Kinji Uchida

Aspartate Decarboxylation Encoded on the Plasmid in the Soy Sauce Lactic Acid Bacterium, Tetragenococcus halophila D10

Tetragenococcus halophila D10 decarboxylates aspartate to alanine, but T. halophila D10 derivatives generated by a curing treatment could not (Asd(-) derivatives). We observed by electrophoresis three plasmid bands in T. halophila D10; all Asd(-) derivatives lost the largest of these bands. This plasmid, pD1, has two SalI sites. We cloned and sequenced the 10 kb SalI fragment. The DNA sequence suggests that this fragment contains the aspartate decarboxylating trait.

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate: mannose phosphotransferase system and in phosphofructokinase.

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTSd) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTSd phosphofructokinase defective (PFK−) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N′-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (ΔP) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTSd strain.

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EIIman), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (Km=11 μM) and a low-affinity proton motive force driven glucose permease (GP) (Km=170 μM). (ii) Both 9R-4 and X-160 had only the low-affinity system (Km=181 μM for 9R-4, 278 μM for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EIIman) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.

Esterase activity in soy sauce Moromi as a factor hydrolyzing flavor esters

Abstract In order to elucidate the reason for the meager occurrence of volatile esters in soy sauce, the ester-decomposing activities of microorganisms concerned in soy sauce fermentation were examined. Soy yeasts showed at least 10 times higher esterase activity than the other yeasts used for fermented beverages. The yeast esterase was not greatly affected by the pH or NaCl concentration. Soy koji cultured with Aspergillus sojae or A. oryzae showed very high ester-splitting activity. By gel-filtration of koji esterase, the i -amylacetate ( i -AmAc) decomposing fraction was obtained. This fraction showed a decrease of activity at lower pH or higher NaCl concentration. Koji esterase decreased its activity in moromi but remained over the entire moromi period. Koji esterase exhibited a higher activity than yeast esterase in fermenting moromi . These strong esterase activities are thought to be one of the causes of the low concentration of ester flavor in soy sauce.

Preparation of Phage-insensitive Strains of Tetragenococcus halophila and Its Application for Soy Sauce Fermentation

We attempted to breed phage-insensitive strains of Tetragenococcus halophila D10. Phage contact during selection initially caused the occurrence of lysogeny. Subsequently, we screened phage-insensitive mutants by replica plating so that mutant cells did not touch the phage during selection. Two strains were selected from about 150,000 strains. They grew normally in soy sauce mash (moromi) in the presence of phage φD-10, although they had a similar extent of adsorption of φD-10 as did the parent strain.

Inhibitory Effect of Sodium Chloride on Methylammonium Uptake by a Salt-Tolerant Yeast : Zygosaccharomyces rouxii

Abstract Uptake of methylammonium, an analogue of ammonium, by Zygosaccharomyces rouxii cells grown without sodium chloride (NaCl) was found to be reduced by the presence of NaCl in the uptake-reaction mixtures. The reduced uptake was almost inversely proportional to NaCl concentration, up to 15%. As for Saccharomyces cerevisiae , in Z. rouxii cells grown with NaCl, however, high activities of methylammonium uptake and ammonium consumption even in the presence of NaCl was found. Kinetic analysis of methylammonium uptake by Z. rouxii showed that the cells grown with NaCl provided an uptake system with a large V max value. It was thought that an enhanced uptake system enabld Z. rouxii to incorporate enough ammonium to support its vigorous growth in the presence of NaCl. Attempts were made to discover the mechanism for the increased in V max , but no evidence for the induction by NaCl of a new transport system for methylammonium or ammonium was obtained.