More than 99% of the microbes that exist in the environment can not be cultivated easily. Thus, most of the microbes in the environment have not been described and accessed for biotechnology or basic research. "Metagenomics," which is the culture-independent genomic analysis of the microbial community, attempts to overcome these difficulties. This technology has the potential for providing insight into the functional dimensions of environmental genomic datasets and will help to achieve a major goal of environmental microbiology, the complexities of microbial community function and interaction among these microbes. This article gives an overview of the current knowledge of metagenomic technologies and presents examples of studies that have used these techniques. Furthermore, future technologies related to understanding the metagenome are discussed.

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The growth and survival of a rifampicin-resistant strain (22R6) of Ralstonia solanacearum were monitored after incorporation of cruciferous plants into infested soil. Air-dried residues of eighteen cruciferous plants (including two amphidiploid lines) were individually added to soil at a rate of 1.6% (w/w) and the water condition maintained at −10 kPa. Within one month of incubation at 24°C, nine of the 18 residues reduced the population of the strain 22R6; B. rapa sp. rapifera `Ayumi', Brassica oxyrrhina, and an amphidiploid line of Brassicoraphanus, were most inhibitory. In contrast, total soil bacteria increased, regardless of the nature of the residue. After infested soil was incorporated with cruciferous plant residues and pre-incubated for one month in glasshouse, wilt-susceptible tomato were transplanted and monitored for 34-days on residue-treated soil. The pathogen was detected in shoots of only 2-7% of tomato grown respectively in soil with residue of B. rapa sp. rapifera `Ayumi' and Diplotaxis muralis against 47% of tomato in non-residue soil. Data indicated that suppressive effects of the residues might depend on nutritional status and undefined factor in the soil but were not directly dependent on the concentration of glucosinolates (7.2×10^-3-1.1×10¹ μmole g^-1 tissue) in the residues and in vitro tests showed that authentic isothiocyanates were inhibitory to the pathogen only at high concentrations (more than 2 μmole g^-1 soil).

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The simultaneous detection of different bacterial strains carrying a certain functional gene is absolutely imperative in order to detect bacteria on the basis of functional gene expression products (mRNA) in situ. Functional genes are possessed by bacterial strains with different types of cell walls; therefore, the optimization of conditions for digesting different types of cell walls is significant. In this study, we defined the optimal conditions for permeabilization in eight bacterial strains belonging to different phylogenetic divisions using solutions containing different concentrations of lysozyme and/or achromopeptidase, for conventional fluorescence in situ hybridization (FISH), digoxigenin (DIG)-FISH, and catalyzed reporter deposition (CARD)-FISH with a rRNA-targeting oligonucleotide probe. Most bacterial strains were successfully detected using CARD-FISH with lysozyme 10 mg/ml pretreatment. Additionally, achromopeptidase pretreatment combined with lysozyme pretreatment was a highly effective means of permeabilizing bacterial strains that were unable to be detected using lysozyme pretreatment. However, this additional pretreatment resulted in a loss of cell morphology in some bacterial strains due to excessive permeabilization. Consequently, permeabilizing conditions for applying highly sensitive FISH to the well-defined target bacterial strains used in this study were optimized. The results of this study will contribute to the optimization of permeabilizing conditions, which is one of the most important factors for the successful application of highly sensitive FISH.

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This paper describes a plasmid transformation system that permits genetic manipulation of the genus Sphingomonas. A cryptic indigenous plasmid, pAMI-1, of 10 kb from Sphingobium amiense was isolated and characterized. Nucleotide sequencing revealed that pAMI-1 contains five open reading frames, which are predicted to encode proteins associated with integration, recombination, conjugation and replication. Escherichia coli-S. amiense shuttle vectors were successfully introduced into Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis strains by electroporation. The copy number of the shuttle vector was estimated to be 1-2 per chromosome in the Sphingobium yanoikuyae cell.

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Pathways for aerobic degradation of naturally occurring aromatics were estimated from the entire genome sequence of Bradyrhizobium japonicum strain USDA110, a symbiotic nitrogen-fixing bacterium in soil. Many homologs for the genes encoding various oxygenases and enzymes for the β-ketoadipate pathway in the degradation of vanillin, vanillate, protocatechuate, and 4-hydroxybenzoate were scattered over nine loci of the genome. Using a macroarray developed for B. japonicum strain USDA110, we compared gene expression profiles in cells grown in each of these aromatic compounds as a sole carbon source with those of succinate-fed cells. One set of oxygenase genes homologous to pcaGH, pobA, and vanAB and structurally accompanied by transcriptional regulator homologs was markedly upregulated in their expression by one or more of the four aromatics, whereas no marked change was observed in the expression levels of pcaBCDIJF genes for the β-ketoadipate pathway. In addition, cells fed vanillin and vanillate showed high levels of expression of genes for a glutathione-dependent pathway of formaldehyde oxidation, suggesting that the formaldehyde generated from vanillate's demethylation is oxidized via C1 metabolism in B. japonicum. The expression of the above genes was confirmed by quantitative reverse transcription PCR. The implications of these results are discussed in terms of degradation pathways, gene regulation, and the soil environment.

