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Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate. Here, we show that this growth defect could be restored by silencing redundant or less important genes affecting exponential growth by manipulating the global transcription factor AbrB. Comparative transcriptome analysis revealed that AbrB-regulated genes were upregulated and those involved in central metabolic pathway and synthetic pathways of amino acids and purine/pyrimidine nucleotides were downregulated in MGB874 compared with the wild-type strain, which we speculated were the cause of the growth defects. By constitutively expressing high levels of AbrB, AbrB regulon genes were repressed, while glycolytic flux increased, thereby restoring the growth rate to wild-type levels. This manipulation also enhanced the productivity of metabolites including γ-polyglutamic acid. This study provides the first evidence that undesired features induced by genome reduction can be relieved, at least partly, by manipulating a global transcription regulation system. A similar strategy could be applied to other genome engineering-based challenges aiming toward efficient material production in bacteria.

BACKGROUND AND AIMS The Glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide (LIRA) has cardioprotective effects and may reduce the development of kidney disease in type 2 diabetic patients. We here evaluated the effects of LIRA on both kidney and heart function in the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic cardiorenal model. METHOD SDT fatty male rats were treated subcutaneously with vehicle or LIRA 0.4 mg/kg QD for 10 weeks. To measure the effects of LIRA on glomerular hyperfiltration, rats were injected with FITC-sinistrin to measure glomerular filtration rate (GFR) at 4 weeks of treatment. At 5 weeks of treatment, rats underwent unilateral nephrectomy and were put on a 0.3% salt diet to induce a GFR decline. GFR was then measured at 10 weeks of treatment, before hemodynamics measurement and echocardiography. RESULTS Compared with vehicle, LIRA induced significant body weight loss, as well as blood glucose levels reduction by up to ∼20%. During the hyperfiltration phase, LIRA attenuated hyperfiltration, with a 19% lower GFR versus vehicle (P CONCLUSION In the SDT fatty rat, LIRA shows significant benefits by reducing renal hyperfiltration, preventing GFR decline, and improving cardiac hypertrophy, blood pressure and diastolic dysfunction. This preclinical model will be useful to evaluate drugs targeting the cardiorenal axis in type 2 diabetes.

The Glucagon-Like Peptide 1 (GLP-1) receptor agonist liraglutide (LIRA) may reduce the rate of development and progression of diabetic kidney disease in type 2 diabetic patients. To evaluate its impact on kidney function, we evaluated the effects of LIRA on glomerular filtration rate (GFR), in both the hyperfiltration and GFR decline phases in the Spontaneously Diabetic Torii (SDT) fatty rat, a preclinical model of type 2 diabetes. SDT fatty male rats were treated subcutaneously, once daily with vehicle or LIRA 0.4mg/kg for 10 weeks. To measure the effects of LIRA on glomerular hyperfiltration, rats were injected with FITC-sinistrin at 4 weeks of treatment to measure GFR. After 5 weeks of treatment, rats underwent unilateral nephrectomy and were put on a 0.3% salt diet to induce a GFR decline for the last weeks of treatment, then GFR was measured at 10 weeks of treatment. Urine biochemistry and kidney histology analysis were also performed. Compared with vehicle, LIRA resulted in significant body weight loss and lower body weight. LIRA also reduced blood glucose levels significantly by up to ∼20%, as expected. During the hyperfiltration phase, LIRA attenuated hyperfiltration, with a 19% lower GFR vs. vehicle (vehicle: 21 ± 1.1 mL/min/kg; LIRA: 17.1 ± 1.0 mL/min/kg, p In conclusion, LIRA shows significant benefits on GFR, renal inflammation and fibrosis in the SDT fatty rat model. Disclosure F. Briand: Employee; Self; Physiogenex. Stock/Shareholder; Self; Physiogenex. M. Shinohara: None. E. Brousseau: Employee; Self; Physiogenex. Y. Kageyama: None. T. Sulpice: Employee; Self; Physiogenex. Stock/Shareholder; Self; Physiogenex.

