One of the most astonishing findings of whole-genome sequencing is the massive degree of horizontal gene transfer, which implies that gene exchange among organisms is not a laboratory peculiarity but a constitutive factor in microbial consortia. Although the rRNA operon is highly conserved, Escherichia coli has an extremely variable genome size ranging from 4.5 to 5.5 Mb, of which ca 10% consists of acquired genes as the result of horizontal gene transfer over 100 million years. Direct evidence of such horizontal gene transfer is provided by a huge amount of prophage or prophage remnant sequences. It is often assumed that viruses do not intrude on the genus barrier, but this concept has been questioned since host range can vary greatly between marine phages even with insufficient knowledge of transduction in natural microbial communities. Novel "broad-host-range vector particles", with a variety of recipients and an extremely high transduction frequency of up to 2.6×10^-3 per particle, have been discovered among virus-like particles (VLPs) in marine and thermal environments. The discovery might provide a clue as to the role of environmental gene mediators contributing to evolutionary diversification and speciation.

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Bacillus cereus is a gram-positive, rod-shaped, endospore-forming bacterium that occurs ubiquitously and is frequently isolated from soil and food products. When B. cereus is present in foods, it can cause spoilage and poisoning. The work of our group is focussed on several properties of B. cereus that are of key importance in the food-industry, e. g. sporulation, spore properties, biofilm formation and stress response. This review describes our progress and current research with B. cereus isolates from natural and industrial sources that will utilize the fully sequenced and annotated genome of the model strain, B. cereus ATCC 14579.

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Microbes with which ruminants have established symbiotic relationships include bacteria, protozoa and fungi. Microbiological studies in Japan have emphasized the ecology of the ruminal system including the physiological and biochemical interrelationships between individual microbes. The difference in the distribution of various types of microbes in the rumen was studied first and the importance of the attachment of microbes to solid feed substrates was clarified. The existence of substrate-binding factors in bacteria has also been demonstrated. Many genes encoding fibrolytic enzymes have been cloned and characterized from ruminal microbes to improve the digestion of fiber. Some highly fibrolytic bacteria were produced and the survival of recombinant bacteria in the rumen was studied. The manipulation of ruminal bacteria has not been successful and is still at a challenging stage. Both microbial genetics and ecological approaches are needed to improve ruminant productivity through microbial manipulation. As for the study of microbial protein and amino acid metabolism, a particular emphasis was given to the role of ciliate protozoa. The synthesis of lysine from diaminopimelate by protozoa was demonstrated. The metabolic pathways for many kinds of amino acids were well studied, and it was shown that protozoa actively decompose feed protein and excrete amino acids as metabolic products. Therefore, protozoa tended to reduce the microbial amino acid flow to the intestine. A specific role for pipecolic acid in the physiology and behavior of the host animal was postulated. Protozoal function is still an important problem and deserves further research. Concerning the metabolism of carbohydrate, the reduction of ruminal methane production has been studied because methane is viewed as a pollutant greenhouse gas. Some possible methods of reducing methane production with the least adverse effect have been developed. In this review, some recent topics in ruminal microbiology are described.

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The gastro-intestinal (GI) tract is the primary site of food intake, perception and conversion. It represents one of the most important metabolic organs of the body and is colonised by a myriad of microbes that contribute to nutrient processing, affect immune function, and stimulate a variety of other host activities. Genomes of GI-tract resident microbial species have been sequenced, e.g. the Lactobacillus plantarum WCFS1 genome sequence has been published and an ever improving annotation database is in facilitating ongoing research to provide a model for specific and mechanistically predictable interactions of host and microbe. Bifidobacteria are of particular interest as they are dominantly present in the human GI-tract. The genome of Bifidobacterium longum has been sequenced and its analysis has provided insights into the interactions of Bifidobacteria with their hosts. Research into novel species, such as the recently identified Akkermansia muciniphila, which grows solely on mucin, will help to unravel the species-specific functionality of microbes. This article provides an overview of current research initiatives undertaken to elucidate microbial functionality in the human GI-tract.

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The cellular slime mold Dictyostelium discoideum expresses a RecA homolog that is distributed in mitochondria. A gene disruption mutant is hypersensitive to DNA-damaging agents, indicating that RecA is required for the repair of mitochondrial DNA. We analyzed the process by which a multicellular structure of recA^- cells is formed. Four hours after starvation, the internal glucose level of recA^- cells (11.8±0.9 nmol/mg protein) was higher than that of wild-type cells (6.7±0.1 nmol/mg protein). The degree of cell-cell adhesion of recA ^- cells was greater than that of wild-type cells, while the migration speed of recA^- cells (3.5±0.2 μm/min) was less than that of wild-type cells (4.3±0.3 μm/min). The increase in cell-cell adhesion and decrease in motility of recA^- cells gave rise to the formation of large multicellular structures. The diameters of mounds formed by recA^- and wild-type cells were 0.30±0.01 mm and 0.17±0.01 mm, respectively.

