A novel colorimetric method for the detection of Escherichia coli using cytochrome c peroxidase-encoding bacteriophage.

A new rapid and simple method was developed for the detection of Escherichia coli by constructing a recombinant T4 phage carrying the cytochrome c peroxidase gene derived from Saccharomyces cerevisiae (T4ccp) using which, the colorimetric detection of E. coli K12 was examined. The oxidation activity toward the chromogenic substrate cytochrome c was demonstrated by the cytochrome c peroxidase (CCP) produced from the T4ccp genome. The color change caused by the oxidation of the substrate could be visually perceived. The possibility of interference in the detection by the coexistence of other bacteria was assessed using Pseudomonas aeruginosa as a nontarget bacterium, and it was confirmed that the coexistence of P. aeruginosa caused no interference in the detection of E. coli K12.

Production of triacylglycerol and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by the toluene-degrading bacterium Rhodococcus aetherivorans IAR1.

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) produced by various bacteria has been intensively investigated as a promising biodegradable plastic, but required a supply of an expensive precursor as a secondary carbon source for its production. In a previous study, we identified a new bacterial strain, Rhodococcus aetherivorans IAR1, which synthesizes PHBV from toluene without the supply of a precursor. Toluene is the volatile organic compound most abundantly emitted to the environment. In the present paper, we show that R. aetherivorans IAR1 produces triacylglycerols (TAGs) simultaneously with PHBV. Both PHBV and TAGs were synthesized before the nitrogen source is completely exhausted. The cellular content of PHBV reached 10% of cell dry weight (CDW) and its synthesis ceased even during intermittent supply of toluene. However, accumulation of TAGs continued during cultivation and their cellular content reached 24% of CDW at the end of cultivation. Cerulenin inhibited TAG production and increased PHBV cellular content up to 30% of CDW. The mole fraction of 3-hydroxyvalerate (3HV) in PHBV produced from toluene increased from 60% to 80% during its accumulation. Fatty acid compositions of TAGs produced from acetate and toluene were different. At the end of cultivation, the mole fraction of C17:0, one of odd-carbon number fatty acids, was 5% on toluene or 10% on acetate while the mole fraction of 3HV in PHBV from toluene was as high as that in PHBV from acetate, suggesting that a C5 intermediate of toluene degradation might directly become a precursor of 3HV whereas propionyl-CoA is required for the incorporation of C17:0 into TAGs.

Comparison of organic matter degradation and microbial community during thermophilic composting of two different types of anaerobic sludge.

Changes in organic matter degradation and microbial communities during thermophilic composting were compared using two different types of anaerobic sludge, one from mesophilic methane fermentation, containing a high concentration of proteins (S-sludge), and the other from thermophilic methane fermentation, containing high concentrations of lipids and fibers (K-sludge). The difference in the organic matter degradation rate corresponded to the difference in the organic matter constituents; the CO(2) evolution rate was greater in the composting of S-sludge than of K-sludge; moreover, the NH(3) evolution resulting from the protein degradation was especially higher in the composting of S-sludge. Then the differences in the microbial communities that contributed to each composting were determined by the PCR-DGGE method. Ureibacillus sp., which is known as a degrader with high organic matter degradation activity, was observed during the composting of S-sludge, whereas Thermobifida fusca, which is a well known thermophilic actinomycete that produces enzymes for lignocellulose degradation, were observed during the composting of K-sludge.

Characteristic fungi observed in the fermentation process for Puer tea.

The fermentation process for Puer tea, a unique Chinese tea produced by microbial activities, was investigated by physicochemical and microbial analyses. The temperature of a windrow-shaped pile of tea leaves increased instantly at the beginning of fermentation and stayed at around 50 degrees C until day 35, then decreased gradually to room temperature at the end of fermentation, at day 50. Water content was approximately 30% or less, and pH value was maintained at a weakly acidic level of 5 to 6 throughout the fermentation, conditions that are favorable for propagation of fungi including yeasts. Polyphenol, the characteristic component of tea leaves, decreased continually from day 10 to day 50 of fermentation, corresponding well with the fact that the total concentration of fungi steadily increased during the same period. PCR followed by denaturing gradient gel electrophoresis (DGGE) analysis revealed that there were at least two major fungi: Aspergillus niger, which has been well known among Puer tea manufacturers, and Blastobotrys adeninivorans, which is newly recognized in the present study. Furthermore, both of these fungi were observed in the DGGE fingerprint when other commercial Puer tea products were analyzed. These results prompted us to deduce that both A. niger and B. adeninivorans play important roles in the nutritional enhancement of tea leaves during Puer tea fermentation.

Phenotypic transformation including host-range transition through superinfection of T-even phages.

Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination.

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7.

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.