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author:("haruna, Shin")
1.  Differences in Survivability under Starvation Conditions Among Four Species of Purple Nonsulfur Phototrophic Bacteria 
Microbes and Environments  2014;29(3):326-328.
Survivability under carbon-starvation conditions was investigated in four species of purple phototrophic bacteria: Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodospirillum rubrum, and Rubrivivax gelatinosus. All these test organisms survived longer in the light than in the dark. ATP levels in the cultures were maintained in the light, which indicated that survivability was supported by photosynthesis. Survivability and tolerance against hypertonic stress in the dark was higher in Rhodopseudomonas palustris, which is widely distributed in natural environments including soils, than in the three other species.
doi:10.1264/jsme2.ME14013
PMCID: PMC4159045  PMID: 24941957
starvation; survivability; purple phototrophic bacteria
2.  Genome Sequence of the Thermophilic Cyanobacterium Thermosynechococcus sp. Strain NK55a 
Genome Announcements  2014;2(1):e01060-13.
The genome of the unicellular cyanobacterium Thermosynechococcus sp. strain NK55a, isolated from the Nakabusa hot spring, Nagano Prefecture, Japan, comprises a single, circular, 2.5-Mb chromosome. The genome is predicted to contain 2,358 protein-encoding genes, including genes for all typical cyanobacterial photosynthetic and metabolic functions. No genes encoding hydrogenases or nitrogenase were identified.
doi:10.1128/genomeA.01060-13
PMCID: PMC3907722  PMID: 24482507
3.  Rediscovery of the Microbial World in Microbial Ecology 
Microbes and Environments  2013;28(3):281-284.
doi:10.1264/jsme2.ME2803rh
PMCID: PMC4070962  PMID: 24064954
4.  Challenges for Complex Microbial Ecosystems: Combination of Experimental Approaches with Mathematical Modeling 
Microbes and Environments  2013;28(3):285-294.
In the past couple of decades, molecular ecological techniques have been developed to elucidate microbial diversity and distribution in microbial ecosystems. Currently, modern techniques, represented by meta-omics and single cell observations, are revealing the incredible complexity of microbial ecosystems and the large degree of phenotypic variation. These studies propound that microbiological techniques are insufficient to untangle the complex microbial network. This minireview introduces the application of advanced mathematical approaches in combination with microbiological experiments to microbial ecological studies. These combinational approaches have successfully elucidated novel microbial behaviors that had not been recognized previously. Furthermore, the theoretical perspective also provides an understanding of the plasticity, robustness and stability of complex microbial ecosystems in nature.
doi:10.1264/jsme2.ME13034
PMCID: PMC4070964  PMID: 23995424
complex systems; mathematical modeling; plasticity; interaction; diversification
5.  Candidatus Methanogranum caenicola: a Novel Methanogen from the Anaerobic Digested Sludge, and Proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a Methanogenic Lineage of the Class Thermoplasmata 
Microbes and Environments  2013;28(2):244-250.
The class Thermoplasmata harbors huge uncultured archaeal lineages at the order level, so-called Groups E2 and E3. A novel archaeon Kjm51a affiliated with Group E2 was enriched from anaerobic sludge in the present study. Clone library analysis of the archaeal 16S rRNA and mcrA genes confirmed a unique archaeal population in the enrichment culture. The 16S rRNA gene-based phylogeny revealed that the enriched archaeon Kjm51a formed a distinct cluster within Group E2 in the class Thermoplasmata together with Methanomassiliicoccus luminyensis B10T and environmental clone sequences derived from anaerobic digesters, bovine rumen, and landfill leachate. Archaeon Kjm51a showed 87.7% 16S rRNA gene sequence identity to the closest cultured species, M. luminyensis B10T, indicating that archaeon Kjm51a might be phylogenetically novel at least at the genus level. In fluorescence in situ hybridization analysis, archaeon Kjm51a was observed as coccoid cells completely corresponding to the archaeal cells detected, although bacterial rod cells still coexisted. The growth of archaeon Kjm51a was dependent on the presence of methanol and yeast extract, and hydrogen and methane were produced in the enrichment culture. The addition of 2-bromo ethanesulfonate to the enrichment culture completely inhibited methane production and increased hydrogen concentration, which suggested that archaeon Kjm51a is a methanol-reducing hydrogenotrophic methanogen. Taken together, we propose the provisional taxonomic assignment, named Candidatus Methanogranum caenicola, for the enriched archaeon Kjm51a belonging to Group E2. We also propose to place the methanogenic lineage of the class Thermoplasmata in a novel order, Methanomassiliicoccales ord. nov.
doi:10.1264/jsme2.ME12189
PMCID: PMC4070666  PMID: 23524372
Methanogranum caenicola; methanogen; Thermoplasmata; rice cluster III; anaerobic digested sludge
6.  Diversification of Bacterial Community Composition along a Temperature Gradient at a Thermal Spring 
Microbes and Environments  2012;27(4):374-381.
To better understand the biogeography and relationship between temperature and community structure within microbial mats, the bacterial diversity of mats at a slightly alkaline, sulfide-containing hot spring was explored. Microbial mats that developed at temperatures between 75–52°C were collected from an area of approximately 1 m2 in Nakabusa, Nagano, Japan. Bacterial 16S rRNA genes from these samples were examined by terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. T-RFLP profiles revealed 66 unique fragments (T-RFs). Based on total T-RFs observed in environmental profiles and clone libraries, a temperature effect on diversity was determined, with complexity in the community increasing as temperature decreased. The T-RF pattern indicated four distinct community assemblages related to temperature. Members of the Aquificales and particularly the sulfuroxidizing bacterium Sulfurihydrogenibium were present at all temperatures and were the dominant component of mats taken at 75–67°C. Sulfide oxidation, which persisted throughout the temperature gradient, was the presumed dominant pathway of primary production above 67°C. As temperature decreased, successive additions of anoxygenic and oxygenic phototrophs increased primary productivity, allowing for diversification of the community.
doi:10.1264/jsme2.ME11350
PMCID: PMC4103544  PMID: 22673306
biogeography; sulfur oxidizing bacteria; photosynthetic bacteria; thermophile; Aquificales
7.  Diversity of Purple Phototrophic Bacteria, Inferred from pufM Gene, within Epilithic Biofilm in Tama River, Japan 
Microbes and Environments  2012;27(3):327-329.
