A unique consortium of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria was obtained via a long-term, 3-step cultivation of isolates from organically-enriched marine sediment. We developed this microbial consortium for possible applications in the remediation of degraded habitats in closed aquaculture or other aquatic environments via microbial degradation. Analysis of media components found definitive evidence of nitrogen removal via the coupling of ammonia-oxidation and denitrification. The phylogenetic diversity of the consortium was investigated by performing polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) that targeted the 16S rRNA gene, and the functional genes involved in ammonia-oxidation (amoA) and denitrification (nirS, nirK, norB and nosZ). Consequently, no significant divergence was observed, and thus it is suggested microbial populations were selected via a long-term, 3-step incubation process. All of the 16S rRNA clones fell into three phylogenetic groups, namely, γ -proteobacteria, Actinobacteria and Flavobacteria. For almost half of the clones, the closest relatives in the database were identified as Alcanivorax spp. and these clones were present at all cultivation stages. The presence of these species as the dominant clones is significant since these bacterial species are known to reduce nitrate to nitrite.Accordingly, their abundance in our microbial consortium may have been responsible for the observed stepwise denitrification. All sequences of the amoA gene were identified to be Nitrosomonas lineage. Half of the nirS clones were identified to be from one major group of well-known denitrifying bacteria, Pseudomonas sp. Furthermore, 70% of the nirK clones were closely related to the nirK sequences of uncultured bacterial clones isolated from arable soil. The gnorB clones consisted of clusters exclusively, and formed a distinct cluster from the novel sequences of cultivated species. The nosZ clones also were not found in any of the closest relatives in the database including the uncultured bacterium from marine sediment. The unique clones obtained from the functional genes were related to each denitrification step.
To isolate bacteria capable of producing enzymes that lyse cell wall, we screened soil samples from Rokko Mo zuntain where is situated in west Japan. The isolated strains KH1, KH2 and KH3 that secrete yeast cell wall lytic enzymes are described herein. The activity of the enzymes, after isolation from culture supernatants, were examined. The lytic enzyme produced by strain KH3 was the most active in both cell wall degradation and β-1, 3 glucanase activity in comparison with commercially available enzymes. This enzyme also had the highest thermo- and pH-stability. On the baisis of partial 16S rDNA sequence analyses, KH1 and KH3 were identified as Cellulosimicrobium sp. and KH2 as Bacillus sp. KH3 was tested by DNADNA hybridization and GC content in an attempt to precisely classify it, because the 16S rDNA sequence similarities to other strains were very high at 99.4%. These chemotaxonomic experiments indicate that KH3 belongs to Cellulosimicrobium cellulans.
Narezushi is one of Japanese traditional foods and is made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of samma-narezushi (narezushi using saury, Cobolabis saira) was analyzed by the 16S ribosomal RNA gene (rDNA) clone library. Randomly selected 89 clones were sequenced and phylogenetically analyzed. The sequences were classified into 12 operational taxonomy units (OTUs) at the 97% identity threshold. Most of the clones (89%) were lactic acid bacteria (LAB) and included Lactobacillus sakei, Leuconostoc gelidum, Lactococcus lactis subsp. lactis and L. pisium. The most predominant clone was L. sakei at 72% of the clones. The second most abundant clone was L. gelidum. These results suggest that L. sakei mainly contributes to the lactic acid fermentation process, taste and flavor of samma-narezushi. The present study showed that the culture independent method is useful for the microbial diversity analysis of narezushi.
Anti-VRE and anti-MRSA activities of new quinolone derivatives [The two quinolone derivatives are 8- [3- [(ethylamino) methyl] -1-pyrrodinyl] -7-fluoro-9, 1- [(N-methylimino) methano] -5-oxo-5H-thiazolo [3, 2-a] quinolone-4-carboxylic acid (compound A) and 7-fluoro-8-morpholino - 9, 1- [(N-methylimino) methano] - 5 - oxo - 5H - thiazolo [3, 2-a] quinolone-4-carboxylic acid (compound B)] and their synergism with commercial antibiotics were investigated. Compound A exhibited potent antibacterial activity against VRE and MRSA among the five new quinolone compounds tested, and showed superior activity to pefloxacin, ofloxacin and levofloxacin, which are clinically in use these days. With respect to the anti-VRE activity, compound A showed synergism with fosfomycin (FOM), and partial synergism with ampicillin (ABPC), gentaicin (GM), minocycline (MINO) and vancomycin hydrochloride (VCM). Partial synergism in anti-VRE activity was also observed between compound B and GM, MINO, FOM and VCM. Compound A also showed synergism with MINO and FOM in anti-MASA activity. Partial synergism was observed with ABPC, GM and VCM. Synergism with ABPC was not detected in anti-MRSA activity. On the other hand, the synergism of compound B with FOM, and the partial synergisms with ABPC, GM and MINO were also found against MRSA. No synergism with ABPC was found against MRSA. These results suggested that compound A and B could possibly reduce the daily administration dose of these antibiotics in the treatment of nosocomial infections, and also reduce the possibility of the occurrence of nosocomial infections caused by VRE and/or MRSA.