A major goal of microbial ecology is to study the abundance, localization, and activities of microorganisms in situ in order to understand ecophysiological roles that the microorganisms play in complex natural ecosystems. In fact, in typical microbial habitats such as biofilms, sediments, and microbial aggregates, resources and physicochemical conditions are dynamically changing with time and across even a very tiny distance because of metabolic activities and substrate transport limitation. To directly correlate microbial identity (16S rRNA-based phylogeny) to the specific metabolic function of individual cells within such complex and heterogeneous microbial habitats, several new molecular-based techniques have been developed in the last decade. These techniques exploit in situ simultaneous phylogenetic identification and metabolic capabilities of even uncultured microorganisms without the need to isolate them in culture. Microautoradiography is a powerful but "rather old" tool, with which the in situ uptake of specific radiolabeled substrates by individual cells can be determined. Fluorescence in situ hybridization (FISH) is a new molecular-based technique that allows the in situ phylogenetic identitification of individual cells. However, FISH cannot provide sufficient information on metabolic capabilities, because phylogeny and phenotype are rarely congruent. Recently, microautoradiography and FISH have been successfully combined to further improve the complementary strengths of the two methods. Microautoradiography combined with FISH (MAR-FISH) can be used to simultaneously examine the phylogenetic identity and the relative or actual specific activity of microorganisms within a complex microbial community at a single-cell level. This article overviews the principle, experimental protocol and application of the MAR-FISH technique, as well as current developments of other analytical techniques for in situ microbial functions (metabolic activities) from a single-cell level to community levels.

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Nitrogen-fixing nodules are formed as a result of a series of interactions between rhizobia and leguminous plants. Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process and nodulation competitiveness by inhibiting ethylene biosynthesis in host roots. In addition, the gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase was recently found in some rhizobia, such as Mesorhizobium loti, Bradyrhizobium japonicum and Rhizobium sp. ACC deaminase also facilitates symbiosis by decreasing ethylene levels in host roots. The cumulative evidence reveals general strategies by which rhizobia produce an inhibitor and an enzyme to decrease ethylene levels in host roots and thereby enhance nodulation. In this review, we compare these strategies and discuss how they function and have evolved in terms of genetics, biochemistry, and ecology. These rhizobial strategies might be utilized as tools in agriculture and biotechnology.

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Since most of the anaerobic bacterial isolates from rice plant residue in irrigated rice field soil grew slowly or weakly in the medium (PY medium) used, growth factors for the isolates were investigated. Plant residue extract (RE) was prepared by autoclaving plant residue collected from the soil, and RE was added to the medium as a possible source of growth factors. With the addition, growth of the slowly-growing, propionate-producing strains of Propionicimonas paludicola was considerably improved. Lactate was the dominant product of these strains in the presence of lower concentrations of RE. Moreover, amounts of acetate and propionate produced increased in proportion to the RE concentration added. The factor in RE affecting the growth of these strains appeared to be cobalamin, and addition of cobalamin to the medium remarkably improved their growth. With a sufficient amount of cobalamin added, propionate was the dominant product from the onset of fermentation, while lactate was only a minor product. Concentrations of cobalamin in RE were determined using the microbiological method. Almost exactly the same amount of cobalamin was detected in RE prepared from plant residue samples collected in different years. Cobalamin was also detected in extracts of the rice field soil, although the concentrations were much lower than those in RE. It was suggested that these cobalamin-requiring, propionate-producing bacteria survive in rice field soil by using cobalamin supplied by other microbes, which endogenously produce cobalamin and release it into the environment.

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Two species of cellulolytic protist, Dinenympha parva and Microjoenia, living in the guts of the lower termite Reticulitermes speratus are known to harbor endosymbiotic methanogens detectable with an epifluorescent microscope. DNA isolated from the guts of worker termites in a colony of R. speratus was amplified using archaea-specific primers and cloned, and partial 16S rRNA gene sequences were obtained. Archaeal PCR clones obtained from the guts of xylophagous insects in this and previous works formed four subgroups within the Methanobrevibacter branch of a phylogenetic tree; the sequences of the clones obtained in this report belonged to subgroups, designated XSAT1A and XSAT1D. Using a probe specific to each of the subgroups, 50 and 10 endosymbiotic Methanobrevibacter cells per protist respectively were detected with a probe specific to the subgroup XSAT1A by fluorescent in situ hybridization analysis in D. parva and Microjoenia. There was no observed hybridization to the endosymbiont with other subtype-specific probes including XSAT1D. Based on these results, endosymbionts in D. parva and Microjoenia sp. are proposed to belong to the Methanobrevibacter subgroup XSAT1A.

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To test the hypothesis that different phytoplankton cellular components undergo distinct decomposition processes, we examined bacterial decomposition of extracted dissolved organic matter (EX-DOM) and cell debris from the diatom (Skeletonema costatum) using long-term incubation experiments. The decay rates indicated that EX-DOM is extremely bioreactive (1.2-1.7 d^-1), and that particulate organic carbon (POC) from cell debris is resistant to rapid microbial degradation (0.047-0.055 d^-1). In the experiment with grazers, the percentage of accumulated dissolved organic carbon (DOC) in the EX-DOM and cell debris experiments was 5% and 64%, respectively, of the initial DOC concentrations derived from phytoplankton cells. The initial C/N ratio of DOM in the cell debris experiments was 19.2-19.7, higher than those in the EX-DOM experiments (5.5-6.0). The percentage of accumulated DOC in the cell debris experiment with grazers was 35% at day 13. Thereafter DOC accumulated until it reached 64%. Our data indicate that these two distinct phytoplankton-derived organic materials (EX-DOM and cell debris) decay and accumulate differently. These results suggest that structural components of phytoplankton cells may persist as semi-labile POC, and that DOC produced by solubilization of structural components of cell and phytoplankton-derived DOM with high C/N ratios may accumulate as semi-labile DOC in seawater.

