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

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

A Novel Integron in the Genome of Escherichia Coli Isolated From Indonesian Monitor Lizard (Varanus Spp).

The genotype of antibiotic resistance in natural isolates of Escherichia coli was determined through integron detection and characterization of the associated antibiotic resistance. E. coli SG2 isolated from Varanus salvator of Java demonstrated resistance to spectinomycin (50ng/ml) and streptomycin (SOng/ml). Integron detection indicated that eight isolates out of nine E. coli isolates possessed a conserved segment of the integron. Amplification of the inserted cassette of the integron in this SG2 isolate yielded a 1-kb DNA fragment. Sequence analyses indicated that this fragment was homologous with aad gene, which confirmed the resistance to spectinomycin/streptomycin. This is the first report on the presence of integron in the E. coli isolated from the environment

Heterologous Expression of a Chitinase Gene From Aeromonas Caviaein Pseudomonas Fluorescens

A transcriptional fusion for an Aeromonas caviae chitinase gene was constructed under the control of a constitutive promoter of the kanaraycin resistance gene (PKmR). The construct was inserted into a medium copy number broad host range plasmid vector to yield recombinant plasmid pAM340, which harbored transcriptional fusion PKmR- chi. Another transcriptional fusion, Ptac-chi, in a recombinant plasmid pAM630, was conducted as comparison. Triparental mating of E. coli carrying the recombinant plasmids with Pseudomotws fluorescens 5100, a phyllosphere bacterium, was performed. Pseudomonas fluorescens 5100 exconjugants were examined for constitutive expression of chitinase employing a spectrophotometric assay; they showed stronger chitin degradation activity than Escherichia coli transformants. Using a fungal antagonism plate assay, this chitinolytic P. fluorescens, however, could not inhibit selected phytopathogenic fungi

Adherence and Pathogenicity Assay of Vibrio Harveyi in Tiger Shrimp (Penaeus Monodon) Larvae for Screening Biocontrol Agent

Rifampicin-resistant marker was employed as a reporter to detect the adherence and colonization of V. harveyi in shrimp larvae. Vibrio harveyi P1B and YA32.2 were isolated from dead shrimp larvae in Besuki, Northern Coast of East Java, while V. harveyi HB3, was isolated from pristine sea water in Pacitan, Southern Coast of East Java. Vibrio metschnikovii used as biocontrol agent was isolated from healthy shrimp larvae in Serang, West Java. Spontaneous mutation was conducted to generate V. harveyi P1B, YA32.2 and HB3 resistant to rifampicin. These mutants exhibited similar survival ability to their parental (wild type) strains. Significant larval mortality was observed in shrimp larvae inoculated with YA32.2 than that of larvae inoculated with P1B. Larvae inoculated with HB3 showed the lowest mortality. Bacterial cell count of Vibrio Rf* in dead larvae were 103-104 cells/larvae. Isolates of Vibrio metschnikovii Z and M as biocontrol candidates effectively reduced the growth and adherence ability of YA32.2 to shrimp larvae. Larval mortality in rearing water inoculated simultaneously with YA32.2 and V. metschnikovii was lower than the one inoculated with YA32.2 alone. Therefore, Vibrio metschnikovii Z or M could be developed as an effective probiotic or biocontrol agent for V. harveyi in shrimp hatcheries

Isolation and Characterization of Mannanolytic Thermophilic Bacteria From Palm Oil Shell and Their Mannanase Enzyme Production Properties

A mannanolytic thermophilic bacterium (L-07) was isolated from palm oil shell after 2 days ofenrichment in liquid medium supplemented with 1% palm kernel meal as mannan source. Sequenceanalysis of 16S-rRNA indicated that L-07 was similar (98%) to Geobacillus stearothermophilus, aspecies of thermophilic aerobic bacteria. We found that G. stearothermophilus L-07 producedextracellular β-1,4-mannanases, but no β-manosidase and α-galactosidase activities. The growth of L-07reached its maximum (3.0 x 106 cell/ml) at 12-20 hours, while the highest β-mannanase activity (0.52U/ml) was observed in culture medium after 36 hours of cultivation at 60oC. The medium containinglocust bean gum was the best for producing extracellular β-1,4-mannanases compared with kolang kaling,konjak, and palm kernel meal. SDS-PAGE and zymogram analysis demonstrated that crude mannanasecomplex of L-07 from locust bean gum containing medium comprised three active bands with molecularweight of 85, 73 and 50 kDa

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

Keragaman Genetika Xanthomonas Axonopodis Pv. Glycines Asal Kedelai Varietas Edamame di Indonesia

Xanthomonas axonopodis pv. glycines cause bacterial pustule disease caused a serious disease in Edamame cultivation in Indonesia. We collected a total of 29 X. axonopodis pv. glycines isolates from Edamame fields at Jember, Ciawi, Cipanas and Bogor. The genetic diversity analysis of all isolates employing ARDRA and ISR technique showed six and seven different DNA profile, respectively. Therefore there are at least seven strains of X. axonopodis pv. glycines infected Edamame in Indonesia. Both CPI from Cipanas and JA4 from Sukorejo Jember isolates possess unique DNA profle and genetically are not closely related to other isolates