Mitsuo Takano

Characterization of starch-hydrolyzing lactic acid bacteria isolated from a fermented fish and rice food, “burong isda”, and its amylolytic enzyme

Abstract Nine strains of lactic acid bacteria that hydrolyze starch were isolated from burong isda, an indigenous fermented food made from fish and rice in the Philippines. Conventional taxonomic and DNA-DNA reassociation studies indicated that all these isolates belong to Lactobacillus plantarum . Each of these isolates harbored more than ten plasmid species with molecular sizes of 2 to 60 kb. The amylolytic activity of L137, one of the isolates, was lost by treatment with novobiocin at 43% frequency, concomitant with curing of a 33-kb plasmid, pLTK13; this suggested that pLTK13 carries a gene necessary for synthesis of amylolytic enzyme. An acidophilic starch-hydrolyzing enzyme secreted from L137 cells was purified 46-fold with specific activity of 44 units per mg protein. The enzyme was shown to have a molecular mass of about 230 kDa and the optimum temperature and pH for the enzyme reaction with soluble starch were 35°C and 3.8–4.0, respectively. The enzyme hydrolyzed soluble starch, amylopectin, glycogen, and pullulan, and to a small extent amylose, while exerted no activity on dextran and cyclodextrins. The major reaction products from soluble starch were maltotriose, maltotetraose and maltopentaose, but no panose was detected, and maltotriose was the sole product from pullulan. The K m values for soluble starch, pullulan, and amylose were 4.0, 5.1 and 33 g per liter, respectively. These observations suggest that this enzyme hydrolyzes both α-1,6- and α-1,4-glucosidic linkages.

Wound-induced expression of horseradish peroxidase.

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

Genomic DNA structure of two new horseradish-peroxidase-encoding genes.

Genomic DNAs encoding the horseradish peroxidase (HRP) isozymes, prxC2 and prxC3, were cloned and sequenced. By comparing the sequences of the HRP isozyme-encoding genes, prxC1a and prxC1b and their cDNA [Fujiyama et al., Eur. J. Biochem. 173 (1988) 681-687], , it was concluded that prxC2 and prxC3 consisted of four exons and three introns as in the prxC1 gene family. The position of introns in coding regions were the same in all four prx genes. Genes prxC2 and prxC3 coded for 347 and 349 amino acid (aa) residues, respectively, including putative signal sequences at the N termini. In the flanking regions of both genes, putative promoters and polyadenylation signals were found. Nucleotide sequence homology in the coding region was 71% between prxC1a and prxC2, and 66% between prxC1a and prxC3. The aa sequence homologies in plant and microbial peroxidases were compared.

Effect of preincubation temperature on the heat resistance of Escherichia coli having different fatty acid compositions.

The heat resistance of Escherichia coli depends on the temperature of incubation prior to heat treatment at 50°C. This dependence is affected by the fatty acid composition in bacterial membranes. The results are discussed in relation to the change in fluidity of membrane lipids.

Interaction of the Fluorescent Dye 1-N-Phenylnaphthylamine with Escherichia coli Cells during Heat Stress and Recovery from Heat Stress

The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.

Lactic acid bacteria in fermented fishery product, “burong bangus”

Abstract Analyses of the microflora of “burong bangus”, a traditional fermented fish and rice product of the Philippines, revealed that a sequential type of fermentation with overlapping growth takes place. Streptococcus initiated the fermentation process and generally persisted up to the latter part of the fermentation. Pediococcus appeared next, but comprised only a small percentage of the microflora. Both Leuconostoc and Lactobacillus appeared on the 3rd day and were generally present up to the end of the fermentation, with Lactobacillus predominating among the microflora in the final days. In the course of characterizing the lactic acid bacteria involved in the fermenting rice-fish mixture, some isolates were found to be capable of hydrolyzing starch. These were revealed to be Gram positive, rod-shaped and catalase negative. Tentative identification of one of the isolates, L137, showed that this strain possesses very similar characteristics to those of Lactobacillus plantarum and Lactobacillus coryniformis subsp. coryniformis . The % G+C of L137 was 45.2 while those of L. plantarum and L. coryniformis subsp. coryniformis are 45.1 and 45.0 respectively. However, L137 differs from the other two in its ability to utilize starch.