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We investigated the population dynamics of the free-living microcystin-degrading isolate Y2 (MCD-isolate) in an eutrophic lake (Lake Suwa, Japan) using fluorescence in situ hybridization (FISH) with a specific probe. Free-living MCD-isolate was successfully visualized by direct viable count method combined with FISH analysis. The highest concentration of MCD-isolate existed in 1999 when high concentrations of dissolved microcystin (Mcyst) and Microcystis populations were present. Mcyst degradation experiments with free-living bacteria collected at different times during a Microcystis bloom in 2000 indicated that three samples of free-living bacteria completely degraded Mcyst-LR and its isomer. The lag time before the degradation was however different for the free-living bacteria. Free-living bacteria present during the mid-bloom of Microcystis completely degraded Mcysts with the shortest lag time. The number of free-living cells of MCD-isolate detected by the FISH method significantly increased when Mcyst was degraded with the fastest degradation rate in the mid-bloom sample. Other bacterial populations collected at mid-and late-bloom increased during the experiments with the exception of the δ-Proteobacteria. However, the community structure was stable. Our findings suggest that MCD-isolate exists with various bacterial consortia in water and degrades Mcysts, the function of which is considered to be induced by exposure to Mcyst.

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Microbes of the Cytophaga-Flavobacterium (CF) group, within the phylum Bacteroidetes, have been considered contributors to the early decomposition of particulate organic matter (POM) in sediments because of their abundance in the ecosystem and their great ability to degrade macromolecules on artificial media. However, there is no report on the ability of members of this group to decompose POM in sediment. We investigated the POM-decomposition capabilities of members of the CF group, Flavobacterium limicola strains ST-82^T, ST-10 and ST-92 isolated from freshwater sediment as proteolytic psychrophiles, in sterilized sediment slurries. All strains of F. limicola grew and survived at over 10⁸ cfu per ml for more than 30 days in the sediment slurries at 5°C. Protease activity levels in the slurries inoculated with the strains were 3-5 times higher than the control level for over 30 days of incubation at 5°C. Concentrations of total dissolved nitrogen (TDN) released by the hydrolysis of particulate organic nitrogen (PON), significantly increased only in the slurries inoculated with F. limicola strains. Approximately 70-80% of the TDN released was converted to NH₄⁺-N in these inoculated slurries. The results clearly demonstrate that F. limicola strains ST-82^T, ST-10 and ST-92 are able to secrete an extracellular protease and hydrolyze PON to TDN and thereby mineralize TDN to NH₄⁺-N in freshwater sediment. This is the first report on the decomposition and mineralization of PON by members of the CF group in benthic environments.

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This study was conducted to gain knowledge about how microbial communities are affected by burning, and to know their present conditions at various periods after the occurrence of forest fires. It aimed to determine the microbial community diversity, biomass carbon and microbial abundance in soil. Six different sites in Hiroshima prefecture, Japan were chosen for this study, 5 sites in burned forest areas (2 months, and 3, 6, 9 and 25 years, after fire) and a control site (an undisturbed forest). From a simple count of the number of TRFs (Terminal Restriction Fragments), the burned areas showed low community diversity even many years after fire. The biomass carbon in burned areas was low, even 25 years post fire. In terms of microbial abundance, the overall result showed that the undisturbed forest (control) had the largest number of gram-positive and gram-negative bacteria, actinomycetes and fungi, while of all the burned sites, the area burned 2 months before being studied had the highest microbial count. These results show that forest wildfires can have a long-term effect on microbial biomass, abundance and diversity in soil.

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The Mai Po Nature Reserve and Inner Deep Bay Ramsar site are an important area of unique ecology and high biodiversity. In this study, environmental strains of Aeromonas hydrophila MP-2, A. salmonicida MP-3, Vibrio vulnificus MP-4 and V. cholerae MP-1 were isolated and examined for their responses to UV exposure, ferric (Fe³⁺) ions and H₂O ₂ challenge under laboratory conditions. In addition, microbial growth data were used to obtain important parameters of bacterial growth including lag phase, specific growth rate and maximum biomass yield for comparison. It was found that V. vulnificus MP-4 was the most sensitive to UV and Fe³⁺ treatments. A. salmonicida MP-3 was only inhibited by 100 mM H₂O₂ while the other three strains did not show any growth at 10 mM H₂O ₂. Our experimental results indicated that environmental isolates of Aeromonas spp. and Vibrio spp. possess different responses for survival under these treatment conditions.

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Aerobic chemoorganotrophic bacteria in soils polluted with different levels of polychlorinated dioxins (6.8 to 4,600 pg-TEQ g^-1 dry weight) were isolated by the quantitative agar-plating method and tested for their ability to degrade dibenzofuran (DF) using DF-overlaid agar and DF-containing liquid media. For comparison, bacteria isolated from river sediments were also tested. Out of the 5,069 strains thus isolated, 23 strains were found to be able to degrade DF, and the majority produced soluble yellow metabolites during the degradation. A higher isolation frequency for DF degraders was obtained with samples containing higher concentrations of polychlorinated dioxins. Most of the DF-degrading isolates were identified as members of the class Actinobacteria, particularly of the genera Nocardioides and Rhodococcus. These results suggest that particular actinobacterial species constitute the major populations of culturable DF-degrading bacteria in dioxin-polluted environments.

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