The diversity of diazotrophic bacteria in the rhizosphere of Melastoma malabathricum L. was investigated by cloning-sequencing of the nifH gene directly amplified from DNA extracted from soil. Samples were obtained from the rhizosphere and bulk soil of M. malabathricum growing in three different soil types (acid sulfate, peat and sandy clay soils) located very close to each other in south Kalimantan, Indonesia. Six clone libraries were constructed, generated from bulk and rhizosphere soil samples, and 300 nifH clones were produced, then assembled into 29 operational taxonomic units (OTUs) based on percent identity values. Our results suggested that nifH gene diversity is mainly dependent on soil properties, and did not differ remarkably between the rhizosphere and bulk soil of M. malabathricum except in acid sulfate soil. In acid sulfate soil, as the Shannon diversity index was lower in rhizosphere than in bulk soil, it is suggested that particular bacterial species might accumulate in the rhizosphere.

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A comprehensive survey of bacterial and archaeal community structures within granular sludges taken from twelve different types of full-scale, food-processing wastewater-treating, upflow anaerobic sludge blanket (UASB) reactors was performed with a 16S rRNA gene-based clone library method. In total, 1,282 bacterial 16S rRNA gene clones and 722 archaeal clones were analyzed, and their identities were determined by phylogenetic analyses. Overall, clones belonging to the bacterial phyla Proteobacteria (the class Deltaproteobacteria in particular), Firmicutes, Spirochaetes, and Bacteroidetes were observed in abundance within the bacterial clone libraries examined, indicating common bacterial denominators in such treatment systems. Within the domain Archaea, clones affiliated with the classes Methanomicrobia and Methanobacteria were found to be abundant in the archaeal libraries. In relation to features of reactor performance (such as chemical oxygen demand removal, fatty acid accumulation, and sludge bulking), possible representative phylotypes likely to be associated with process failures, such as sludge bulking and the accumulation of propionate, were found in comparative analyses of the distribution of phylotypes in the sludge libraries.

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In upflow anaerobic sludge blanket (UASB) reactors treating high-strength organic wastewaters, uncultured phylotypes belonging to the phylum Nitrospirae were assumed to play a key role in the treatment processes because of their high abundance as determined by 16S rRNA gene-based clone library analyses (Narihiro et al. (27)). A comparative nucleotide sequence analysis of the phylotypes, commonly found in anaerobic mesophilic sludges, revealed that these phylotypes formed a coherent clade at the family to genus levels with no cultured representatives. To determine the abundance and distribution in methanogenic waste/wastewater treatment sludges, 16S rRNA gene-based quantitative PCR measurements and 16S rRNA-targeted fluorescence in situ hybridization (FISH) were carried out. A specific gene primer/probe for the uncultured clade was developed and used for quantitative PCR and in situ detection of the targeted cells within the sludge granules. In all the six UASB sludges treating food-processing organic wastewaters, the phylotypes were detected at a range of 0.1 to 10.9% of total 16S rRNA genes. FISH revealed that the gene probe reacted with coccoid or irregular coccoid-like cells in the middle and inner layers of sludge granules of a UASB reactor, in which the Nitrospirae phylotypes were found in abundance by real-time PCR analyses.

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The phylogenetic diversity of cowpea root-nodulating bacteria in the South-West of Japan was investigated using 60 isolates. Seeds of cowpea were aseptically sown in vermiculite and inoculated with a suspension of Cowpea Soil (CS) or Bean Soil (BS) or without a soil suspension as a control. CS and BS were collected from the Kyushu University's farm (Japan) at sites where cowpea and bean, respectively, have been cultivated previously. Based on an analysis of the 16S rRNA gene and the Internal Transcribed Spacer (ITS) sequence between the 16S and 23S rRNA genes, 56 isolates were assigned to the genus Bradyrhizobium, while one isolate was found to be closely related to the genus Ralstonia. The ITS-based phylogeny showed 53 isolates, 2 isolates, and 1 isolate, to be closely related to B. yuanmingense, B. elkanii and B. japonicum, respectively, suggesting that B. yuanmingense strains predominated in the soils. Among the isolates tested, B. yuanmingense TSC10 and TTC9 exhibited a greater symbiotic activity and could be considered efficient inoculants for cowpea.

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The nucleotide sequences of two endopolygalacturonase genes (pg1 and pg5) and two exopolygalacturonase genes (pgx1 and pgx4), which encode members of a major family of secreted cell-wall-degrading enzymes (CWDEs), were compared to detect the extent of genetic variation among isolates of Fusarium oxysporum. The nucleotide variation rate in exons was 0.23-0.93%, higher than that in introns (0.01-0.64%) and untranslated regions (UTRs) (0.07-0.25%), resulting in 0.05-0.31% variation in amino acids. pgx1 exhibited the most genetic diversity. Phylogenetic analysis of the four genes, which reside on different chromosomes, revealed different evolutionary patterns for each. Our results suggest a biased evolution of the polygalacturonase genes of F. oxysporum, or alternatively, that the genes were acquired at different times during evolution.