Accumulation of compatible solutes, ectoine and hydroxyectoine, in a moderate halophile, Halomonas elongata KS3 isolated from dry salty land in Thailand

Abstract A moderately halophilic and halotolerant eubacterium, strain KS3, was isolated from a salty soil in Thailand and identified as Halomonas elongata based on the results of conventional taxonomic and DNA-DNA reassociation studies. H. elongata KS3 grows in the presence of 0.3 to 21% NaCl and at temperatures of 5 to 45°C. The optimum concentration of NaCl and temperature for growth were 3 to 5% and 37°C, respectively. Under hyperosmotic stress in a glucose-mineral medium, the accumulation of several low-molecular weight nitrogenous organic compounds was observed. These compounds were identified as 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), 1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid (hydroxyectoine), γ-N-acetyl-α,γ-diaminobutyric acid (ADABA), and l -alanine. Ectoine production was induced immediately by the addition of NaCl and the amount accumulated increased with an increasing external concentration of NaCl and reached approximately 120 μg·mg−1 of dry cells at a concentration of 2.56 M NaCl. Hydroxyectoine was detected with a lag in time after the addition of NaCl at a concentration greater than 1.71 M, and the amount accumulated increased and reached 45 μg·mg−1 of dry cells at a concentration of 2.56 M NaCl. Thus, hyperosmotic stress induced by salt appears to cause the accumulation of ectoine and hydroxyectoine as compatible solutes in H. elongata KS3.

Structure of the genomic DNA encoding cucumber ascorbate oxidase and its expression in transgenic plants.

SummaryGenomic DNA (Aso1) encoding the ascorbate oxidase (ASO) of cucumber (Cucumis sativas) was cloned and sequenced. From a comparison of the sequences of Aso1 and its cDNA [Ohkawa et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1239–1243], it was concluded that Aso1 consisted of four exons and three introns. In the 5′-flanking region, sequence motif resembling a TATA box and a CAAT box, were found 124 and 218 bp upstream, respectively, from the start codon. Sites of initiation of transcription, determined by primer extension, were the A and G residues located 90 and 93 bp, respectively, upstream from the initiation codon. ASO was abundant in cucumber fruits and stems. The 5′-flanking region, placed upstream of the structural gene of β-glucuronidase (GUS), expressed the GUS activity in cucumber plants in a transient expression assay after introduction of DNA with a particle gun. High-level GUS expression was found in stems of transgenic tobacco plants harboring Aso1 promoter-GUS chimeric gene.

Cell death of Bacillus subtilis caused by surfactants at low concentrations results from induced cell autolysis

Abstract A cationic surfactant, palmityltrimethylammonium iodide at 6 to 40 μM caused the death and lysis of Bacillus subtilis 168 cells growing logarithmically in a chemically defined medium. The relationship between the induced death and lysis was investigated by using an autolytic enzyme-defective mutant and by exposure of cells to several treatments. While pretreatment with erythromycin rendered cells resistant to the surfactant, pretreatment with cerulenin caused the sensitization of cells. Addition of cardiolipin neutralized the lytic and killing action of the surfactant. The results obtained suggest that the cell death caused by the surfactant at low concentrations results from the induction of autolysis. At higher concentrations of the surfactant, however, no substantial cell lysis occurred, although much cell death was observed. Similar situations were observed with other surfactants tested.

Regulatory sequences involved in the peroxidase gene expression in Arabidopsis thaliana

SummaryOrgan-specific expression of two peroxidase genes (prxCa and prxEa) from Arabidopsis thaliana was studied. The prxCa gene showed non-specific expression with relatively high levels of mRNA accumulation in the roots, stems and leaves of A. thaliana. The prxEa gene, on the other hand, accumulated high levels of mRNA only in roots. Promoter fragments from each gene were fused to the coding region of β-glucuronidase (gusA) reporter gene introduced into tobacco. Promoter/gusA contructs were transferred to tobacco (Nicotiana tabacum BY-2) protoplasts by electroporation or to N. tabacum SR-1 by Agrobacterium tumefaciens-mediated leaf disk transformation. Transient expression in tobacco protoplasts showed that the 580 fragment from prxEa (Ea-580) expressed thirteen-fold and eight-fold higher GUS activity than prxCa (Ca-622) fragment and CaMV35S promoter, respectively. Tobacco plants transformed with the gusA gene, fused to the -580 deletion (Ea-580), exhibited high GUS expression in roots. The root-specific expression of GUS gene was also observed when the -281 bp deletion end point was used. Although the GUS activity in transgenic tobacco under the control of Ca-622 was low, the activity was found in all organs examined. Histochemical analyses of stem and root tissues of Ea-580 showed that the GUS gene was expressed specifically in phloem and pith parenchyma cells. For Ca-622, high level, specific expression of the gusA gene was observed in the xylem of roots. The results of this study implicate multiple cis-elements in the control of transcription from the prxEa promoter.