Keietsu Abe

Exchange of Aspartate and Alanine MECHANISM FOR DEVELOPMENT OF A PROTON-MOTIVE FORCE IN BACTERIA

We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0.4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (K = 0.4 mM) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na, K, or Mg. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37°C prior to reconstitution was prevented by the presence of either aspartate (K(aspartate) = 0.3 mM) or alanine (K(alanine) ≥ 10 mM), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton-motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3.

Efficient gene disruption in the koji -mold Aspergillus sojae using a novel variation of the positive-negative method

When no phenotypic screen is available, gene disruption in the koji -mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji -mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in A. sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A. sojae.

Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae)

Abstract. Ten strains isolated from industrial soy sauce producing koji mold were identified as Aspergillus sojae and distinguished from Aspergillus parasiticus morphologically and physiologically. There was no detectable aflatoxin in any culture extracts of A. sojae strains. Strain 477 was chosen as a representative strain of industrial A. sojae for further molecular analysis. All enzymatic activities associated with the aflatoxin biosynthesis were not detected or negligible in strain 477 compared with that of the A. parasiticus strain. Southern analysis suggested that the genomic DNA of strain 477 contained aflatoxin biosynthetic pathway genes. In contrast, all industrial strains lacked detectable transcripts of aflR, the main regulatory gene for aflatoxin biosynthesis, under the aflatoxin-inducing condition. Our data suggest that defects in aflR expression cause the lack of expression of aflatoxin-related genes which results in the absence of aflatoxin biosynthesis in A. sojae strains.

Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis.

Abstract. The aflR gene product is the main transcriptional regulator of aflatoxin biosynthesis in Aspergillus parasiticus and Aspergillus flavus. Although A. sojae strains do not produce aflatoxins, they do have an aflR homologue. When compared with the aflR of A. parasiticus, the A. sojae gene contains two mutations: an HAHA motif and a premature stop codon. To investigate the functionality of the A. sojae aflR gene product, we used a GAL4 one-hybrid system in yeast. The transcription-activating activity of AflR from A. sojae was 15% of that from A. parasiticus. The introduction of an additional aflR from A. sojae into an A. parasiticus strain did not affect aflatoxin productivity. A hybrid aflR comprising the amino-terminal region of A. sojae aflR and the carboxy-terminal region of A. parasiticus aflR suppressed the effect associated with pre-termination of the A. sojae AflR. We conclude that the premature stop codon of the A. sojae aflR is the key to its functionality and leads to prevention of aflatoxin biosynthesis through loss of the transcription of aflatoxin biosynthesis-related genes.

Cryptococcus nodaensis sp nov, a yeast isolated from soil in Japan that produces a salt-tolerant and thermostable glutaminase

An anamorphic basidiomycetous yeast, which produced a salt-tolerant and thermostable glutaminase, was isolated from soil in Japan and classified in the genus Cryptococcus. Its substrate specificity suggests that this enzyme is an L-glutaminase asparaginase (EC 3.5.1.38). The strain, G60, resembles Cryptococcus laurentii in the taxonomic criteria traditionally employed for yeasts, however it can be distinguished as a separate species based on DNA–DNA reassociation experiments and sequence analysis of the large sub-unit rDNA. Phenotypically, the isolate can be differentiated from C. laurentii by the inability to utilize arbutin as a sole source of carbon. Based on sequence analysis, the strain is related to a group of hymenomycetous yeasts including Bulleromyces albus, Bullera unica, C. laurentii and C. skinneri. The strain, which is formally described as Cryptococcus nodaensis, is industrially important for the formation of the umami taste during production of proteolytic seasonings.

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.

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.