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Development of applied microbiology to modern biotechnology in Japan.

Development of modern biotechnology in Japan is characterized by unique contributions from applied microbiology and bioindustry. This review tries to summarize these original contributions with special emphasis on industrial production of useful substances by microorganisms. In the first part, development of applied microbiology and bioindustry in the last half of the twentieth century is summarized with a brief overview of the traditional background. In the second part, recent progress is reviewed with citation of typical achievements in biotechnology, applied enzymology, secondary metabolites, genetic engineering, and screening of microbial diversity, respectively.

Amino Acids↗

Dental plaque as a biofilm.

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogenous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.

Biofilms↗

Microbial growth in a steady-state model of ethylene glycol-contaminated soil.

Biodegradation of ethylene glycol was tested in a laboratory-scale, steady-state infiltration system of two arid region soil types by monitoring indigenous microbial growth after the infiltration of three concentrations of ethylene glycol. Microorganisms in the soils were able to adapt to the ethylene glycol in several cases, resulting in higher numbers of microorganisms and lower pHs in the effluents. These microorganisms were identified and were able to use ethylene glycol as a sole carbon source. The adaptation was seen best with high-moisture-content soils when the ethylene glycol concentrations were 1% or 10%. However, acclimation to 0.1% and 10% ethylene glycol did not occur in low-moisture-content clay soil, but did occur in low-moisture-content silt soil, indicating that soil type and moisture content are important factors. In all cases, microbial diversity decreased over time.

Bacteria↗

Nitrogenase activity and nifH expression in a marine intertidal microbial mat.

N(2) fixation, diazotrophic community composition, and organisms actively expressing genes for N(2) fixation were examined over at 3-year period (1997-1999) for intertidal microbial mats on a sand flat located in the Rachel Carson National Estuarine Research Reserve (RCNERR) (Beaufort, NC, USA). Specifically, diel variations of N(2) fixation in the mats from the RCNERR were examined. Three distinct diel patterns of nitrogenase activity (NA) were observed. NA responses to short-term inhibitions of photosynthesis corresponded to one of the three patterns. High rates of NA were observed during peak O(2) production periods for diel experiments during summer months. Different types of NA diel variations correspond to different stages of mat development. Chloramphenicol treatments indicated that the mechanism of protein synthesis supporting NA changed throughout the day. Analysis of mat DNA and RNA gave further evidence suggesting that in addition to cyanobacteria, other functional groups were responsible for the NA observed in the RCNERR mats. The role of microbial diversity in the N(2) fixation dynamics of these mats is discussed.

Bacteria↗

Effects of culturing on the population structure of a hyperthermophilic virus.

The existence of a culturing bias has long been known when sampling organisms from the environment. This bias underestimates microbial diversity and does not accurately reflect the most ecologically relevant species. Until now no study has examined the effects of culture bias on viral populations. We have employed culture-independent methods to assess the diversity of Sulfolobus spindle-shaped viruses (SSVs) from extremely hyperthermal environments. This diversity is then compared to the viral diversity of cultured samples. We detected a clear culturing bias between environmental samples and cultured isolates. This is the first study identifying a culture bias in a viral population.

Archaeal Viruses↗

FISH shows that Desulfotomaculum spp. are the dominating sulfate-reducing bacteria in a pristine aquifer.

The hydrochemistry and the microbial diversity of a pristine aquifer system near Garzweiler, Germany, were characterized. Hydrogeochemical and isotopic data indicate a recent activity of sulfate-reducing bacteria in the Tertiary marine sands. The community structure in the aquifer was studied by fluorescence in situ hybridization (FISH). Up to 7.3 x 10(5) cells/mL were detected by DAPI-staining. Bacteria (identified by the probe EUB338) were dominant, representing 51.9% of the total cell number (DAPI). Another 25.7% of total cell were affiliated with the domain Archaea as identified by the probe ARCH915. Within the domain Bacteria, the beta-Proteobacteria were most abundant (21.0% of total cell counts). Using genus-specific probes for sulfate-reducing bacteria (SRB), 2.5% of the total cells were identified as members of the genus Desulfotomaculum. This reflects the predominant role these microorganisms have been found to play in sulfate-reducing zones of aquifers at other sites. Previously, all SRB cultured from this site were from the spore-forming genera Desulfotomaculum and Desulfosporosinus.

Archaea↗

Drugs from the seas - current status and microbiological implications.

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

Animals↗

Characterization of a phosphate solubilizing and antagonistic strain of Pseudomonas putida (B0) isolated from a sub-alpine location in the Indian Central Himalaya.