The diversity of purple phototrophic bacteria in algae-dominated biofilm of a streambed in Tama River, Japan was investigated. Clone library analysis of the pufM gene encoding a subunit of the photochemical reaction center of purple bacteria detected 18 operational taxonomic units (OTUs) in several classes of Proteobacteria. Most of the OTUs showed less than 85% identity to the PufM amino acid sequences of known phototrophic bacteria. These results suggest that phylogenetically divergent and unknown purple phototrophic bacteria are present in the epilithic biofilm of the river.
doi:10.1264/jsme2.ME11306
PMCID: PMC4036046  PMID: 22446305
Phototrophic bacteria; river; epilithic biofilm; pufM; purple bacteria
8.  Complete Genome Sequence of Bradyrhizobium sp. S23321: Insights into Symbiosis Evolution in Soil Oligotrophs 
Microbes and Environments  2012;27(3):306-315.
Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.
doi:10.1264/jsme2.ME11321
PMCID: PMC4036050  PMID: 22452844
Bradyrhizobium sp. S23321; comparative genomics; symbiosis evolution; photosynthesis; oligotrophic soil bacterium
9.  Production and Consumption of Hydrogen in Hot Spring Microbial Mats Dominated by a Filamentous Anoxygenic Photosynthetic Bacterium 
Microbes and Environments  2012;27(3):293-299.
Microbial mats containing the filamentous anoxygenic photosynthetic bacterium Chloroflexus aggregans develop at Nakabusa hot spring in Japan. Under anaerobic conditions in these mats, interspecies interaction between sulfate-reducing bacteria as sulfide producers and C. aggregans as a sulfide consumer has been proposed to constitute a sulfur cycle; however, the electron donor utilized for microbial sulfide production at Nakabusa remains to be identified. In order to determine this electron donor and its source, ex situ experimental incubation of mats was explored. In the presence of molybdate, which inhibits biological sulfate reduction, hydrogen gas was released from mat samples, indicating that this hydrogen is normally consumed as an electron donor by sulfate-reducing bacteria. Hydrogen production decreased under illumination, indicating that C. aggregans also functions as a hydrogen consumer. Small amounts of hydrogen may have also been consumed for sulfur reduction. Clone library analysis of 16S rRNA genes amplified from the mats indicated the existence of several species of hydrogen-producing fermentative bacteria. Among them, the most dominant fermenter, Fervidobacterium sp., was successfully isolated. This isolate produced hydrogen through the fermentation of organic carbon. Dispersion of microbial cells in the mats resulted in hydrogen production without the addition of molybdate, suggesting that simultaneous production and consumption of hydrogen in the mats requires dense packing of cells. We propose a cyclic electron flow within the microbial mats, i.e., electron flow occurs through three elements: S (elemental sulfur, sulfide, sulfate), C (carbon dioxide, organic carbon) and H (di-hydrogen, protons).
doi:10.1264/jsme2.ME11348
PMCID: PMC4036054  PMID: 22446313
microbial mats; hydrogen; electron cycling; ecosystem
10.  Coexistence of Antibiotic-Producing and Antibiotic-Sensitive Bacteria in Biofilms Is Mediated by Resistant Bacteria▿ †  
Applied and Environmental Microbiology  2008;74(12):3887-3894.
Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.
doi:10.1128/AEM.02497-07
PMCID: PMC2446560  PMID: 18441106
11.  Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester 
In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.
doi:10.1128/AEM.72.2.1623-1630.2006
PMCID: PMC1392901  PMID: 16461718
12.  Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community 
Applied and Environmental Microbiology  2005;71(11):7099-7106.
A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.
doi:10.1128/AEM.71.11.7099-7106.2005
PMCID: PMC1287685  PMID: 16269746
13.  Enzyme Production-Based Approach for Determining the Functions of Microorganisms within a Community 
The functions of specific microorganisms in a microbial community were investigated during the composting process. Cerasibacillus quisquiliarum strain BLxT and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system. Strain BLxT degrades gelatin, while strain BTa degrades starch. We hypothesized that these strains play roles in gelatinase and amylase production, respectively. The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis. The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLxT. Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLxT was similar to that from the compost. Gelatinases from both BLxT and compost were partially purified and compared. Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLxT and that of compost. These results indicate strain BLxT produces gelatinases during the composting process. Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost. Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample. These results imply that strain BTa may not produce amylases during the composting process. To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches.
doi:10.1128/AEM.70.6.3329-3337.2004
PMCID: PMC427761  PMID: 15184128
14.  Functional Analysis of the Active Site of a Metallo-β-Lactamase Proliferating in Japan 
An R-plasmid-mediated metallo-β-lactamase was found in Klebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene of Serratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.
PMCID: PMC90062  PMID: 10952572
15.  Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase 
In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene blaIMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene blaIMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of blaIMP-3 was identical to that of blaIMP-1, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from blaIMP-3 and blaIMP-1 by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the blaIMP-3 gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene blaIMP-3 is an ancestor of blaIMP-1.
PMCID: PMC90008  PMID: 10898670

Results 1-15 (15)