Katsumi Doi

Adhesion of Escherichia coli onto quartz, hematite and corundum: Extended DLVO theory and flotation behavior

The adhesion of Escherichia coli onto quartz, hematite and corundum was experimentally investigated. A strain of E. coli was used that had the genes for expressing protein for silica precipitation. The maximum cell adhesion was observed at pH <4.3 for quartz and at pH 4.5-8.5 for corundum. For hematite, cell adhesion remained low at all pH values. The microbe-mineral adhesion was assessed by the extended DLVO theory approach. The essential parameters for calculation of microbe-mineral interaction energy (Hamaker constants and acid-base components) were experimentally determined. The extended DLVO approach could be used to explain the results of the adhesion experiments. The effect of E. coli on the floatability of three oxide minerals was determined and the results showed that E. coli can act as a selective collector for quartz at acidic pH values, with 90% of the quartz floated at 1.5 x 10(9)cells/ml. However, only 9% hematite and 30% corundum could be floated under similar conditions. By using E. coli and no reagents, it was possible to separate quartz from a hematite-quartz mixture with Newtons efficiency of 0.70. Removal of quartz from the corundum mixture was achieved by E. coli with Newtons efficiency of 0.62.

Highly stable meso-diaminopimelate dehydrogenase from an Ureibacillus thermosphaericus strain A1 isolated from a Japanese compost: purification, characterization and sequencing

We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The Km values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible.

Bio-deposition of Amorphous Silica by an Extremely Thermophilic Bacterium, Thermus spp.

The bio-deposition of amorphous silica, which occurred in vitro by exposure to the extremely thermophilic bacterium Thermus spp. began from the latter part of the exponential phase of growth of the bacteria. The concentration with which the deposition occurred exceeded the solubility of amorphous silica of neutral pH at the temperature 60~85°C. Our observations suggest that Thermus spp. promotes the formation of siliceous minerals in a geothermal environment.

Amino acid components of lees in salmon fish sauce are tyrosine and phenylalanine

We report that the lees in salmon fish sauce consist of Tyr and Phe. The concentration of free l-Tyr (2.0mM) was almost same as the saturated concentration (2.4mM) in water at 20°C. This result shows that lees are formed by Tyr precipitation due to its saturation in the sauce.

A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Pediococcus lolii sp. nov., isolated from ryegrass silage

A Gram-positive, coccus-shaped, lactic acid bacterium, strain NGRI 0510Q(T), was isolated from ryegrass silage produced in Okinawa Prefecture, Japan. The cell is non-spore-forming, non-motile, and occurs in pairs or tetrads. The strain is homofermentative and produces d- and l-lactic acid from glucose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NGRI 0510Q(T) belongs to the genus Pediococcus and clusters within the Pediococcus acidilactici and Pediococcus pentosaceus group, with 98.2 and 96.9 % sequence identity, respectively. DNA-DNA relatedness between strain NGRI 0510Q(T) and P. acidilactici JCM 8797(T) and P. pentosaceus JCM 5890(T) was 19.3 and 17.3 %, respectively. Based on its phenotypic characteristics, phylogenetic relationship and DNA-DNA relatedness, NGRI 0510Q(T) (=JCM 15055(T)=DSM 19927(T)) represents the type strain of a novel species, for which the name Pediococcus lolii sp. nov. is proposed.

Effects of bacitracin and excess Mg2+ on submerged mycelial growth of Streptomyces azureus

Effects of bacitracin (BC) and excess Mg2+ on pellet formation and mycelial growth of Streptomyces azureus were studied in a liquid culture using Bennett medium. The addition of Mg2+ at concentrations above 0.2 mM to the medium resulted in the promotion of pellet formation, a distinct inhibition of growth and decrease in cell mass. BC changed the growth type of mycelia from the compact pellet-type to the dispersed-type, and stimulated mycelial growth after a long lag period, accompanied by an increase in cell mass. In the presence of less than 0.2 mM Mg2+, BC completely inhibited mycelial growth. Ca2+ showed an effect similar to Mg2+. EDTA inhibited pellet formation, but never stimulated growth. Furthermore, EDTA suppressed the growth inhibitory and stimulatory actions of BC. From these results, we speculated that: (i) excess Mg2+ induced pellet formation; (ii) BC, similar to EDTA chelated with excess Mg2+ in the medium, leading to the inhibition of pellet formation; and (iii) BC-induced stimulation of growth might be due to its chelating and antimicrobial activities. It was also said that the growth inhibitory action of BC on S. azureus was antagonized by excess Mg2+ or Ca2+ and EDTA.

Distribution of D-amino acids in vinegars and involvement of lactic acid bacteria in the production of D-amino acids

Levels of free D-amino acids were compared in 11 vinegars produced from different sources or through different manufacturing processes. To analyze the D- and L-amino acids, the enantiomers were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine. This was followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using ultra-performance liquid chromatography. The analyses showed that the total D-amino acid level in lactic fermented tomato vinegar was very high. Furthermore, analysis of the amino acids in tomato juice samples collected after alcoholic, lactic and acetic fermentation during the production of lactic fermented tomato vinegar showed clearly that lactic fermentation is responsible for the D-amino acids production; marked increases in D-amino acids were seen during lactic fermentation, but not during alcoholic or acetic fermentation. This suggests lactic acid bacteria have a greater ability to produce D-amino acids than yeast or acetic acid bacteria.

Stimulation of Expression of a Silica-Induced Protein (Sip) in Thermus thermophilus by Supersaturated Silicic Acid

ABSTRACT The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75°C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO2), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe3+ ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe3+-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.

Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant

Aims:  To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains.