DNA microarray technology has been successfully used in numerous medical and clinical applications, but its application in environmental studies for bacterial detection and microbial community analysis has faced many challenges due to the highly diverse and complex nature of environmental samples. Only a limited number of studies have been published, mainly showing the "proof of principle" of the method in this field of research. More rigorous and systematic assessments and improvements are required to extend the microarray technique to its full potential. Here, some fundamental concepts involved in the microarray technique are discussed, and recent developments in microarray-based technology in microbial ecology applications are reviewed.

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A 5'-nuclease PCR assay, targeting 16S rDNA, was developed to detect a group of gliding bacteria that digest Skeletonema costatum cells and are phylogenetically close to Cytophaga latercula. The detection limit was 15 molecules of the target DNA in one reaction mixture. The assay is so strict that the probe did not hybridize to DNA fragments with one nucleotide mismatch, even though the amount of DNA fragments was increased to the order of 10¹⁰ molecules. The assay was applied to DNA extracted from natural seawater of Yoshimi Bay, Hibiki-nada Sea, Japan, during the period from August 1998 to February 1999. A positive result was obtained only for a seawater sample of September 10, 1998. Among 30 PCR clones obtained from the 5'-nuclease PCR product of the positive sample, 25 clones gave positive results and 4 clones negative results in the assay. The positive clones examined were identical in the structure of the probe region, whereas negative clones had one nucleotide mismatch or deletion. The results indicate that the assay detects only the target sequence even in natural seawater DNA.

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Vibriosis caused by Vibrio anguillarum seriously injures freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen needs NaCl for its growth and survival. When the pathogen was directly exposed to sterilized aged lake water (ALW) at room temperature, it suddenly lost its culturability and pathogenicity, and died within half a day due to the low osmolarity. In this report, the survival of the pathogen as biofilms formed on air-solid and liquid-solid (agar or polystyrene) interfaces in ALW was investigated. When the biofilms formed at air-solid and liquid-solid (agar or polystyrene) interfaces were exposed to ALW at 4-5°C in the dark, the pathogen survived for more than 2 and 4 weeks, respectively. The biofilms at both interfaces at 4-5°C in the dark enhanced the production of a mucous polymer matrix. The main constituent of the polymer was exopolysaccharide. The polymer was produced only in the dark at low temperature. At 20°C, there was no production of the polymer and the survival of the pathogen was shortened. The biofilm seemed to provide a functional consortium to support the survival of V. anguillarum in freshwater.

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The metabolic characteristics of the NO₂ ^- transforming activities of Burkholderia cepacia NH-17, which was isolated as a heterotrophic nitrifying bacterium with O₂ tolerant denitrification activity, were characterized. The conversion of NO₂^- to N₂O and NO₃^- occurred concomitantly with a decrease in NO₂^- under aerobic conditions in growing cultures. In an in vivo assay, production of N₂O and NO₃^- was induced by NO₂^- as an inducer for denitrification, and nitrite reductase activity in sonicated fraction (NiR) assay indicated that in vitro nitrite reductase activity was also induced by NO₂^-. These results suggested that nitrification and denitrification in Burkholderia cepacia NH-17 might be closely related. Therefore, we constructed a nirS knockout mutant of Burkholderia cepacia NH-17. The mutant had no in vitro nitrite reductase activity and did not convert NO₂ ^- to N₂O and NO₃^-. These properties were restored by introducing the intact nirS gene into the mutant strain, indicating that reduction of NO₂ ^- to NO is necessary for the conversion of NO₂ ^- to NO₃^- in Burkholderia cepacia NH-17.

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A new most-probable-number (MPN) method using 96-well microtiter plates was developed to enumerate denitrifying bacteria. In this method, dilutions of samples are added to microtiter plates with a medium and incubated anaerobically using the AnaeroPouch culture system (Mitsubishi Gas Chemical, Inc., Tokyo, Japan). The microtiter plate MPN method gave precise estimates of the population densities of a culture of Paracoccus denitrificans, similar to the estimates obtained using a test tube MPN method and a colony count method. The population densities of denitrifying bacteria in soil samples estimated by the microtiter plate MPN method were higher than the population densities estimated by the test tube MPN method. Because the new method requires less equipment, labor, and time than the conventional test tube method. It will be valuable for estimating the biomass of denitrifying bacteria in natural samples.

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Algal exposure assays revealed that all tested strains of H. circularisquama and H. illdefina were markedly toxic to juvenile pearl oysters. H. triquetra, and Heterocapsa sp. 3 and sp. 5 caused low levels of mortality in juveniles (1.7-7.5%), whereas H. lanceolata, H. horiguchii and Heterocapsa sp. 4 caused no mortality. Three strains of H. circularisquama alone exhibited strong toxicity against bivalves among Heterocapsa species isolated in Japan.
The sequencing of internal transcribed spacer (ITS) regions including 5.8S rDNA and phylogenetic analysis were performed for isolates of Heterocapsa species. Alignment of the sequences demonstrated that ITS regions were highly conserved among H. circularisquama strains, while the distance between H. circularisquama and other species ranged from 0.165 to 0.254. Except for H. horiguchii and Heterocapsa sp. 4, the phylogenetic tree obtained was congruent with morphological identification. Heterocapsa species were subjected to PCR amplification with a primer set designed based on a specific signature sequence for H. circularisquama in the ITS 2 region. A specific band was detected for all strains of H. circularisquama but not other Heterocapsa species, indicating that the ITS region is suitable as a species-specific marker for H. circularisquama.

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In the cellular slime mold Dictyostelium discoideum, Countin and Countin2 proteins are thought to control the size of the multicellular structure since the countin^- strain forms a huge fruiting body and countin2^- strain forms a small fruiting body. Recently, the countin3 gene encoding a polypeptide homologous to Countin and Countin2 was identified in the D. discoideum genome. The countin3^- strain formed a 1.8-fold larger number of aggregates, resulting in smaller fruiting bodies compared to those formed by the wild-type. The extent of cell-cell adhesion was reduced in the mutant, indicating that Countin3 protein regulates size by controlling the amounts of proteins responsible for the adhesion.

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We examined the effect of a transient temperature up-shift on Vibrio parahaemolyticus cells that had been stressed by cold and nutrient-deficient conditions. Under these stressful conditions, V. parahaemolyticus entered into a viable but non-culturable (VBNC) state. The number of colony forming units (CFU) decreased exponentially with time, whereas the total cell count and the viable cell count remained almost constant. When the cells were exposed to a temperature of 30°C for 20 min, the decrease in the number of CFU was suppressed. This effect was dependent on the growth phase. Addition of nalidixic acid, rifampicin, or erythromycin negated the effect.

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