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1.  Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis 
Many deaths of wild birds that have drunk water contaminated with hepatotoxic microcystin-producing cyanobacteria have been reported. A mass death of egrets and herons with steatitis were found at the agricultural reservoir occurring cyanobacterial waterblooms. This study aimed to verify a hypothesis that the egrets and herons which died in the reservoir drink microcystin-producing cyanobacteria and microcystin involves in the cause of death as well as the symptoms of steatitis. The cyanobacterial community in gastric contents of egrets and herons that died from steatitis was assessed using cyanobacterial 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) profiling and a cyanobacterial 16S rRNA-based clone library analysis. In addition, PCR amplification of the mcyB–C region and the mcyG gene, involved in microcystin biosynthesis, was examined. The cyanobacterial community in the gastric contents of two birds showed a simplistic composition. A comparison of cyanobacterial T-RFLP profiling and cloned sequences suggested that the genus Microcystis predominated in both samples of egrets died. Although we confirmed that two egrets which died in the reservoir have taken in cyanobacterial waterblooms containing the genus Microcystis, no mcy gene was detected in both samples according to the mcy gene-based PCR analysis. This study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds by molecular analysis. Additionally, we consider causing symptoms of steatitis in the dead egrets.
PMCID: PMC4676040  PMID: 26668668
Agricultural reservoir; cyanobacterial community; gastric content; Microcystis; microcystin biosynthesis (mcy) gene; T-RFLP profiling.
2.  Draft Genome Sequence of the Betaproteobacterial Endosymbiont Associated with the Fungus Mortierella elongata FMR23-6 
Genome Announcements  2014;2(6):e01272-14.
The fungus Mortierella elongata FMR23-6 harbors an endobacterium inside its mycelium. Attempts to isolate the endobacterium from the fungus were not yet successful, but a highly purified bacterial fraction was prepared. Here, we report the draft genome sequence of the endobacterium.
PMCID: PMC4263831  PMID: 25502669
3.  Complete Genome Sequence of Leptospirillum ferrooxidans Strain C2-3, Isolated from a Fresh Volcanic Ash Deposit on the Island of Miyake, Japan 
Journal of Bacteriology  2012;194(15):4122-4123.
A diazotrophic, acidophilic, iron-oxidizing bacterium, Leptospirillum ferrooxidans, known to be difficult to cultivate, was isolated from a fresh volcanic ash deposit on the island of Miyake, Japan. Here, we report the complete genome sequence of a cultured strain, C2-3.
PMCID: PMC3416516  PMID: 22815442
4.  Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil 
AMB Express  2012;2:50.
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
PMCID: PMC3488030  PMID: 22985609
16S rRNA gene; Denitrifying bacteria; Functional single-cell isolation; Phylogenetic analysis; Rice paddy soil
5.  Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Azoarcus sp. Strain KH32C 
Journal of Bacteriology  2012;194(5):1255.
We report the finished and annotated genome sequence of a denitrifying and N2O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.
PMCID: PMC3294784  PMID: 22328754
6.  Detection of Anammox Activity and 16S rRNA Genes in Ravine Paddy Field Soil 
Microbes and Environments  2012;27(3):316-319.
An anammox assay involving a 15N tracer and gas chromatography-mass spectrometry revealed that the potential anammox activity accounted for 1 to 5% of total N2 production in a ravine paddy field, Japan. Among four 4-cm-deep layers, the top layer showed the highest activity. Clone libraries showed that the DNA in the top layer contained sequences related to those of Candidatus ‘Brocadia fulgida’, Ca. ‘B. anammoxidans’, and Ca. ‘Kuenenia stuttgartiensis’. These results suggest that a specific population of anammox bacteria was present in paddy soils, although a small part of dinitrogen gas was emitted from the soil via anammox.
PMCID: PMC4036040  PMID: 22353769
anaerobic ammonium oxidation; ravine paddy field soil; nitrogen removal
7.  Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Pseudogulbenkiania sp. Strain NH8B 
Journal of Bacteriology  2011;193(22):6395-6396.
Pseudogulbenkiania sp. strain NH8B is a Neisseriales bacterium isolated from an agricultural field. This strain has strong denitrification and N2O reduction activities. Here, we report the finished and annotated genome sequence of this organism.
PMCID: PMC3209224  PMID: 22038961
8.  Analysis of Early Bacterial Communities on Volcanic Deposits on the Island of Miyake (Miyake-jima), Japan: a 6-year Study at a Fixed Site 
Microbes and Environments  2011;27(1):19-29.
Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0–3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)−1 of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit.
PMCID: PMC4036035  PMID: 22075623
volcanic deposit; chemolithotrophs; Acidithiobacillus; Leptospirillum; early ecosystem
9.  Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus 
Journal of Bacteriology  2006;188(20):7111-7122.
Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel+; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNASer aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).
PMCID: PMC1636220  PMID: 17015650
10.  Molecular Analysis of the Rebeccamycin l-Amino Acid Oxidase from Lechevalieria aerocolonigenes ATCC 39243 
Journal of Bacteriology  2005;187(6):2084-2092.
Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an l-amino acid oxidase (l-AAO) that has 27% identity to an l-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-l-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.
PMCID: PMC1064027  PMID: 15743957

Results 1-10 (10)