The morphological, biochemical, and physiological characteristics of a phosphate solubilizing and antagonistic bacterial strain, designated as B0, isolated from a sub-alpine Himalayan forest site have been described. The isolate is gram negative, rod shaped, 0.8 x 1.6 microm in size, and psychrotrophic in nature that could grow from 0 to 35 degrees C (optimum temp. 25 degrees C). It exhibited tolerance to a wide pH range (3-12; optimum 8.0) and salt concentration up to 4% (w/v). Although it was sensitive to kanamycin, gentamicin, and streptomycin (<10 microg mL(-1)), it showed resistance to higher concentrations of ampicillin, penicillin, and carbenicillin (>1000 microg mL(-1)). The isolate showed maximum similarity with Pseudomonas putida based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (4-28 degrees C), and maximum activity (247 microg mL(-1)) was recorded at 21 degrees C after 15 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced chitinase, ss-l,3-glucanase, salicylic acid, siderophore, and hydrogen cyanide. The plant growth promotion and antifungal properties were demonstrated through a maize-based bioassay under greenhouse conditions. Although the bacterial inoculation was found to result in significant increment in plant biomass, it stimulated bacterial and suppressed fungal counts in the rhizosphere. The present study is important with respect to enumerating microbial diversity of the colder regions as well as understanding the potential biotechnological applications of native microbes.

Alternaria↗

A quantitative study of the morphological development and bacterial colonisation of the gut of the tammar wallaby Macropus eugenii eugenii and brushtail possum Trichosurus vulpecula during in-pouch development.

We compared the rates of change of various morphological parameters of the stomach, small intestine, caecum and colon of tammar wallabies and brushtail possums with body mass during in-pouch development. These were correlated with changes in the numbers of bacterial species in the various gut segments. In the pouch-young of both species, the wet tissue masses of all gut segments increased with body mass in a positively allometric manner (i.e. with a body mass exponent > 1), suggesting that the mass of each component was disproportionately low at birth, but increased disproportionately rapidly postnatally. However, the lengths of the wallaby stomach and small intestine scaled isometrically with respect to body mass (i.e. with a body mass exponent around 0.33), which may indicate that the shape of these components changes to the adult form during early neonatal development. Conversely, the length of the caecum and colon of both wallabies and possums scaled in a positively allometric manner with respect to body mass, showing area to volume compensation. This may indicate a more general pattern of disproportionately rapid postnatal enlargement in areas that are distal to the principal sites of neonatal digestion (i.e. the stomach). The numbers of bacterial species present in the various gastrointestinal segments of both species were low in animals aged 100 days or less but there was a significant increase in microbial diversity in the caecum of brushtail possums aged over 100 days. The possum caecum also showed the greatest rate of increase in wet tissue mass relative to body mass. It is postulated that caecal development may act as a nidus for establishment of communities of commensal microflora in the developing marsupial.

Animals↗

History and services of culture collections.

Microbial culture collections aim at collecting, maintaining and distributing microbial strains among microbiologists, and are considered to be a means to preserve microbial diversity ex situ. This article reviews the early history of culture collections, which were first set up in the late nineteenth century, with the introduction of pure culture techniques. It also describes the international connections between culture collections, which led to the foundation of the World Federation for Culture Collections (WFCC) and the International Congress of Culture Collections (ICCC). An overview of the Spanish Type Culture Collection (CECT) is included, as well as a description of the services that culture collections can offer.

History, 19th Century↗

Phytoalexin synthesis in soybean cells: elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation.

A glucan elicitor from cell walls of the fungus Phytophthora megasperma f. sp. glycinea, a pathogen of soybean (Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspension-cultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.

Acyltransferases↗

The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen.

The superantigens are receiving a great deal of attention as a new group of potent immunomodulatory molecules. They are produced by diverse microbial agents including staphylococci, streptococci and mycoplasmas and are also encoded by murine tumor viruses (the Mls antigens). Superantigens activate T cells by a unique pathway which can lead to modification of the T-cell repertoire and induction of autoimmunity. Here, Barry Cole and Curtis Atkin review their observations on the Mycoplasma arthritidis superantigen, MAM, and discuss how MAM might contribute to the acute and chronic inflammatory disease mediated by this organism.

Animals↗

Comparison of long-term performances and final microbial compositions of anaerobic reactors treating landfill leachate.

Laboratory scale anaerobic upflow filter, sludge blanket and hybrid bed reactors were operated for 860 days in the treatment of high ammonia landfill leachate. Organic loading was gradually increased from 1.3 to 23.5 kg COD/m3 day in the start-up period and then fluctuated according to the COD concentration of raw leachate. To prevent free ammonia inhibition, influent pH was reduced to 4.5 after Day 181 and consequently COD removal efficiencies above 80% were achieved in all reactors. However, the anaerobic filter and hybrid bed reactor were generally found slightly more efficient and stable than the UASB reactor. In addition to conventional anaerobic reactor control parameters, the complementary techniques of denaturing gradient gel electrophoresis (DGGE), cloning and fluorescent in situ hybridization (FISH) were used to identify and compare the microbial profiles in the reactors at Day 830. Molecular analyses revealed that acetoclastic Methanosaeta species were prevalent in all reactors and configuration did not have an impact on microbial diversity in the long-term.

