Transposition and expression of GEP gene in the genome of Vibrio harveyi to monitor its adherence in shrimp larvae

Expression of green fluorescent protein encoded by GFP gene in Vibrio harveyi was investigated to understand the ability of the gene as a molecular marker for adherence of this pathogenic Vibrio in shrimp larvae. The GFP gene was inserted into pUC18Not and pUTmini-Tn5 to generate a recombinant plasmid pWGO2 and pWGO3, respectively, which was transferred into the three isolates of V. harveyi employing diparental mating. Recombinant E. coli carrying pWGO2 and pWGO3 resulted in green-fluorescent colonies and cells due to the production of GFP. However, al1 of mini-Tn5, including mini-Tn5-gfp were not successful1y transferred to V. harveyi. Therefore, we used mini-Tn10 (pLOFKm-gfp) for inserting of gfp gene into V. harveyi genome. Although we could obtain relatively high (l0 pangkat -8) transconjugans employing Tn10, only one of Tnl0 derived isolate of V. harveyi G3 (G3-Tn1Ogfp) showed gfp expression and was further employed for adherence assay. Fluorescent 03-TnlOgfp cells could be observed inside the digestive tract of shrimp larvae and could be distinguished from vibrio that naturally exist in shrimp larvae

GENETIC DIVERSITY OF AMPICILLIN-RESISTANT Vibrio ISOLATED FROM VARIOUS STAGES OF TIGER SHRIMP LARVAE DEVELOPMENT

This research was carried  out to  study  genetic  diversity of ampicillin-resistant Vibrio  from various  stages of tiger  shrimp larvae  (Penaeus Monodon) development from,Tambak Inti Rakyat hatchery, near Labuan, West Java, Indonesia. A total of 25 ampicillin-resistant Vibrio isolates were isolated using thiosulphate  citrate bile-salt  sucrose  agar (TCBS-Agar) and seawater  complete agar (SWC-Agar). Physiological and biochemical characterization showed that the isolates could be grouped into only two species, i.e. V. harveyi from the egg stage; and V. metschnikovii from larvae and post-larval stage (i.e nauplius, zoea, mysis, PLi, PL5, PL,0, and PL,5). These isolates were also present in their respective rearing water  of  each stage  and  some natural feed.  Schizotyping analysis employing restriction endonuclease Noll (5'-GC4GGCCGC) indicated that the isolates could be grouped into  at least  13 different  genotypes.  Therefore,  schizotyping  was  more discriminative than physiological characterization. This study showed that particular groups of Vibrio colonized all stages of shrimp larvae and demonstrated closed phylogenetic relationship. These groups of Vibrio might be  the dominant microbiota which could suppress the development of other Vibrio including the pathogenic Vibrio. Key words : Shrimp/ampicillin-resistant K/fcno/schizotypin

SOLATION AND CHARACTERIZATION OF A NOVEL BENZOATE- UTILIZING Serratia marcescens

A new benzoate-utilizing strain, Serratia marcescens DS-8, isolated from the environment was characterized. The strain was enterobacilli, Gram negative, mesophilic, non halophilic, and aerobic bacterium that showed motile ovale-rod shaped cells. The isolate produced extracellular chitinase, protease, and prodigiosin (a red pigment produced by several Serratia strains yielding bright red or pink colonies). A physiological assay using Microbact* test showed that the strain was closely related to Klebsiella ozaenae  (49.85%) and Serratia liquefaciens  (24.42%), respectively. However, 16S rRNA sequence analysis indicated that the strain was closely related to  S. marcescens  DSM 30121 with similarity level of 98%. DS-8 strain was able to synthesize its own vitamins. Optimum growth in benzoate was obtained at pH between 7-8.5 and NaCl concentration of 1-1.5% (w/v). The isolate could grow in benzoate-containing medium up to 10 mM. Other carbon sources that could support the growth of DS-8 were casamino acid, glutamate, glucose, acetate, potato starch, and ethanol. Keywords: Serratia marcescens/aromatic degradation/168 rRNA sequenc

PRESENCE OF hemA-LIKE AND hemT-LlKE GENES IN A JSUMBER OF ANOXYGENIC PHOTOSYNTHETIC BACTERIAL ISOLATES FROM INDONESIA AND SOIL SAMPLES FROM BOGOR AREA

The Rhodobacter sphaeroides hemA and hemT are known to encode a distinct 5-aminolevulinic acid (ALA)-synthase isozyme. This enzyme catalyzes the first and rate limiting step in ALA biosynthesis through the C4 pathway. This study was carried out to detect hemA-\\ke and hemT-\\ke genes in twenty Anoxygenic Photosynthetic Bacterial (APB) isolates from several wetland areas in Indonesia, and four DNA samples that were isolated from four soil samples obtained from Bogor area. Hybridization techniques of Southern and dot blot were used, using hemA and hemT fragment as probes. Southern hybridization analyses indicated the presence of hemA-\\ke gene in five of APB isolates, i.e., MB15, MB16, MB21.2, MB55 and MB6, whereas hemT-\\ke gene was detected only in MB15. Dot blot hybridization analyses suggested that the soil samples from waterlogged paddy-field, dry paddy-field as well as a mud pond were predominantly occupied by prokaryotic organisms which harboured hemA-]\ke gene. However, /iem7"-like sequences were also found in soil sample from dry paddy-field. Key words:    hemA-\\ks gene / hemT-\\ke gene / Southern hybridization analysis / dot blot hybridization analysis

