Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Eleven carbazole (CAR)-degrading bacterial strains were isolated from seawater collected off the coast of Japan using two different media. Seven isolates were shown to be most closely related to the genera Erythrobacter, Hyphomonas, Sphingosinicella, Caulobacter, and Lysobacter. Meanwhile, strains OC3, OC6S, OC9, and OC11S showed low similarity to known bacteria, the closest relative being Kordiimonas gwangyangensis GW14-5 (90% similarity). Southern hybridization analysis revealed that only five isolates carried car genes similar to those reported in Pseudomonas resinovorans CA10 (car(CA10)) or Sphingomonas sp. strain KA1 (car(KA1)). The isolates were subjected to GC-MS and the results indicated that these strains degrade CAR to anthranilic acid.

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibioticproducing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.

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The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

A method for bioremediation of chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) by a carbazole-utilizing bacterium, Pseudomonas sp. strain CA10, was developed. CA10 cells transferred to carbon- and nitrogen-free mineral medium supplemented with 1 mg carbazole (CAR)/ml grew rapidly during the first 2 days; and the cells at the end of this rapid growth period showed the highest 2,3-dichlorodibenzo-p-dioxin (2,3-Cl2DD)-degrading activity. The CA10 cells pregrown for 2 days efficiently degraded 2,3-Cl2DD in aqueous solution at either 1 ppm or 10 ppm. The effect of inoculum density on the efficiency of 2,3-Cl2DD degradation was investigated in a soil slurry microcosm [ratio of soil:water = 1:5 (w/v)]. The results showed that a single inoculation with CA10 cells at densities of 10(7) CFU/g soil and 10(9) CFU/g soil degraded 46% and 80% of 2,3-Cl2DD, respectively, during the 7-day incubation. The rate of degradation of each CDD congener, 2-ClDD, 2,3-Cl2DD, and 1,2,3-Cl3DD (1 ppm each) by strain CA10 in the soil slurry system was not significantly influenced by the coexistence of the other congeners. Using this soil slurry system, we tried an experimental bioremediation of the actual dioxin-contaminated soil, which contained mainly tetra- to octochlorinated dioxins. Although the degradation rate of total CDD and CDF congeners by a single inoculation with CA10 cells was 8.3% after a 7-day incubation, it was shown that strain CA10 had a potential to degrade tetra- to hepta-chlorinated congeners including the most toxic compound, 2,3,7,8-tetrachlorinated dibenzo-p-dioxin.

Streptomyces are a group of Gram-positive bacteria belonging to the Actinobacteria class, which are among the most important bacteria for producing secondary bioactive metabolites such as antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. Recently, we have isolated the Streptomyces sp. GMR22 from Cajuput rhizospheric soil at Wanagama Forest, Indonesia. GMR22 produced a secondary metabolite that inhibited Candida albicans with IC 50 of 62.5μg/mL. The objective of this work was to reveal the novel secondary metabolites from GMR22 by genome mining approach. The antiSMASH 3.0 was used to predict gene clusters that encode the biosynthetic pathways of secondary metabolites in the genome of GMR22, and their core chemical structures. The phylogenomic analysis showed that GMR22 was closely related to Streptomyces bingchenggensis BCW1, as well as to the large genome size (9.5-12.7Mbp) groups of Streptomyces. AntiSMASH 3.0 analysis revealed that the genome of Streptomyces sp. GMR22 harbored at least 63 gene clusters that encode the biosynthetic pathways of secondary metabolites. It was the highest number of gene clusters had been observed among the members of Streptomyces groups, with polyketide synthetase (PKS) was predicted as the major groups of the identified gene cluster products. The results suggested that GMR22 could be a strong potential candidate for secondary bioactive metabolites source.

Lactobacillus plantarum Dad-13 is a probiotic candidate from dadih, a traditional fermented buffalo milk from West Sumatera, Indonesia. This study aimed to evaluate safety aspects of L. plantarum Dad-13 in high dose consumption on Sprague Dawley rats. Two treatment groups were fed with 10 11 CFU/mL/day of L. plantarum Dad-13 for 14 and 28 days respectively. Two placebo groups were fed with 1 mL skimmed milk per day for 14 and 28 days and a fifth, untreated group used as control. Feed intake and body weight were monitored, while blood samples and mesenteric lymph node (MPN) organs were dissected. Organ weight, leukocyte profiles, glutamic-oxaloacetic transaminase (GOT) activity, plasma malondialdehyde (MDA) concentration and intestinal morphology were measured. Microbial analyses were conducted on fecal matter, digesta, blood and organs. Results showed that consumption did not negatively affect general health, organ weight, leukocyte profiles, GOT activity, MDA concentration and intestinal morphology. Numbers of L. plantarum in treated rats' feces were significantly increased, indicating its survival in gastrointestinal tracts. Bacteria in the blood and organs of both groups were identified using rep-PCR with BOX A1R primer, which revealed that it was not identical to L. plantarum Dad-13. Thus, L. plantarum Dad-13 did not translocate in the organs and blood of rats. Therefore, L. plantarum Dad-13 is suggested as likely to be safe for human consumption.