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Environmental isolates of Salmonella enterica serover Enteritidis (S. Enteritidis) clones were grown to the logarithmic phase, washed and re-suspended in saline or Luria-Bertani (LB) medium, and then 10-μL aliquots of the suspensions were dried overnight at room temperature. The dried bacteria were mixed with 1 mL of ice-cold PBS, suspended and examined for colony-forming activity. All of the pathogenic clones with high levels of SEp22, identical to Salmonella Dps, maintained good viability if suspended in LB medium prior to drying. However, none of the non-virulent strains, exhibiting low levels of SEp22, survived. Similar results were obtained with sep22-knocked out mutants, suggesting that SEp22 is important for the acquisition of dry-resistance. Nutritional factors, such as LB medium, cabbage extracts, and egg yolk but not egg white, were shown to be necessary for the acquisition of dry-resistance, because none of the clones remained viable irrespective of SEp22 expression if suspended in saline. Scanning electron micrograms also supported the importance of nutrition, showing re-growth of the bacteria after drying in LB but not in saline. These results suggest the importance of both SEp22 expression and nutrients for the acquisition of dry-resistance by S. Enteritidis.

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In the model leguminous plant Lotus japonicus, the reception of a symbiotic signal called Nod factor (NF), which is secreted by the symbiont bacterium Mesorhizobium loti, induces wavy shaped root hairs. This is called root hair deformation. To dissect the root hair deformation process, we studied symbiosis- deficient mutants of L. japonicus, castor, nup85, ccamk and nsp2. The CASTOR, NUP85, and CCaMK genes are also required for mycorrhizal infection and thus called common symbiotic genes. On the global application of NF, all the mutants except nsp2 exhibited extensive branching of root hairs. The actin cytoskeleton was also observed as a marker for NF-dependent responses in mutant root hairs. At 2 hours of NF treatment, the ccamk mutant showed exaggerated swelling compared with the other mutants, indicating CCaMK to be required to terminate the swelling. In the nsp2 mutant, two hours of NF treatment remarkably induced swelling at root hair tips, although root hair deformation was not apparent at 24 hours of NF treatment. These results showed that common symbiotic components are involved in root hair deformation, which is regulated by a fine tuning mechanism early in the symbiosis between leguminous plants and rhizobia.

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Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thailand. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and considered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization.

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This study investigated the active microbial community in a full-scale granular activated carbon-anaerobic fluidized bed (GAC-AFB) reactor treating wastewater from the manufacturing of phenolic resin, using 16S rRNA-based molecular analyses. The results of cDNA from 16S rRNA revealed that Methanosaeta-related (83.9% of archaeal clones) and Syntrophorhabdaceae (formerly named Deltaproteobacteria group TA)-related (68.9% of bacterial clones) microorganisms were as the most predominant populations in the phenol-degrading GAC-AFB reactor. The high abundance of Syntrophorhabdaceae was supported by a terminal restriction fragment length polymorphism (T-RFLP) analysis, which showed that a Syntrophorhabdaceae-like fragment of 119 bp (~80% of total fragments) was the most predominant phylotype. Furthermore, fluorescence in situ hybridization (FISH) analyses suggested that Syntrophus- and Chloroflexi-like cells were also in high abundance in the GAC biofilm. A non-layered structure of microorganisms was found in the GAC biofilm, where Methanosaeta (thick filamentous), Syntrophorhabdaceae (oval-shaped), Syntrophus (small rods) and Chloroflexi (thin-filamentous) were randomly distributed with high abundance. These findings greatly improve our understanding of the diversity and distribution of microbial populations in a full-scale mesophilic bioreactor treating an actual phenol-containing waste stream.

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Endophytic bacteria are considered to originate from the external environment. To examine the hypothesis that rice (Oryza sativa, cultivar Kinuhikari) seeds are a source of endophytic bacteria, we isolated endophytic bacteria from the shoots, remains of the seeds, and roots of rice seedlings that were aseptically cultivated in vitro from surface-disinfected seeds. Of the various bacterial strains isolated, the closest relatives, identified by 16S rRNA gene sequencing, were: Bacillus firmus, B. fusiformis, B. pumilus, Caulobacter crescentus, Kocuria palustris, Micrococcus luteus, Methylobacterium fujisawaense, Me. radiotolerans, and Pantoea ananatis. The latter three species have been detected frequently inside both rice seedlings and mature rice plants. These results indicate that rice seeds are an important source of endophytic bacteria. The bacteria that colonize the seed interior appear to infect the subsequent generation via rice seeds and become the dominant endophytic species in the mature plant.

