Some microorganisms, such as Escherichia coli, harbor transhydrogenases that catalyze the interconversion between NADPH and NADH. However, such transhydrogenase genes have not been found in the genome of a glutamic acid-producing bacterium Corynebacterium glutamicum. In this study, the E. coli transhydrogenase genes udhA and pntAB were introduced into the C. glutamicum wild-type strain ATCC 13032, and the metabolic characteristics of the recombinant strains under aerobic and microaerobic conditions were examined. No major metabolic changes were observed following the introduction of the E. coli transhydrogenase genes under aerobic conditions. Under microaerobic conditions, significant metabolic change was not observed following the introduction of the udhA gene. However, the specific production rates of lactic acid, acetic acid, and succinic acid, and the overall production levels of acetic acid and succinic acid were increased by introducing the E. coli pntAB gene. Moreover, the NADH/NAD + ratio was increased by introduction of pntAB. Our results suggest that the E. coli PntAB transhydrogenase enhances the conversion of NADPH to NADH in C. glutamicum under microaerobic conditions, and the increased NADH/NAD + ratio results in increased succinic acid production. In addition, acetic acid production might be enhanced to supply ATP to the anaplerotic reaction catalyzed by pyruvate carboxylase.Key words: acetic acid; Corynebacterium glutamicum; pntAB; redox balance; succinic acid; transhydrogenases IntroductionMany oxidation and reduction reactions are involved in cellular metabolism, most of which require pyrimidine nucleotide cofactors, such as nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) and their reduced forms NADH and NADPH, to supply oxidizing and reducing equivalents. Generally, NADH is produced via catabolism, and is oxidized in the respiratory chain under aerobic conditions or by fermentation under anaerobic conditions. Anabolic reactions to produce cell biomass components, such as fatty acid and amino acid biosynthesis, require NADPH as a reducing equivalent. Therefore, maintaining the intracellular balance of NAD(P) + and NAD(P)H is very important for most intracellular metabolic reactions to occur.Some organisms contain the enzymes catalyzing the following interconversion of NADH and NADPH, called transhydrogenase:NADPH + NAD + NADP + + NADH. To date, two types of transhydrogenases have been identified; the cytoplasmic-soluble and membrane-bound types. Membrane-bound transhydrogenases have been found in the inner mitochondrial membrane of eukaryotic cells and in the cytoplasmic membrane of many bacteria, and simultaneously catalyze proton translocation across a membrane. Full PaperEnhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB (Received April 3, 2014; Accepted April 8, 2014) Yuto Yamauchi, None of the authors of this manuscript ha...

Three halophilic archaeal strains, MH1-34-1 T , MH1-16-1 and MH1-224-5 were isolated from commercial salt samples produced from seawater in Indonesia, the Philippines and Japan, respectively. Cells of the three strains were pleomorphic and stained Gram-negative. Strain MH1-34-1 T was orange-red pigmented, while MH1-16-1 and MH1-224-5 were pink-pigmented.Strain MH1-34-1 T was able to grow at 12-30 % (w/v) NaCl (with optimum at 18 % NaCl, w/v) at Many haloarchaeal strains grow well at neutral to slightly alkaline pH, and alkaliphilic haloarchaea have also been isolated from various sources. Recently, Minegishi et al. (2008) showed the presence of moderately acidophilic haloarchaeal strains in many solar salt samples. They isolated more than 50 strains capable of growth in a medium adjusted to pH 4.5, from 28 out of 240 commercially available salt samples. Most strains grew at pH range of 4.5-6.0, and none of them showed growth at pH higher than 6.5. As judged from partial (about 500 bp, corresponding to nucleotides 21-520 of Halobacterium salinarum DSM 3754 T ) 16S rRNA gene sequences, they formed four clusters consisting of four to 25 strains. Strain MH1-52-1, a representative of the most acidophilic strains was classified as Halarchaeum acidiphilum gen. nov., sp. nov. (Minegishi et al., 2010). Unfortunately most of the isolates died during storage for a year, except for strain MH1-34-1T and a few others. In this study we repeated the isolation of acidophilic haloarchaeal strains using the same medium as Minegishi et al. (2008), and obtained 28 strains capable of growth at pH 4.5 from 583 salt samples. Full-length 16S rRNA gene sequences of the 28 strains showed that two isolates, MH1-16-1 and MH1-224-5, were almost identical to strain MH1-34-1 T . In the present study, we report on the phenotypic andThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strains MH1-34-1 T , MH1-16-1 and MH1-224-5 are AB372514.2, AB693106 and AB693105, respectively.

