Hisayo Ono

Characterization of Acetic Acid Bacteria in Traditional Acetic Acid Fermentation of Rice Vinegar (Komesu) and Unpolished Rice Vinegar (Kurosu) Produced in Japan

ABSTRACT Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.

A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia

A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp. rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus. The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene. The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd(2+) and Cu(2+), under the low oxygen condition. The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd(2+), but not Cu(2+) in free-living cells. The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd(2+) in the bacterial cells. The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A. sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene. Moreover the MTL4 protein was detected by immunostaining. By infection of the recombinant B3, A. sinicus established symbiosis with the recombinant B3 that was grown in Cd(2+) and Cu(2+)-polluted soils. The symbionts increased Cd(2+) accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu(2+) accumulation was noted.

Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase

ABSTRACT We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCSAt) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCSAt gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCSAt gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(γ-Glu-Cys)n-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCSAt gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl2 activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd2+/mg (dry weight) of cells. The expression of the PCSAt gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd2+ approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCSAt gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd2+ accumulation in nodules 1.5-fold.

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.

Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii

Since the chemical synthesis of vitamin B12 requires more than 70 steps, the production of vitamin B12 has been achieved by microorganism fermentation with additional brief chemical modifications. In an effort to increase the productivity of vitamin B12, we tried to express 10 genes belonging to the hem, cob and cbi gene families involved in the synthesis of vitamin B12 in Propionibacterium freudenreichii, which is a known producer of vitamin B12. In a recombinant P. freudenreichii clone that harbored the expression vector containing a cobA, cbiLF, or cbiEGH, we obtained an increase in vitamin B12 production of 1.7-, 1.9-, and 1.5-fold higher, respectively, than that in the microorganism without any cloned genes in the expression vector pPK705. The cobU and cobS genes caused a slight increase in the production of vitamin B12. Furthermore, we achieved multigene expression in P. freudenreichii. In a recombinant P. freudenreichii clone that harbored an exogenous gene, hemA, from Rhodobacter sphaeroides and endogenous hemB and cobA genes, we successfully achieved the production of about 1.7 mg/l vitamin B12, 2.2-fold higher than that produced by P. freudenreichii harboring pPK705.

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria

ABSTRACT The complete nucleotide sequence of pRGO1, a cryptic plasmid fromPropionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4,orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containingorf1 (repA), orf2(repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichiisubsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp.freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

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.

Characterization of Gene Encoding Amylopullulanase from Plant-Originated Lactic Acid Bacterium, Lactobacillus plantarum L137

A starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, was isolated from traditional fermented food made from fish and rice in the Philippines. A gene (apuA) encoding an amylolytic enzyme from Lactobacillus plantarum L137 was cloned, and its nucleotide sequence was determined. The apuA gene consisted of an open reading frame of 6171 bp encoding a protein of 2056 amino acids, the molecular mass of which was calculated to be 215,625 Da. The catalytic domains of amylase and pullulanase were located in the same region within the middle of the N-terminal region. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the N-terminal region, a six-amino-acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) is repeated 39 times, and a three-amino-acid sequence (Gln-Pro-Thr) is repeated 50 times in the C-terminal region. The apuA gene was subcloned in L. plantarum NCL21, which is a plasmid-cured derivative of the wild-type L137 strain and has no amylopullulanase activity, and the gene was overexpressed under the control of its own promoter. The ApuA enzyme from this recombinant L. plantarum NCL21 harboring apuA gene was purified. The enzyme has both alpha-amylase and pullulanase activities. The N-terminal sequence of the purified enzyme showed that the signal peptide was cleaved at Ala(36) and the molecular mass of the mature extracellular enzyme is 211,537 Da. The major reaction products from soluble starch were maltotriose (G3) and maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. From these results, we concluded that ApuA is an amylolytic enzyme belonging to the amylopullulanase family.

Molecular Cloning and Characterization of the srdBCA Operon, Encoding the Respiratory Selenate Reductase Complex, from the Selenate-Reducing Bacterium Bacillus selenatarsenatis SF-1

Previously, we isolated a selenate- and arsenate-reducing bacterium, designated strain SF-1, from selenium-contaminated sediment and identified it as a novel species, Bacillus selenatarsenatis. B. selenatarsenatis strain SF-1 independently reduces selenate to selenite, arsenate to arsenite, and nitrate to nitrite by anaerobic respiration. To identify the genes involved in selenate reduction, 17 selenate reduction-defective mutant strains were isolated from a mutant library generated by random insertion of transposon Tn916. Tn916 was inserted into the same genome position in eight mutants, and the representative strain SF-1AM4 did not reduce selenate but did reduce nitrate and arsenate to the same extent as the wild-type strain. The disrupted gene was located in an operon composed of three genes designated srdBCA, which were predicted to encode a putative oxidoreductase complex by the BLASTX program. The plasmid vector pGEMsrdBCA, containing the srdBCA operon with its own promoter, conferred the phenotype of selenate reduction in Escherichia coli DH5α, although E. coli strains containing plasmids lacking any one or two of the open reading frames from srdBCA did not exhibit the selenate-reducing phenotype. Domain structure analysis of the deduced amino acid sequence revealed that SrdBCA had typical features of membrane-bound and molybdopterin-containing oxidoreductases. It was therefore proposed that the srdBCA operon encoded a respiratory selenate reductase complex. This is the first report of genes encoding selenate reductase in gram-positive bacteria.