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The source of Salmonella cross-contamination in 15 retail chicken outlets in a residential area of Coimbatore City, southern India, was studied. Chopping boards (18.75%) and the butcher's hands (14.29%) were predominant for Salmonella followed by knives and the weighing balance tray. Serotyping of the Salmonella strains revealed that all the strains were Salmonella enteritis, except one, which was found to be Salmonella cerro. The antibacterial activity of commonly used spices, such as garlic, onion, ginger, turmeric, clove, cinnamon and dry ginger against S. enteritidis, was evaluated. While garlic showed very good antibacterial activity even at very low concentrations, onion and ginger showed only moderate levels of inhibitory effect. The efficacy of the spice extracts in controlling the growth of S. enteritidis on various surfaces such as wood, plastic and stainless steel was also studied. The results indicated that garlic extract was very efficient in removing the organism from different contaminated surfaces at various concentrations.

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Crude extracts of extracellular enzymes were prepared from a solid waste-compost mixture (SCM) taken from two mesophilic fed-batch composting (FBC) reactors for garbage treatment, and were tested for protease activity with azocasein as the substrate. The protease activity was highest at a reaction temperature of 70°C and remained relatively high even at 90°C. The optimum pH for the activity was 7 to 10. There were a significant negative correlation between the protease activity and the moisture content in the FBC reactors when the extracts were prepared. Seventy-six strains of proteolytic bacteria were isolated quantitatively by the agar-plating method and identified phylogenetically by 16S rRNA gene sequencing. Most of the isolates were assigned to members of the phyla Actinobacteria, Bacteroidetes, and Firmicutes, especially those of the genera Bacillus, Cellulosimicrobium, and Ornithinococcus and an unaffiliated Cytophaga-like group. Inhibitor and zymography experiments showed that the proteolytic activities of the isolates belonging to the above-noted Gram-positive genera as well as of the SCM sample were inhibited by phenylmethylsulfonyl fluoride and that the isolates of Bacillus, Cellulosimicrobium, and the Cytophaga-like group excreted a protease similar to those from the SCM sample in electrophoretic mobility. The results of this study suggest that bacteria belonging to the phyla Actinobacteria and Firmicutes and producing alkaline serine protease play primary roles in protein digestion in the mesophilic FBC process.

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Using a widely used commercial DNA extraction kit and a newly modified direct DNA extraction method proposed in this report, soil DNA was extracted from arable land in diverse geological locations in Japan and the quality and quantity of the DNA were examined. A modified direct DNA extraction method was developed, consisting of one-step extraction and two-step purification using potassium acetate and a DEAE-cellulose column, respectively, and designated as a DSPD (Differential Salt Precipitation and DEAE-cellulose) method. The total time needed to process six soil samples with this method was less than two hours, including the time taken to set up the DEAE-cellulose columns. Other advantages of our DSPD method are the use of non-hazardous reagents and running costs only 5-10% of the commercial kits currently used in soil microbiological research. This DSPD method was tested using 24 soil samples collected from diverse locations in Japan and showed that it is a reliable technique for DNA extraction from a wide range of soil types when compared with commercial DNA extraction kits. This DSPD method will therefore contribute to the molecular and genomic analyses of microbial populations and ecosystems that require numerous samples of soil DNA.

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Six different soils that have received different organic matter amendments at different rates for more than 7 years (OM-soils) were collected and their suppressiveness against bacterial wilt in tomato was compared with that of corresponding control soils, that is, those amended with only chemical fertilizers (CF-soils). Disease incidence was lower in a yellow soil (Toyohashi, Aichi, Japan) and a brown lowland soil (Ayabe, Kyoto, Japan) with repeated application of cow manure (OM-soils) than in the corresponding CF-soils. The dynamics of the pathogen in non-rhizosphere soils after inoculation showed a quite similar fate in both the OM-soils and CF-soils. However, lux activity tended to be lower in tomato roots grown in the OM-soils than CF-soils when a lux-marked pathogen was inoculated, suggesting that disease incidence is suppressed in the OM-soils because of the reduced growth of the pathogen in the rhizosphere. In contrast, there was no significant difference in disease incidence in four soils between the OM-soils and CF-soils. These results suggested that repeated application of organic matter does not always induce suppression of bacterial wilt, and that the kind of organic matter and application rate may affect the suppressiveness.

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Microbes and Environments volume 19, No. 1 (March 2004), p. 13, "Yuko Takada-Hoshino" should read "Yuko Takada Hoshino". Wrong:Yuko Takada-Hoshino
Right:Yuko Takada Hoshino

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