lose its viability in distillery waste water (DWW) of beet molasses mash after ethanol fermentation. Through acclimations of strain H-1 in DWW, strain W-9, resistant to DWW, was isolated. Strain M-9, resistant to 2-deoxyglucose was further isolated through acclimations of strain W-9 in medium containing 150 ppm 2-deoxyglucose. A fermentation test of beet molasses indicated that the ethanol productivity and sugar consumption were improved by strain M-9 compared to the parental strain H-1 and strain W-9. The concentration of ethanol produced by strain M-9 was 107.2 g/l, and the concentration of residual sugars, which were mainly composed of sucrose and fructose, were lower than those produced by the parental strain H-1 and strain W-9 at the end of fermentation of beet molasses.     

小鼠血清中杀O139霍乱弧菌抗体检测方法的初步建立

目的研究探索O139霍乱弧菌杀弧菌抗体的检测方法。方法用微孔板培养和琼脂平板克隆计数相结合的杀弧菌抗体检测方法,对实验菌株及稀释度、补体浓度等关键参数进行筛选;对50份小鼠免疫血清进行杀弧菌抗体滴度检测,并与O139群霍乱弧菌LPS Ig G抗体滴度进行相关分析;对该方法的特异性、线性和精密性进行了验证。结果筛选出最佳菌株为20100603菌株,最佳稀释度倍数为2 000倍,补体最佳稀释倍数为16倍。O139群霍乱弧菌小鼠免疫血清检测到较高的杀弧菌抗体滴度而PBS小鼠免疫血清未检测到杀弧菌抗体滴度。小鼠免疫血清杀弧菌抗体滴度与O139群霍乱弧菌LPS Ig G抗体滴度之间存在正相关关系。验证结果显示,在抑制剂浓度达到1.0~2.0 A600时,抑制率100%;线性回归方程为y=-1.093x+5.058,其相关系数为-0.999,P0.05;方法批内CV值为15.72%,批间CV值为23.47%。结论初步建立了O139霍乱弧菌杀弧菌抗体的检测方法,该方法具有较高的特异性、线性和精密度。