Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family.

Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth ß-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis.

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 â†’ 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 â†’ 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 â†’ 4)-> α-(1 â†’ 2)- > α-(1 â†’ 3)- > α-(1 â†’ 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 â†’ 4) to α-(1 â†’ 6) transglucosylation activity. Ps6GT31A elongated α-(1 â†’ 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 â†’ 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells.

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Evidence for cycloisomaltooligosaccharide production from starch by Bacillus circulans T-3040.

Bacillus circulans T-3040 produces cycloisomaltooligosaccharide glucanotransferase (CITase) and cycloisomaltooligosaccharides (cyclodextrans, CIs) when it is grown in media containing dextran as the carbon source. To investigate the effects of carbon sources on CITase activity, B. circulans T-3040 was cultured with glucose; sucrose; a mixture of isomaltose, isomaltotriose, and panose (IMOs); a mixture of maltohexaose and maltoheptaose (G67); dextrin (average degree of polymerization = 36); dextran 40; and soluble starch. In addition to dextran 40, CIs were produced when the T-3040 strain was grown in media containing soluble starch as the sole carbon source. CITase production was induced by dextran 40, IMOs, and soluble starch but not by G67 or dextrin, which suggests that α-1,6 glucosidic linkages are required for CITase induction. Although CITase was induced by IMOs, no CIs were produced in the culture. CI-producing activity in the presence of soluble starch as the substrate (SS-CITase activity) was observed only in cultures containing dextran 40 or soluble starch. The production of CITase was significantly unaffected by glucose addition, but SS-CITase activity almost completely disappeared after glucose addition. A 135-kDa protein was found to contribute to CI formation from starch in the presence of CITase. This protein had a disproportionation activity with maltooligosaccharides, and its induction and inhibition system may be different from those of CITase.

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria.

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K.

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.

Crystal structure of bacteriophage ϕNIT1 zinc peptidase PghP that hydrolyzes γ-glutamyl linkage of bacterial poly-γ-glutamate.

Poly-γ-glutamate hydrolase P (PghP) of Bacillus subtilis bacteriophage ΦNIT1 hydrolyzes the γ-glutamyl peptide linkage of extracellular poly-γ-glutamate produced by bacilli, which facilitates infection and propagation of phage progenies. Crystal structure of PghP was determined at a resolution of 1.9 Å. Structure of PghP was elucidated as a globular protein with an open α/ß mixed core structure and a seven-stranded parallel/anti-parallel ß-sheet. The ß-sheet contained a core four-stranded parallel ß-sheet. A zinc-binding motif, His-Glu-His, was identified at the C-terminal end of the ß-sheet. Structure analysis demonstrated that PghP, which had not been previously classified into any peptidase/protease family due to lack of amino acid sequence similarity with known enzymes, had a catalytic center containing a zinc ion and an overall topology resembling mammalian carboxypeptidase A and related enzymes. Structural comparisons indicated important amino acid residues of PghP for catalysis and recognition of the γ-peptide bond of poly-γ-glutamate, which was confirmed by site-directed mutagenesis of PghP.

Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient Bacillus subtilis host-vector system.

A cycloisomaltooligosaccharide (CI; cyclodextran) production system was developed using a Bacillus subtilis expression system for the cycloisomaltooligosaccharide glucanotransferase (CITase) gene. The CITase gene of Bacillus circulans T-3040, along with the α-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, was cloned into the Bacillus expression vector pUB110 and subsequently expressed in B. subtilis strain 168 and its alkaline (aprE) and neutral (nprE) protease-deficient strains. The recombinant CITase produced by the protease-deficient strains reached 1 U/mL in the culture supernatant within 48 h of cultivation, which was approximately 7.5 times more than that produced by the industrial CITase-producing strain B. circulans G22-10 derived from B. circulans T-3040. When aprE- and nprE-deficient B. subtilis 168 harboring the CITase gene was cultured with 10% dextran 40 for 48 h, 17% of the dextran in the culture was converted to CIs (CI-7 to CI-12), which was approximately three times more than that converted by B. circulans G22-10 under the same dextran concentration. The B. subtilis host-vector system enabled us to produce CIs by direct fermentation of dextran along with high CITase production, which was not possible in B. circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase.

Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production.

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis.

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

Comparative Genome Analysis of Three Komagataeibacter Strains Used for Practical Production of Nata-de-Coco.

We determined the whole genome sequences of three bacterial strains, designated as FNDCR1, FNDCF1, and FNDCR2, isolated from a practical nata-de-coco producing bacterial culture. Only FNDCR1 and FNDCR2 strains had the ability to produce cellulose. The 16S rDNA sequence and phylogenetic analysis revealed that all strains belonged to the Komagataeibacter genus but belonged to a different clade within the genus. Comparative genomic analysis revealed cross-strain distribution of duplicated sequences in Komagataeibacter genomes. It is particularly interesting that FNDCR1 has many duplicated sequences within the genome independently of the phylogenetic clade, suggesting that these duplications might have been obtained specifically for this strain. Analysis of the cellulose biosynthesis operon of the three determined strain genomes indicated that several cellulose synthesis-related genes, which are present in FNDCR1 and FNDCR2, were lost in the FNDCF1 strain. These findings reveal important genetic insights into practical nata de coco-producing bacteria that can be used in food development. Furthermore, our results also shed light on the variation in their cellulose-producing abilities and illustrate why genetic traits are unstable for Komagataeibacter and Komagataeibacter-related acetic acid bacteria.

