[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to investigate the bacterial community structures of deep-sea water (DSW) and surface seawater (SSW) samples in Japan by molecular biological techniques. DGGE analyses and pyrosequencing analysis revealed that bacterial community structures of DSW were diverse and differed from those of SSW. This is the first report on the horizontal variation of bacterial community structures of DSW throughout Japan. In addition, pyrosequencing analysis revealed that the number of phyla in DSW was larger than that in SSW, and specific phyla, such as Firmicutes and Planctomycetes, were characterized by a higher proportion of the bacterial community structure in DSW than in SSW. Taken together, these results indicate that a variety of bacteria that are specifically adapted to the DSW environments can be expected to be found in DSW, and DSW would thus be a potential resource for novel or unique microorganisms and compounds.
[Show abstract][Hide abstract] ABSTRACT: The screening of malachite green (MG)-degrading microorganisms was carried out using various sources, namely, fish farms and a traditional fermented fishery product in Myanmar and Thailand. The enrichment culture method was performed using MG-containing broth media, and colonies that showed the decolorization of MG on plate media were isolated as MG-degrading candidates. From the results of the sequencing of the D1/D2 domain of the 26S rRNA gene, strain M3, a representative strain of MG-degrading candidates was identified as a halotolerant yeast, Debaryomyces
nepalensis. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis indicated that during the incubation of this strain, the MG concentration gradually decreased and eventually reached undetectable levels. Conversely, the concentration of leucomalachite green (LMG) increased, and the final amount of LMG in the broth culture was estimated to be approximately 40 % of the initial MG amount. In addition, results of proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis also showed that MG and the tautomer of MG or other aromatic decomposition products of MG were not detected as a major component at the end of incubation. These results suggest that strain M3 removed MG and changed approximately 40 % to LMG and 60 % to some metabolites other than LMG.
[Show abstract][Hide abstract] ABSTRACT: An antibacterial-substance-producing bacterium, namely, strain F412, was isolated from a traditional Myanmar shrimp product fermented with boiled rice. It was a gram-positive, spore-forming, and rod-shaped bacterium, and identified as Bacillus mojavensis on the basis of the gyrA sequence. The antibacterial substance of this strain was partially purified from a culture supernatant using two steps of column chromatography. This substance was found to be widely effective against gram-positive bacteria, including Listeria monocytogenes. The antibacterial activity of this substance was not susceptible to treatments with several proteolytic enzymes. The antibacterial activity gradually decreased with increasing treatment temperature, but it remained even after heating for 15 min at 121 °C. This antibacterial substance showed different molecular weights, as shown by the results of gel filtration and electrophoresis analyses. Staining results after electrophoresis suggest that the antibacterial substance might be a glycopeptide with an estimated molecular weight between 3.5 and 8.5 kDa. From the decrease in optical density of a culture of the L. monocytogenes treated with this antibacterial substance, it was suggested that this substance might have bacteriolytic activity.
[Show abstract][Hide abstract] ABSTRACT: Journal of Antibiotics, official journal of the Japan Antibiotics Research Association, is a print and online publication that focuses on research on antibiotics and related types of medicinal substances
No preview · Article · Apr 2015 · The Journal of Antibiotics
[Show abstract][Hide abstract] ABSTRACT: Bioremediation technologies have strong potential use in the less costly and more environmentally friendly removal of highly toxic hexavalent-chromium (Cr(VI)) compared with physicochemical technologies. Several Cr(VI)-reducing bacteria have been isolated; however, there are few studies on Cr(VI)-resistant and Cr(VI)-reducing actinomycetes. In this study, Cr(VI)-reducing actinomycetes were screened from estuarine, marine, and terrestrial samples on the basis of Cr(VI)-resistant and Cr(VI)-reducing ability. Of the 80 Streptomyces-like strains isolated, 20 strains were found to be resistant to 50 mg/l of Cr(VI). In addition, two strains isolated from the estuarine sediment of Tokyo Bay were found to be resistant to a concentration of 150 mg/l of Cr(VI). Furthermore, one Cr(VI)-reducing strain was found to remove 60 mg/l of Cr(VI) within 1 week and was identified as Streptomyces thermocarboxydus based on 16S rRNA gene analysis. The comparative evaluation with the type strain S. thermocarboxydus NBRC 16323 showed that our isolated strain had higher ability to grow at 27 °C and reduce Cr(VI) at a NaCl concentration of 6.0 % at 27 °C compared with the type strain NBRC 16323. These results indicate that our isolated strain have a potential ability to remove Cr(VI) from contaminated, highly saline sources without heating.
Full-text · Article · Feb 2015 · Applied Biochemistry and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: This study is a detailed description of the microflora of a traditional fishery product in Myanmar which is fermented with boiled rice. We approached this analysis from two viewpoints; namely, the culture-dependent and culture-independent methods.