Two halophilic moderately acidophilic archaeal strains, MH1-136-2 T and MH1-370-1 were isolated from commercial salt samples made from seawater in Japan and Indonesia, respectively. Cells of the two strains were pleomorphic and Gram-stain-negative. Strain MH1-136-2 T was pink pigmented, while MH1-370-1 was orange-red pigmented. Strain MH1-136-2 T was able to grow at 9-30 % (w/v) NaCl (with optimum, 21 % NaCl, w/v) at pH 4.5-6.2 (optimum, pH 5.2-5.5) and at 18-55 6C (optimum, 45 6C). Strain MH1-370-1 was able to grow at 12-30 % (w/v) NaCl (optimum, 18 %, w/v) at pH 4.2-6.0 (optimum, pH 5.2-5.5) and 20-50 6C (optimum, 45 6C). Strain MH1-136-2 T required at least 1 mM Mg 2+ , while MH1-370-1 required at least 10 mM for growth. Both strains reduced nitrate and nitrite under aerobic conditions. The 16S rRNA gene sequences of strains MH1-136-2 T and MH1-370-1 were identical, and the closest relative wasHalarchaeum rubridurum MH1-16-3 T with 98.3 % similarity. The level of DNA-DNA relatedness between these strains was 90.9 % and 92.4 % (reciprocally), while that between MH1-136-2 T and Halarchaeum acidiphilum MH1-52-1 T , Halarchaeum salinum MH1-34-1 T and Halarchaeum rubridurum MH1-16-3 T was 37.7 %, 44.3 % and 41.1 % (each an average), respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates represent a novel species of the genus Halarchaeum, for which the name Halarchaeum nitratireducens sp. nov. is proposed. The type strain is MH1-136-2 T (5JCM 16331 T 5CECT 7573 T ) isolated from solar salt produced in Japan.Members of the family Halobacteriaceae, 143 species accommodated in 40 genera (as of June 2013), are known to grow at neutral to alkaline pH (Oren, 2007). We have, however, demonstrated that moderately acidophilic haloarchaeal strains able to grow at acidic pH 4.5 can be isolated from many solar salt samples (Minegishi et al., 2008) and the most acidophilic strain MH1-52-1 T was classified as Halarchaeum acidiphilum gen. nov., sp. nov. (Minegishi et al., 2010). Subsequently, we proposed Halarchaeum salinum (Yamauchi et al., 2013a) and Halarchaeum rubridurum (Yamauchi et al., 2013b). In this study, we repeated the isolation of acidophilic haloarchaeal strains using medium adjusted to pH 4. 3 , pH adjusted to 4.0 with HCl. The medium was autoclaved for 20 min at 121 uC. After incubation for 2-4 weeks at 37 uC, colonies were picked up and transferred to new plates. A total of 74 strains were purified by repeated streaking, and 16S rRNA gene sequences were determined according to the method of Minegishi et al. (2012). Two strains selected for further study, MH1-136-2 T and MH1-370-1, were isolated from commercial solar salt samples made from seawater in Japan and Indonesia, respectively.Phenotypic tests were performed according to the methods of Minegishi et al. (2010) and Oren et al. (1997). The analyses were conducted using liquid or solidified MH1 medium at 37 u C. Colony morphology was observed on agar medium. Gram-staining was performed according to the ...

Six halo-acidophilic archaeal strains were isolated from four commercial salt samples obtained from seawater in the Philippines, Indonesia (Bali) and Japan (Okinawa) on agar plates at pH 4.5. Cells of the six strains were pleomorphic, and stained Gram-negative. Two strains were pink-red pigmented, while four other strains were orange-pink pigmented. Strain MH1-16-3T was able to grow at 9-30 % (w/v) NaCl [with optimum at 18 % (w/v) NaCl], at pH 4.5-6.8 (optimum, pH 5.5) and at 20-50 6C (optimum, 42 6C). The five other strains grew at slightly different ranges. The six strains required at least 1 mM Mg 2+ for growth. The 16S rRNA gene sequences of the six strains were almost identical, sharing 99.9 (1-2 nt differences) to 100 % similarity. The closest relatives were Halarchaeum acidiphilum MH1-52-1 T and Halarchaeum salinum MH1-34-1 T with 97.7 % similarity. The DNA G+C contents of the six strains were 63.2-63.7 mol%. Levels of DNA-DNA relatedness amongst the six strains were 79-86 %, while those between MH1-16-3 T and H.acidiphilum MH1-52-1 T and H. salinum MH1-34-1 T were both 43 and 45 % (reciprocally), respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the six isolates represent a novel species of the genus Halarchaeum, for which the name Halarchaeum rubridurum sp. nov. is proposed. The type strain is MH1-16-3 T (5JCM 16108Members of the family Halobacteriaceae (Oren, 2012) are, at the time of writing, accommodated in 138 species of 40 genera. Most strains grow at neutral to slightly alkaline pH, or in media of pH 8.5-11.0. Minegishi et al. (2008) showed that moderately acidophilic haloarchaeal strains could be isolated from solar salt samples, and that most strains grew at a pH range of 4.5-6.0. The most acidophilic of these strains, MH1-52-1, was classified later as representing a novel species of a new genus, Halarchaeum acidiphilum (Minegishi et al., 2010). In a previous study, we repeated the isolation of acidophilic haloarchaeal strains using the same medium used by Minegishi et al. (2008), and proposed a second species of the genus, Halarchaeum salinum, based on three strains (Yamauchi et al., 2013). In the present study, we report the phenotypic and phylogenetic characterization of six halo-acidophilic strains, MH1-16-3 T , , and propose to classify them as representing a novel species of the genus Halarchaeum.Commercial salt samples (0.3 g each) were dissolved in 1 ml sterile 5 % NaCl solution, and one drop each was 3Present address: Graduate School of Interdisciplinary New Science, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan.Abbreviation: PGP-Me, phosphatidylglycerol phosphate methyl ester; S-TGD-1, sulfated triglycosyl diether-1.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of Halarchaeum rubridurum MH1-16-3