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Thallium (Tl) is a heavy metal found in trace amounts in the earth's crust and has been studied to a much lesser degree than other heavy metals. Since the discovery of high-temperature superconducting components in the Tl-Ca-Ba-Cu-O system, Tl has attracted greater attention as a potential major pollution source of the future. In this study, the response of soil culturable bacterial communities to the addition of Tl was examined with fresh samples of non-polluted garden and arable soils. A preserved air-dried sample from heavy metal-polluted river sediment was also used as a reference. Soil suspension experiments were performed to homogenize the soils to evenly distribute both the microbial populations and Tl and reduce spatial variability. From soil suspensions loaded with Tl at 0.24 to 0.98 mM, bacteria tolerant to 0.49 mM Tl (100 mg Tl l^-1) were isolated and characterized phylogenetically. The background level of culturable Tl-tolerant bacterial populations was 0.0003-0.013% for the non-polluted soils. The Tl-tolerant fraction increased rapidly in response to Tl loading and dominated the bacterial community, which might be attributed mainly to an immediate effect due to the death of Tl-sensitive microorganisms and leaking of nutrients from cell lysis of sensitive species supporting the growth of primarily Tl-tolerant heterotrophs. Such Tl-tolerant culturable bacteria were isolated and classified into 9 groups of Alphaproteobacteria, those from the arable soil were identified as Pseudomonas spp., and those from the contaminated river sediment were identified as Bacillus niacini.

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The impact of overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene on microbial diversity in the rhizosphere of a transgenic tomato was examined using a ribosomal intergenic spacer analysis (RISA) and a terminal restriction fragment length polymorphism (T-RFLP) analysis of two functional bacterial genes (bacterial chitinase and nifH genes). While the overall profiles of both RISA and T-RFLP revealed high similarity between the transgenic and non-trangenic tomato, several polymorphic DNAs were detected as differential bands in both the qualitative and quantitative evaluations. Sequence analysis suggested that differential bands cloned in RISA showed low levels of similarity to known species but were most likely derived from uncultured microbes. The phylogenetic analyses of two differential bands cloned in the T-RFLP analysis of the chitinase and nifH genes indicated that these bands belonged to Streptomyces like chitinase group and cyanobacterial nifH group, respectively.

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A redox dye-staining method using 5-cyano 2,3-ditolyl tetrazolium chloride (CTC) was modified for differential detection of metabolically active gram-positive and gram-negative bacteria. Exponentially growing cells of representative species of the phyla Actinobacteria, Firmicutes and Proteobacteria were stained with CTC in the presence of a substrate mixture, Meldola's Blue and KCN, and post-treated with acetone. Epifluorescence microscopy and spectrophotometric experiments showed that the acetone treatment resulted in the almost complete extraction of CTC formazan crystals from the gram-negative species, whereas it had little or no effect on CTC-stained gram-positive bacteria. Metabolically active gram-positive and gram-negative bacteria in municipal-sewage activated sludge measured by the modified CTC-staining method with microscopic detection composed 24 and 29% of the total count on average, respectively, both of which corresponded to approximately 74% of the viable gram-positive and gram-negative counts detected with a ViaGram Red⁺ Bacterial Gram Stain and Viability kit. Flow cytometric analyses of the CTC-stained and acetone-extracted activated sludge samples gave similar counts to those measured by epifluorescence microscopy. These results indicate that the modified CTC stain procedure with epifluorescence microscopy or flow cytometry can be used as a simple and rapid method for the estimation of metabolically active populations of gram-positive bacteria and gram-negative bacteria in the environment.

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An in-gel kinase assay revealed that homogenates of Closterium ehrenbergii contain Ca²⁺-dependent protein kinases (CDPKs) ranging in relative molecular mass from 47 to 60 kDa, that phosphorylated histone H1, myelin basic protein, and casein as substrates in a Ca²⁺-dependent manner. Immunoblotting and immunoprecipitation assays of Closterium extracts using anti-Dunaliella CDPK serum showed that the antibody cross-reacted with a Closterium CDPK with a relative molecular mass of 55 kDa in the soluble fraction. These results indicate that Closterium possesses CDPK-related polypeptides: one is a 55-kDa CDPK that is immunologically related to Dunaliella CDPK and the others have various molecular masses.

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