In Southeast Asia, there are various types of traditional fermented fishery products. In this study, we isolated and characterized lactic acid bacteria (LAB) from small freshwater fish (tinfoil barb) fermented with boiled rice, a typical Myanmar fermented product, to contribute to the understanding of its fermentation process. Eight fermented fishery products were purchased from different markets in Yangon. Forty-three of the 46 isolates were identified as LAB, and they were classified into two groups: 40 homofermentative and three heterofermentative isolates, on the basis of their phenotypic characteristics. From the results of PCR-restriction fragment length polymorphism (RFLP) analysis and 16S rRNA gene sequencing, our isolates were identified as Lactobacillus plantarum-group, Lactobacillus farciminis, Lactobacillus futsaii, Lactobacillus reuteri,
Weissella paramesenteroides, and Pediococcus pentosaceus. In addition, L. plantarum and L. farciminis were identified as γ-aminobutyric acid (GABA)-producing LAB. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also carried out using DNA samples extracted from these fermented products. In comparison with culture-dependent methods, the results of T-RFLP analysis did not seem to have major contradictions.
Full-text · Article · Jan 2015 · Fisheries Science
[Show abstract][Hide abstract] ABSTRACT: Hyaluromycin (1), a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces sp. as a HAase inhibitor on the basis of HAase activity screening. The structure of 1 was elucidated through the interpretation of NMR data for the compound and its 3″-O-methyl derivative in combination with an incorporation experiment with [1,2-13C2]acetate. The compound's absolute configuration was determined by the comparison of its circular dichroism (CD) spectrum with those of other rubromycins. Hyaluromycin (1) consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2-enone (C5N) unit as an amide substituent of the carboxyl function; both structural units have been reported only from actinomycetes. Hyaluromycin (1) displayed approximately 25-fold more potent hyaluronidase inhibitory activity against hyaluronidase than did glycyrrhizin, a known inhibitor of plant origin.
[Show abstract][Hide abstract] ABSTRACT: A total of 605 actinomycetes were isolated from marine and terrestrial sediments of various Japanese coastal areas using several isolation media. The largest number of alginate lyase (A.L.)-producing strains were isolated from marine sediment samples collected in Otsuchi Bay in Iwate Prefecture. The activities of 18 A.L.-producing strains were measured using liquid culture media and were identified by 16S rDNA sequencing analysis. Among the 18 strains, one species belonging to the genus of Micromonospora (named as strain OB40608, isolated from Otsuchi Bay at a depth of 100 m) showed high A.L. activity and has never been reported as an A.L. producer, therefore it was selected for further study. A.L. was purified from culture supernatant of strain OB40608 by ammonium sulfate precipitation, cation exchange chromatography (Cellufine C-500 m) and gel filtration chromatography (Sephacryl S-200 HR). The final preparation showed a single band on polyacryl amide gel electrophoresis. The molecular weight of purified A.L. was 114 kDa, and the optimum temperature and pH were 50°C and pH 7.0, respectively.
Full-text · Article · Jan 2014 · NIPPON SUISAN GAKKAISHI
[Show abstract][Hide abstract] ABSTRACT: Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.
Full-text · Article · Mar 2013 · World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology)
[Show abstract][Hide abstract] ABSTRACT: Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.
[Show abstract][Hide abstract] ABSTRACT: Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32–80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli. Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p-nitrophenyl acetate, but almost undetectable for p-nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25–70 °C and at pH 8.0–9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA.
No preview · Article · Jul 2010 · Enzyme and Microbial Technology
[Show abstract][Hide abstract] ABSTRACT: The molecular diversity of bacterial chitinases in the bulk soils of arable land was investigated using culture-independent methods. The results demonstrate that bacterial chitinases in arable soils are highly diverse and comprise unique groups when their sequences were compared to those in public databases. The diversity of bacterial chitinases in arable soil was further evaluated using conventional phylogenetic analysis, the UniFrac analysis of the phylogenetic data, and the multidimensional scaling (MDS) analysis of T-RFLP profiles to elucidate the relationship between the diversity of bacterial chitinases and soil characteristics. These analyses indicate that environmental factors such as soil type and pH are responsible for shaping the composition of bacterial chitinases.
No preview · Article · Mar 2009 · Soil Biology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10,000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.
Full-text · Article · May 2008 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification.
cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification.
It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification.
The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.
Full-text · Article · Feb 2005 · Journal of Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: The microbial population dynamics at the start-up stage of a wastewater treatment reactor was investigated using terminal restriction fragment length polymorphism (T-RFLP) analysis based on 16S rDNA and rRNA gene sequences. The results of fragment peaks suggested that the number and activity of nitrifying bacteria increased in association with the start of nitrification, and the relative ratios of 16S rRNA of these bacteria changed prior to those of the 16S rDNA. Furthermore, multidimensional scaling (MDS) analysis revealed that the 16S rRNA exhibited wider dispersion than the 16S rDNA at the start-up stage, indicating that the diversity of 16S rRNA in the microbial communities was strongly affected by environmental changes.
No preview · Article · Feb 2004 · Journal of Bioscience and Bioengineering