Isolasi, Karakterisasi, dan Kloning Gen Penyandi α α α α α-Amilase Bakteri Halofil Moderat asal Bledug Kuwu

A moderately halophilic bacteria, BK-05, was isolated from Bledug Kuwu, a saline terrestrial area at Central Java. Based on partial sequence of 16S-rRNA gene, the isolate was closely related to Halobacillus litoralis. This isolate showed amylolytic activity when grown on saline media [15% (w/v) NaCl] suplemented with starch. A pair of primer was designed based on the sequence of amyH gene from Halomonas meridiana and Pseudoalteromonas haloplanktis. PCR amplification using these primers showed three DNA bands with each size approximately 1.50, 1.00, and 0.75 kb. Partial DNA sequencing analysis based on its deduced protein sequence revealed that the 1.50 kb band was closely related to the sequence of metalloprotease from Bacillus subtilis (approximately 54.3% identity in 184 amino acid overlap). Southern hybridization analysis showed that the 1.50 kb fragment was located within a 4.0 kb fragment of BamHI, 4.8 kb of EcoRI, 4.3 kb of HindIII, and 4.0 kb of XhoI digestion of BK-05 genomic DNA, respectively

CHARACTERIZATION OF THREE BENZOATE DEGRADING ANOXYGENIC PHOTOSYNTHETIC BACTERIA ISOLATED FROM THE ENVIRONMENT

Three  anoxygenic photosynthetic bacteria, DS-1, DS-4 and Cas-13, have been examinated  for  themorphological and physiological  properties. All strains were rod-shape cells with  a swollen terminal  endGram  negative, motile, non-halophilic, non-alkalophilic  and non-acidophilic,  and capable of utilizinbenzoate aerobically and photo-anaerobically. Sequence analysis of part of 16S rRNA genes showed that DS1 and Cas-13 were closely related to Rhodopseudomonas palustris Strain 7 with a similarity of 97%, whereaDS-4 may not be closely related to the former two strains with a similarity of 78% based on the constructephylogenic  tree. Spectral analysis indicated that the three  bacteria  had  bacteriochlorophyl  a  and normaspirilloxanthin series. Growth in medium enriched with vitamin and supplemented with benzoate as their sole C-sources wabetter than in medium without vitamin. Benzoate degradation in medium with vitamin was accelerated. Thability  to grow on benzoate without added vitamins indicated  that  the bacteria were able to synthesize  theown vitamins. Key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16S rRNA gene

Solation and Characterization of a Novel Benzoate- Utilizing Serratia Marcescens

A new benzoate-utilizing strain, Serratia marcescens DS-8, isolated from the environment was characterized. The strain was enterobacilli, Gram negative, mesophilic, non halophilic, and aerobic bacterium that showed motile ovale-rod shaped cells. The isolate produced extracellular chitinase, protease, and prodigiosin (a red pigment produced by several Serratia strains yielding bright red or pink colonies). A physiological assay using Microbact* test showed that the strain was closely related to Klebsiella ozaenae (49.85%) and Serratia liquefaciens (24.42%), respectively. However, 16S rRNA sequence analysis indicated that the strain was closely related to S. marcescens DSM 30121 with similarity level of 98%. DS-8 strain was able to synthesize its own vitamins. Optimum growth in benzoate was obtained at pH between 7-8.5 and NaCl concentration of 1-1.5% (w/v). The isolate could grow in benzoate-containing medium up to 10 mM. Other carbon sources that could support the growth of DS-8 were casamino acid, glutamate, glucose, acetate, potato starch, and ethanol

Cloning of Genomic DNA Flanking Transposon in the Nonpathogenic Mutant of Xanthomonas axonopodis pv. glycines M715

The objective of this work is to clone flanking DNA derived from Tn-5 mutagenesis of wild type strain Xanthomonas axonopodis pv. glycines as first step to clone and to identify the gene involved in pathogenicity mechanism. We have localized the flanking DNA fragment from a nonpathogenic mutant of Xag M715. Southern hybridization analysis using 2.8 kb EcoRI from pYR103 as a probe showed that the fragment is located within 2.0 kb PstI fragment. A 0.7 kb flanking DNA was amplified using inverse PCR technique, and inserted into pGEM-T Easy vector generating a 3.7 kb recombinant plasmid (pAA01). Southern hybridization analysis of the wild type (YR32) with pAA01 as a probe indicated a hybridization signal located at approximately 3.0 kb PstI fragment. DNA sequence analysis revealed that the DNA fragment has a 64% identity to a vir gene of Bacillus anthracis

Population Dynamics of Yeasts and Lactic Acid Bacteria (LAB) During Tempeh Production

Yeasts and lactic acid bacteria (LAB) are commonly found in tempeh and has been studied separately. However, comprehensive study on population dynamics of yeasts and LAB during tempeh production, including the effect of the difference tempeh production methods has not been reported. This research was aimed in studying the effect of different methods of tempeh production applied in tempeh home industry on the dynamics of yeast and LAB communities. Population dynamics was expressed as both changes of colony number and its phylotype. Samples were obtained from five stages and from two different methods of tempeh production. Observations were carried out employing colony counting on selective media followed by Terminal Restriction Fragment Length Polymorphism (T-RFLP). The study indicated that the population of yeasts and LAB during tempeh production were dynamic and different between these methods. Tempeh production methods affected the presence of yeasts and LAB population as indicated by difference in colony number, the number and diversity of phylotype, as well as number of specific phylotypes grew on plates