Ammonia↗

Impact of azadirachtin, an insecticidal allelochemical from neem on soil microflora, enzyme and respiratory activities.

The effect of 10% azadirachtin granules (alcoholic extract of neem seed kernel mixed with China clay) was studied on the population of bacteria, actinomycetes, fungi, Azotobacter and nitrifying bacteria; soil dehydrogenase, phosphatase and respiratory activities on 0, 15th, 30th, 60th and 90th days after application in sandy loam soil collected from the fields. It was observed that baring the Azotobacter sp., azadirachtin at all the doses exerted a suppressive effect on the rest of the microbial communities and enzyme activities in the initial 15 day period. The population of bacteria, actinomycetes besides phosphatase and respiratory activities recovered after 60th day and subsequently increased significantly. The fungi and nitrifiers were most sensitive groups as their numbers were reduced significantly throughout the studies. The two times and five times recommended dose of azadirachtin had very high biocidal effects on the soil microorganisms and its activities. However, analysis of the data by the Shannon Weaver index showed that azadirachtin reduces both the form and functional microbial diversity at all doses.

Azadirachta↗

Regulation of rumen fermentation during seasonal fluctuations in food intake of muskoxen.

We studied rumen fermentation of castrated adult muskoxen (Ovibos moschatus; n=4) during periods of low (May) and high (August) food intake. Turnover time (17+/-1.8 h) and volume (26+/-3.9 L) of rumen fluid were consistent between May and August and among days within each season. Rumen temperature did not vary significantly during the day (38.8+/-0.29 degrees C) in either season. Rumen osmolality (271.9+/-16.4 vs. 245.9+/-11.4 mOsm kg(-1)) and pH (6.81+/-0.31 vs. 6.39+/-0.15) were higher in May than in August indicating a shift in the allostatic set point. Rumen fluid pH was more variable in May than in August both before and after a single meal of fermentable substrate even though fermentation acids were lower in May than in August (101.0+/-11.0 vs. 126.0+/-8.74 mM). Changing proportions of minor fermentation acids indicated a shift in metabolic pathways even though bacterial numbers were similar between seasons (6.4+/-5.8x10(9) mL(-1)). Allostatic set points probably alter the homeostatic range of conditions and the microbial diversity of fermentations in herbivores from highly seasonal environments.

Animals↗

Mining genomes and 'metagenomes' for novel catalysts.

Advances in the field of genomics and 'metagenomics' have dramatically revised our view of microbial biodiversity and its potential for biotechnological applications. Considering the estimation that >99% of microorganisms in most environments are not amenable to culturing, very little is known about their genomes, genes and encoded enzymatic activities. The isolation, archiving and analysis of environmental DNA (or so-called 'metagenomes') has enabled us to mine microbial diversity, allowing us to access their genomes, identify protein coding sequences and even to reconstruct biochemical pathways, providing insights into the properties and functions of these organisms. The generation and analysis of (meta)genomic libraries is thus a powerful approach to harvest and archive environmental genetic resources. It will enable us to identify which organisms are present, what they do, and how their genetic information can be beneficial to mankind.

Biotechnology↗

Analyses of soil fungal communities in adjacent natural forest and hoop pine plantation ecosystems of subtropical Australia using molecular approaches based on 18S rRNA genes.

Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550bp 18S rDNA fragments. TGGE screening of the PCR products showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products. However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.

Ascomycota↗

Boosting kynurenic acid in kombucha via substrate selection: metagenomic and biochemical insights.

Kombucha is gaining global popularity for its health benefits. This study explored the use of chestnut honey, a rich source of kynurenic acid (KYNA), to produce kombucha enriched with this metabolite. Five variants were prepared using different green/black tea blends and carbon sources: white sugar or acacia honey (controls) versus chestnut honey. Samples were analyzed for tryptophan metabolites, physicochemical properties, and microbial diversity. Komagataeibacter and Enterobacter were predominant bacterial genera in SCOBY. Candida and Aspergillus were predominated in the single sample analyzed for fungi. During fermentation, tryptophan decreased, while kynurenine increased. KYNA levels remained largely stable during fermentation and were mainly influenced by the fermentation substrate. No melatonin pathway derivatives were detected. On day 7, chestnut honey yielded kombucha with 381.680-739.915&#xa0;&#x3bc;mol/L KYNA and elevated myricetin. Overall, chestnut honey-based kombucha represents a system in which substrate composition appears to be the main factor influencing KYNA levels in the final beverage.

Kynurenic Acid↗