Twenty-eight strains of aerobic chemoheterotrophic bacteria capable of decomposing methanethiol and hydrogen sulfide were isolated from soil samples obtained from mountains, forests, farm fields, parks, hot springs, riversides, the seashore, pigpens, cowsheds, henhouses and other areas in Japan. Among the strains isolated, TOS-35 and TOS-55 had the greatest activities against both methanethiol and hydrogen sulfide. Based on physiological traits, strain TOS-35 was identified as a member of the genus Alcaligenes and strain TOS-55 as that of the genus Pseudomonas. Some authentic strains such as Pseudomonas putida IFO 3738, Comamonas testosteroni IFO 12048, Brevundimonas diminuta IFO 14213 and Burkholderia cepacia IFO 14595 were found to be capable of oxidizing methanethiol and hydrogen sulfide. Our results showed that many species of chemoheterotrophic bacteria in nature are capable of decomposing organic and inorganic sulfur compounds.

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Respiration-dependent H⁺ and Na⁺ pumps and the driving force for ATP synthesis of a psychrotrophic bacterium, strain 13A, isolated from the bottom layer of Lake Vanda, Antarctica, were examined with intact cells. This bacterium was able to grow well over a pH range of 5 to 10 at 25°C in a synthetic medium containing sodium succinate as a carbon source. The growth at alkaline pH was resistant to a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). Endogenous respiration induced by the O₂-pulsing of an anaerobic cell suspension resulted in transient acidification and CCCP-dependent alkalization of the suspension at both pH 6.5 and 8.5. Strong inhibition of the acidification by CCCP indicated that the acidification is attributable to a respiration-dependent primary H⁺ pump. The inhibition by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) and Na⁺-dependence of the alkalization suggested that a respiration-dependent primary Na⁺ pump is responsible for the CCCP-dependent alkalization. In vivo, the synthesis of ATP through endogenous respiration at pH 6.5 and 8.5 was strongly inhibited by CCCP and HQNO, respectively. Therefore, it is suggested that ATP synthesis in this bacterium at pH 6.5 is driven by an electrochemical potential of H⁺ across membranes (Δμ_H⁺), and at pH 8.5 by Δμ_Na⁺ but not Δμ_H⁺. Even at a Na⁺ concentration as low as 2.1mM, this bacterium grew well and exhibited CCCP-resistant growth at alkaline pH. These results strongly suggest that Na⁺ is indispensable as a coupling ion of energy metabolism to sustain the growth of this bacterium not only in Na⁺-ubiquitous environments but also under alkaline conditions.

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A rapid small-scale DNA extraction method is described for the toxic and/or bloom-forming cyanobacterial genus Microcystis, producing enough genomic DNA for polymerase chain reaction (PCR) amplification. PCR templates from 43 Microcystis strains were extracted and analyzed by PCR amplification. Sonication was needed for some strains before extraction of DNA using InstaGene^TM Matrix with heat treatment at 100°C. DNAs extracted from all strains used in this study by this method could be used as templates for PCR amplification. Depending on the apparatuses used, DNA extraction, PCR amplification and agarose gel electrophoresis analysis can be carried out on about 50 samples of Microcystis one day.

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Biofilms are a ubiquitous life style of microbes. Every surface covered with water will develop microbial biofilms. Biofilms are notorious for strong resistance to antimicrobial agents and for causing serious problems in modern medicine and industry. Biofilms are consortia composed of mushroom-shaped clumps of bacteria bound together by a polysaccharide matrix and have many water channels which deliver nutrients and remove wastes. In experiments using Pseudomonas aeruginosa, the genesis of such biofilm architecture was shown to be under the control of the quorum-sensing system. A mutant defective in the quorum-sensing system established a thin uniform biofilm that was easily disrupted by detergent. Thus, the quorum-sensing (a cell-to-cell communication) system working in a collective behavior of bacteria was shown to be important. With respect to the strong resistance of biofilms to antimicrobial agents, heterogeneous distribution and physiology of biofilm cells under various stresses were indicated as factors to be considered, in addition to the exopolysaccharide barrier to chemicals. Generation of drug resistant mutants from biofilms was suggested by recent arguments on the hypermutability of cells under stress.

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Clubroot incited by Plasmodiophora brassicae Woron. causes serious yield losses in a wide range of brassica crops such as Chinese cabbage (Brassica rapa var. pekinensis). This pathogen is a root-invading organism belonging to the Plasmodiophorales, whose members are mostly obligate parasites. This pathogen does not grow in axenic culture, and can survives in soil as resting spores for a long period of time. These facts imply that the assessment of pathogenic activity of resting spores in soil is essential to study the ecology and control of the disease. The detection method using calcofluor white M2R and ethidium bromide has recently been developed. Other recent advances include in vitro growth of the pathogen and the inclusion of host-DNA in resting spores etc. A review of in vitro growth studies on Plasmodiophora brassicae was discussed here.

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Vesicular-arbuscular (VA) mycorrhizal fungi belonging to order Glomales, Zygomycetes, are obligately associated with plant roots. To culture the fungi, we have to establish the symbiosis by growing host plants inoculated with the fungi. Many trials have been done to culture the fungi asymbiotically, but none have succeeded to date. The fungi can grow asymbiotically from spores or intraradical hyphae for some periods, but do not complete their life cycle; spore are not reproduced under asymbiotic conditions. Possible reasons for the failure in asymbiotic proliferation were discussed. We are not able to exclude the possibility that we have not yet obtained all the compounds essential to the asymbiotic growth, but this possibility is low. During the symbiotic growth phase of the fungi, functional differentiation may occur in the intraradical and extraradical hyphae. This may be one of the keys to solving the problem.

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The “Viable but Nonculturable State” refers to the particular physiological state in which the bacterial cells retain a certain detectable activity, even if conventional culture methods fail to grow colonies. Although both VBNC and VNC are used for abbreviation, the author considers the latter easier for Japanese. Although there have also been proposals for other terms, we should stick to “Viable but Nonculturable” to avoid introducing any confusion at the moment.
The two most important issues in VNC research are; is VNC a programmed phenomenon? and, under what kind of condition do the cells in VNC state resuscitate? To answer these questions, interdisciplinary research especially with microbial ecologists and those in other fields is essential. The author thinks VNC research should be placed in a more comprehensive research field, that is “Death of Microbes”.

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