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The bacterial population dynamics in an industrial scale reverse-osmosis (RO) water purification system were analyzed by fluorescent staining methods and denaturing gradient gel electrophoresis (DGGE). Bacterial numbers increased with storage in a tank, and bacterial diversity changed during the water purification process. A DNA sequence-based analysis of the major bands on the DGGE gel revealed that Simonsiella sp. (Betaproteobacteria) was abundant in the source water (activated sludge-treated waste effluent), while Bosea sp. and Rhizobium sp. (Alphaproteobacteria), which usually exist in an oligotrophic environment, became abundant during the water purification process. These results suggest the importance of microbiological monitoring by culture-independent methods for quality control in RO water purification systems. These methods could provide an early warning of impending problems and clarify critical steps in controlling specific bacteria contributing to the contamination of RO water systems.

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The present study describes the occurrence of a unique archaeal ammonia monooxygenase alpha subunit (amoA) gene in nitrifying acid-sulfate soil microcosms at pH 3.5. The soil was collected from an abandoned paddy field in Thailand. Microcosms were incubated in the dark at 30°C for 372 days with the following three treatments: addition of ammonium sulfate solution once a month (I) or once a week (II), and addition of only sterilized water (III). A quantitative PCR analysis revealed an increase in abundance of the archaeal amoA gene in microcosm soils in which nitrate concentrations increased after incubation. A phylogenetic analysis indicated a predominance of the novel gene, and a predominance of a betaproteobacterial amoA gene affiliated with the genus Nitrosospira. A 16S rRNA gene-based PCR assay revealed that crenarchaeotic Group I.1d was predominant among the Crenarchaeota in microcosms. These results suggest the presence of ammonia-oxidizing archaea corresponding to the unique amoA lineage in nitrifying acid-sulfate soil microcosms at pH 3.5.

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The influence of soil organic matter on DNA adsorption in andosols was investigated using various andosol samples including hydrogen peroxide (H₂O₂)-treated, heated (400°C), and slurry-added soils. Remarkably less DNA was adsorbed in the slurry-added soil than the original soil. The increase in soil organic matter with the addition of slurry had an obvious negative influence on the adsorption. The decrease in organic matter with H₂O₂ treatment slightly raised DNA adsorption per unit weight. Adsorption maxima estimated from a simple Langmuir equation were higher in the samples removed of organic matter by the H₂O₂ treatment and heating at 400°C than in the untreated soil, although surface area was greatly decreased by both treatments. There was no correlation between the total carbon (T-C) content and the estimated DNA adsorption maxima of any of the soil samples. These results suggest little contribution of soil organic matter to DNA adsorption in andosols.

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Phenolics are a dominant class of plant secondary metabolites that have strong effects on various ecosystem processes. The ecological significance of these compounds, however, is still poorly understood. We hypothesized that the inhibitory effects of phenolics on microbial activity could enhance plant nitrogen acquisition by relaxing competition between plants and microbes. To test this hypothesis theoretically, we constructed a novel and simple mechanistic model by unifying two concepts: one is a new paradigm of nitrogen cycling which considers the uptake of organic nitrogen by plants, and the other is that phenolics can regulate nitrogen cycling by inhibiting microbial decomposition processes. Our plant-microbe competition model consists of five compartments (plants, soil microbes, debris, organic nitrogen and inorganic nitrogen) and incorporates the essential processes of nitrogen cycling: plant uptake of monomers, competition between plants and microbes, and the depolymerization process. Our analysis showed that plant nitrogen acquisition was maximized at intermediate levels of phenolics, but only when plants could utilize organic nitrogen. Furthermore, this pattern occurred over a broad range of parameter conditions. Our study successfully demonstrated the potential role of phenolics in plant nitrogen acquisition throughout natural environments.

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Quantitative and qualitative differences were analyzed between planktonic and anode-biofilm bacterial communities developed in a cassette-electrode microbial fuel cell treating starch, peptone, and fish extract. Quantitative analyses based on protein contents and rRNA-gene copy numbers indicated that planktonic microbes were over eight-times more abundant than anode-biofilm microbes. Clone-library analyses of PCR-amplified 16S rRNA gene fragments revealed the presence of bacteria affiliated with the phyla Bacteroidetes, Firmicutes, and Proteobacteria in these two communities. The most abundant sequence was affiliated with the family Porphyromonadaceae, and accounted for over 50% and 20% of all the sequences in the planktonic- and biofilm-microbe libraries, respectively.

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Volume 23, no. 2, p. 109-117, 2008.
Pages 115 and 117, there were errors in references 13 and 75.
The correct version is follows.

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Volume 23, no. 4, p. 277-284, 2008.
Page 279, Table 1, the station names were incorrect. The correct version of the table is shown below.
Page 283, column 1, line 31, Table 4 should read Table 2.
Pages 283 and 284, references 6 and 34 should appear as shown below.

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