Crystallization and preliminary crystallographic analysis of poly-gamma-glutamate hydrolase from bacteriophage PhiNIT1.

Particular Bacillus subtilis strains produce a capsular polypeptide poly-gamma-glutamate (gamma-PGA) that functions as a physical barrier against bacteriophage infection. Bacteriophage PhiNIT1 can infect B. subtilis and produces a novel gamma-PGA hydrolase PghP. PghP was overexpressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.4 A using a synchrotron X-ray source and were found to belong to space group P3(1)21 or P3(2)21.

Expression of the pgsB encoding the poly-gamma-DL-glutamate synthetase of Bacillus subtilis (natto).

An industrial strain of Bacillus subtilis (natto) was used to produce poly-gamma-DL-glutamate (gammaPGA), a polymer of DL-glutamate linked by a gamma-peptide bond. In spite of efforts to improve gammaPGA production by modifying the medium, little attention has been paid to the expression of the gammaPGA synthetase gene. In this study, we investigated the expression of the gammaPGA synthetic gene and the gammaPGA product under various conditions with the LacZ-fusion of the synthetic gene (pgsB-lacZ). The 5' upstream regulatory region of the pgsB gene was also investigated by constructing deletion mutations of lacZ-fusion. The pgsB-lacZ was clearly expressed in the early stationary phase and was abolished by degU gene disruption. The results showed that pgsB-lacZ expression was repressed in rich media, and that gammaPGA production was limited by the substrate supply rather than by the amount of synthetase. Adding D-glutamate to the medium reduced gammaPGA production and synthetic gene expression. The transcription start point was determined by primer extension, and it was found that up to -721 bp (translation start point = +1) of the 5' untranslated region (UTR) was required for optimal pgsB-lacZ fusion gene expression.

Effects of Solution pH and Ions on Suicidal Germination of Bacillus subtilis Spores Induced by Medium High Temperature-Medium High Hydrostatic Pressure Treatment.

Spores of Bacillus subtilis suspended in water or aqueous solution of NaCl, CaCl2, sodium lactate, or calcium lactate at pH 4 - 7 was subjected to spore inactivation by simultaneous combination of medium high hydrostatic pressure (MHHP; 100 MPa) treatment for germination and medium high temperature (MHT; 65℃) treatment for pasteurization of germinated vegetative cells. The spores at pH 4 in NaCl solution and those at pH 5 and 6 in Na lactate solutions were less killed than in water by MHHP+MHT treatment. Spore inactivation was promoted by calcium ion in NaCl solution at pH 4 and in Na lactate solutions at pH 5 and pH 6, while it was more suppressed at pH 5 and pH 6 in Na lactate solutions than at pH 4 in NaCl solution. The spores treated by MHHP+MHT in NaCl or Na lactate solution at pH 4 were further killed by subsequent MHT treatment.

Poly-γ-glutamic acid production of Bacillus subtilis (natto) in the absence of DegQ: A gain-of-function mutation in yabJ gene.

Poly-γ-glutamic acid (γPGA) production by Bacillus subtilis is regulated by the quorum sensing system where DegQ transmits the cell density signal to a DNA-binding protein DegU. A mutation suppressing the γPGA-negative phenotype of degQ gene knock-out mutant (ΔdegQ) was identified through whole genome sequencing. The mutation conferred an amino acid substitution of Ser103 to phenylalanine (S103F) in yabJ that belongs to the highly conserved YjgF/YER057c/UK114 family. Genetic experiments including LacZ-fusion assay of γPGA synthetic operon confirmed that the suppressor mutation (yabJS103F) was responsible for the recovery of γPGA production. The yabJ itself was not essential for the γPGA production and the mutant allele enabled γPGA production of the ΔdegQ strain even in the presence of wild type yabJ. Thus, yabJS103F was a dominant positive allele. degU-lacZ fusion gene was hyper-expressed in cells carrying the yabJS103F, but disruption of yabJ did not affect the transcription level of the degU-lacZ. These observations suggested that YabJ acquired a function to stimulate expression of degU by the S103F mutation which is involved in the regulation of γPGA synthesis.

Metabolome analysis of Escherichia coli ATCC25922 cells treated with high hydrostatic pressure at 400 and 600 MPa.

Escherichia coli cells were treated with high hydrostatic pressure (HHP) at 400 and 600 MPa. Metabolites (70-1027 m/z) extracted from HHP-treated cells were analyzed using capillary electrophoresis-time-of-flight mass spectrometry and were compared with those extracted from control cells (not treated with HHP). A total of 133 metabolites were identified and mapped to metabolic pathways, and many of these (42.1%) decreased due to the HHP treatment, including NAD+, NADP+, ATP, and substrates for DNA synthesis. Principal component analysis suggested that the central sugar and nucleic acid metabolic pathways were strongly influenced by HHP. A bottleneck in the central sugar metabolic pathway was observed in HHP-treated cells, which created a metabolic imbalance; metabolites mapped upstream (glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate) were accumulated and those downstream (3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate) were depleted. Ribonucleotides were decreased, but the reduction was moderate compared with that of substrates for DNA synthesis; the exception was ATP, which also substantially decreased. The bottleneck in the glycolytic pathway partly explained the exhaustion of ATP. NAD+/NADH ratio of HHP treated cells was comparable with that of untreated control cells.