Greedy reduction of Bacillus subtilis genome yields emergent phenotypes of high resistance to a DNA damaging agent and low evolvability.

Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.

Metagenomic gut microbiome analysis of Japanese patients with multiple chemical sensitivity/idiopathic environmental intolerance.

BACKGROUND: Although the pathology of multiple chemical sensitivity (MCS) is unknown, the central nervous system is reportedly involved. The gut microbiota is important in modifying central nervous system diseases. However, the relationship　between the gut microbiota and MCS remains unclear. This study aimed to identify gut microbiota variations associated with MCS using shotgun metagenomic sequencing of fecal samples. METHODS: We prospectively recruited 30 consecutive Japanese female patients with MCS and analyzed their gut microbiomes using shotgun metagenomic sequencing. The data were compared with metagenomic data obtained from 24 age- and sex-matched Japanese healthy controls (HC). RESULTS: We observed no significant difference in alpha and beta diversity of the gut microbiota between the MCS patients and HC. Focusing on the important changes in the literatures, at the genus level, Streptococcus, Veillonella, and Akkermansia were significantly more abundant in MCS patients than in HC (p < 0.01, p < 0.01, p = 0.01, respectively, fold change = 4.03, 1.53, 2.86, respectively). At the species level, Akkermansia muciniphila was significantly more abundant (p = 0.02, fold change = 3.3) and Faecalibacterium prausnitzii significantly less abundant in MCS patients than in HC (p = 0.03, fold change = 0.53). Functional analysis revealed that xylene and dioxin degradation pathways were significantly enriched (p < 0.01, p = 0.01, respectively, fold change = 1.54, 1.46, respectively), whereas pathways involved in amino acid metabolism and synthesis were significantly depleted in MCS (p < 0.01, fold change = 0.96). Pathways related to antimicrobial resistance, including the two-component system and cationic antimicrobial peptide resistance, were also significantly enriched in MCS (p < 0.01, p < 0.01, respectively, fold change = 1.1, 1.2, respectively). CONCLUSIONS: The gut microbiota of patients with MCS shows dysbiosis and alterations in bacterial functions related to exogenous chemicals and amino acid metabolism and synthesis. These findings may contribute to the further development of treatment for MCS. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Clinical Trials Registry as UMIN000031031. The date of first trial registration: 28/01/2018.

The role of p63 in embryonic external genitalia outgrowth in mice.

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh-Wnt/Ctnnb1-Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.

Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation.

BACKGROUND: Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes. RESULTS: Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR. CONCLUSIONS: These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.

A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus.

BACKGROUND: Bacterial strains of the genus Geobacillus grow at high temperatures of 50-75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat. RESULTS: We constructed a transformation system comprising (i) a mobilizable Bacillus subtilis-G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rapLS20 gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rapLS20 increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization. CONCLUSIONS: We describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.

Rapid conjugative mobilization of a 100 kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for self-transfer.

BACKGROUND: The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. RESULTS: In this study, the oriT sequence of pLS20cat (oriTLS20) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriTLS20, after mixing the liquid cultures of the donor and recipient for only 15 min. CONCLUSIONS: The chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.

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

OBJECTIVES: A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. RESULTS: Halotolerant 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. CONCLUSIONS: Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.

Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase.

BACKGROUND: Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD+-dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. RESULTS: Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. CONCLUSIONS: IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.

A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production.

BACKGROUND: A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. RESULTS: When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. CONCLUSIONS: The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.

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.

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

OBJECTIVES: To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). RESULTS: A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126Leu, 132Leu, and 135Lys were implicated in the binding of phosphopantothenic acid, and 100Tyr and 131Lys in that of adenosyl biphosphate. The data supported the participation of 83Glu and 133Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. CONCLUSIONS: 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.

BACKGROUND: Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. RESULTS: The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. CONCLUSION: Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry.

Bacillus subtilis 5'-nucleotidases with various functions and substrate specificities.

BACKGROUND: In 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. RESULTS: We 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. CONCLUSIONS: Accordingly, 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.

BACKGROUND: The 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). RESULTS: Growing 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. CONCLUSIONS: Under 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.

Enhanced secretion of natto phytase by Bacillus subtilis.

Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model.

The nonessential regions in bacterial chromosomes are ill-defined due to incomplete functional information. Here, we establish a comprehensive repertoire of the genome regions that are dispensable for growth of Bacillus subtilis in a variety of media conditions. In complex medium, we attempted deletion of 157 individual regions ranging in size from 2 to 159 kb. A total of 146 deletions were successful in complex medium, whereas the remaining regions were subdivided to identify new essential genes (4) and coessential gene sets (7). Overall, our repertoire covers ~76% of the genome. We screened for viability of mutant strains in rich defined medium and glucose minimal media. Experimental observations were compared with predictions by the iBsu1103 model, revealing discrepancies that led to numerous model changes, including the large-scale application of model reconciliation techniques. We ultimately produced the iBsu1103V2 model and generated predictions of metabolites that could restore the growth of unviable strains. These predictions were experimentally tested and demonstrated to be correct for 27 strains, validating the refinements made to the model. The iBsu1103V2 model has improved considerably at predicting loss of viability, and many insights gained from the model revisions have been integrated into the Model SEED to improve reconstruction of other microbial models.

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.

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

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 Vmax 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).

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

BACKGROUND: Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-ß-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. RESULTS: Quantitative 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. CONCLUSIONS: These 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.

An improved Bacillus subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer's disease.

BACKGROUND: Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. RESULTS: All "useless" genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. CONCLUSIONS: The B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.