Kosei Tanaka

A Single-Batch Fermentation System to Simulate Human Colonic Microbiota for High-Throughput Evaluation of Prebiotics.

We devised a single-batch fermentation system to simulate human colonic microbiota from fecal samples, enabling the complex mixture of microorganisms to achieve densities of up to 1011 cells/mL in 24 h. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that representatives of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. On the earlier stages of fermentation (up to 9 h), trace mixtures of acetate, lactate, and succinate were detectable; on the later stages (after 24 h), larger amounts of acetate accumulated along with some of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human colonic microbiota. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) resulted in an increased population in genus Bifidobacterium, concomitant with significant increases in acetate production. The results suggested that this fermentation system may be useful for in vitro, pre-clinical evaluation of the effects of prebiotics prior to testing in humans.

PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum

BackgroundBradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-β-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression.ResultsQuantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB.ConclusionsThese results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.

Characterization of the native form and the carboxy‐terminally truncated halotolerant form of α‐amylases from Bacillus subtilis strain FP‐133

Two amylases, amylase I and amylase II from Bacillus subtilis strain FP‐133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N‐terminal amino acid sequence, genomic southern blot analysis, and MALDI‐TOFF‐MS analysis indicated that the halotolerant amylase I was produced by limited carboxy‐terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α‐amylases, but their carboxy‐terminal truncation points differed. Three recombinant amylases — full‐length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C‐terminal truncation — were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance.

Bacillus subtilis iolU encodes an additional NADP+-dependent scyllo-inositol dehydrogenase

Bacillus subtilis genes iolG, iolW, iolX, ntdC, yfiI, yrbE, yteT, and yulF belong to the Gfo/Idh/MocA family. The functions of iolG, iolW, iolX, and ntdC are known; however, the functions of the others are unknown. We previously reported the B. subtilis cell factory simultaneously overexpressing iolG and iolW to achieve bioconversion of myo-inositol (MI) into scyllo-inositol (SI). YulF shares a significant similarity with IolW, the NADP+-dependent SI dehydrogenase. Transcriptional abundance of yulF did not correlate to that of iol genes involved in inositol metabolism. However, when yulF was overexpressed instead of iolW in the B. subtilis cell factory, SI was produced from MI, suggesting a similar function to iolW. In addition, we demonstrated that recombinant His6-tagged YulF converted scyllo-inosose into SI in an NADPH-dependent manner. We have thus identified yulF encoding an additional NADP+-dependent SI dehydrogenase, which we propose to rename iolU. Among the Gfo/Idh/MocA family paralogs in B. subtilis, IolU turned out to be an additional scyllo-inositol dehydrogenase.

Genome Sequences of Two Nondomesticated Bacillus subtilis Strains Able To Form Thick Biofilms on Submerged Surfaces

ABSTRACT Genomes of two nondomesticated strains of Bacillus subtilis subspecies subtilis, NDmed and NDfood, have been sequenced. Both strains form very thick and spatially complex biofilms on submerged surfaces. Moreover, biofilms of the NDmed isolate were shown to be highly resistant to antimicrobials action.

Bacillus subtilis 5′-nucleotidases with various functions and substrate specificities

BackgroundIn Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5′-nucleotidases that hydrolyze the phosphate group of 5′-nucleotides. In Bacillus subtilis, genes encoding 5′-nucleotidase have remained to be identified.ResultsWe found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5′-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5′-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not.ConclusionsAccordingly, in B. subtilis, at least YcsE and YktC acted as major 5′-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.

Hyperphosphorylation of DegU cancels CcpA-dependent catabolite repression of rocG in Bacillus subtilis

BackgroundThe two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P).ResultsGrowing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase.ConclusionsUnder physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.

A second-generation Bacillus cell factory for rare inositol production

Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis cell factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the cell factory all “useless” genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the cell factory and its possible applications.

Molecular characterization of a novel N-acetyltransferase from Chryseobacterium sp.

ABSTRACT N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and V max values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).

Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis

ObjectivesA bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential.ResultsHalotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions.ConclusionsChanging the pI of a bacterial serine protease may be an effective strategy to improve the enzyme’s halotolerance.