After initiation with 7,12-dimethylbenz[a]anthracene (DMBA), the promoting potential of 12- O-tetradecanoylphorbol-13-acetate (TPA) on skin tumor development can be detected by an ultra-short-term skin carcinogenicity bioassay using Tg-rasH2 mice. In the present study, 10 chemicals were assessed using this ultra-short-term bioassay as a first step to validate this practical and easy-to-use skin carcinogenicity bioassay. These chemicals belonged to 4 categories: dermal vehicles (acetone, 99.5% ethanol, anhydrous ethanol, and Vaseline), skin noncarcinogens (oleic acid diethanolamine condensate, benzethonium chloride, and diisopropylcarbodiimide), skin tumor promoters (TPA and benzoyl peroxide), and a skin carcinogen (4-vinyl-1-cyclohexene diepoxide). In a first study, DMBA was used as the initiator at a dose of 50 μg according to previous data, but skin tumors were observed in the no-treatment and vehicle groups. Therefore, the dose of DMBA for skin tumor initiation was reevaluated using 12.5 or 25 μg, with 12.5 μg found to be sufficient for initiation activity. In the ultra-short-term assay, the vehicles and skin noncarcinogens were negative while the skin tumor promoters and the skin carcinogen were positive. The detection of skin tumor promotion and carcinogenicity was feasible in only 8 weeks. In conclusion, this carcinogenicity bioassay may represent a useful tool for the assessment of the carcinogenicity potential of topically applied chemicals.

Poly-gamma-glutamic acid (PGA) is a promising bio-based polymer that shares many functions with poly (acrylic acid) and its derivatives. Thus, technologies for efficient production and molecular size control of PGA are required to expand the application of this useful biopolymer. In Bacillus strains, PGA is synthesized by the PgsBCA protein complex, which is encoded by the pgsBCA gene operon, otherwise is known as ywsC and ywtAB operons and/or capBCA operon. Hence, we investigated responsible components of the PgsBCA complex in B. subtilis for over-production of PGA. In particular, we constructed genomic pgsBCA gene-deletion mutants of B. subtilis. And also, we assembled high copy-number plasmids harboring σA-dependent promoter, leading to high-level expression of all combinations of pgsBCA, pgsBC, pgsBA, pgsCA, pgsB, pgsC, and/or pgsA genes. Subsequently, PGA production of the transformed B. subtilis mutant was determined in batch fermentation using medium supplemented with l-glutamate. PGA production by the transformants introduced with pgsBC genes (lacking the genomic pgsBCA genes) was 26.0 ± 3.0 g L−1, and the enantiomeric ratio of d- and l-glutamic acid (d/l-ratio) in the produced PGA was 5/95. In contrast, d/l-ratio of produced PGA by the transformants introduced with pgsBCA genes (control strains) was 75/25. In conclusion, B. subtilis without pgsA gene could over-produce PGA with an l-rich enantiomeric ratio.

Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates. Dipicolinic acid production in B. subtilis during the stationary phase. The deletion of alsSD enhanced the dipicolinic acid productivity by 1.4-fold.

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-β-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

Human interferon-β (hIFN-β) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-β with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-β with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-β in B. subtilis.

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 x 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

In this study, we examined the effects of modifying the C-terminal region of the SecA protein on the production of heterologous proteins in Bacillus subtilis. SecA was selected as a candidate among the components of the Sec system due to its ability to interact directly with both the precursors and membrane translocases. A phylogenetic comparison demonstrated that the C-terminal region is not well conserved among eubacterial SecA proteins. The deletion of the 61 amino acids at the C-terminal region led to an 83% increase in extracellular alkaliphilic Bacillus sp. thermostable alkaline cellulase (Egl-237) activity. Moreover, the productivity of human interferon α (hIFN-α2b) was increased by 2.2-fold compared to the wild-type SecA, by deletion of these 61 amino acids. We indicated that the deletion of the C-terminal domain (CTD) of SecA enhanced the secretion of two different heterologous protein, Egl-237 and hIFN-α2b. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production.

Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.

Thermostability and chelator resistance of the liquefying alkaline α-amylase (AmyK) from alkaliphilic Bacillus sp. strain KSM-1378 were examined by deletion of either Argl81-Glyl82 or Thr 183-G1y184 on a loop in domain B. In the tertiary structure of Bacillus stearothermophilus α-amylase (BSA), Ilel81-Glyl82 (Thr183-G1y184 in AmyK) pushes away a spatially contacting region containing Ca2+-coordinating Asp207 (Asp209 in AmyK). Therefore, the deletion of Ilel81- G1y182 rather than Argl79-G1yl80 was predicted to result in a higher thermostability of BSA. However, our results with AmyK were clearly contrary to this prediction. The resistance to EDTA of both mutant enzymes from AmyK was essentially equal, and the Argl81-Glyl82-deleted mutant was more thermostable than the Thr183-G1y184-deleted one. It strongly implies that the microenvironmental topology around the loop containing these dipeptides in AmyK is different from that in BSA.

Bacillus subtilis has been widely used for the industrial production of useful proteins because of its high protein secretion ability and safety. We focused on genome reduction as a new concept for enhancing production of recombinant enzymes in B. subtilis cells based on detailed analysis of the genome mechanism. First, we reported that a novel B. subtilis strain, MGB874, depleted 20.7 % of the genomic sequence of the wild type by rationally designed deletions to create simplified cells for protein production. When compared with wild-type cells, the productivity of cellulase and protease from transformed plasmids harboring the corresponding genes was markedly enhanced. These results indicate that a bacterial factory specializing in the production of substances can be constructed by deleting the genomic regions unimportant for growth and substance production from B. subtilis. Second, deletion of the rocDEF-rocR region, which is involved in arginine degradation, was found to contribute to the improvement of enzyme production in strain MGB874. The present study indicated that our results demonstrated the effectiveness of a synthetic genomic approach with reduction of genome size to generate novel and useful bacteria for industrial uses. Furthermore, the design of the changes in the transcriptional regulatory network of the nitrogen metabolic pathway in B. subtilis cells could facilitate the generation of improved industrial protein production.

The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.

Background The Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular enzymes under batch fermentation conditions. We predicted that deletion of the gene for RocG, a bi-functional protein that acts as a glutamate dehydrogenase and an indirect repressor of glutamate synthesis, would improve glutamate metabolism, leading to further increased enzyme production. However, deletion of rocG dramatically decreased production of the alkaline cellulase Egl-237 in strain MGB874 (strain 874∆rocG). Results Transcriptome analysis and cultivation profiles suggest that this phenomenon is attributable to impaired secretion of alkaline cellulase Egl-237 and nitrogen starvation, caused by decreased external pH and ammonium depletion, respectively. With NH3-pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874∆rocG was increased, exceeding that in the wild-type-background strain 168∆rocG. Notably, in strain 874∆rocG, high enzyme productivity was observed throughout cultivation, possibly due to enhancement of metabolic flux from 2-oxoglutarate to glutamate and generation of metabolic energy through activation of the tricarboxylic acid (TCA) cycle. The level of alkaline cellulase Egl-237 obtained corresponded to about 5.5 g l-1, the highest level reported so far. Conclusions We found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874∆rocG and using the NH3-pH auxostat. Deletion of the glutamate dehydrogenase gene rocG enhanced enzyme production via a prolonged auxostat fermentation, possibly due to improved glutamate synthesis and enhanced generation of metabolism energy.

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×103 protoplasts per 25 μl bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10-6 M NAA and 10-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.

Bacillus subtilis is a Gram-positive, nonpathogenic organism which is widely used as a host for enzyme production, due to its ability to secrete large amounts of proteins into the growth medium (Simonen et al., 1993; Westers et al., 2004). The secretion of a target protein leads to the natural separation of the product from cell components, which simplifies downstream processing of the protein. Accordingly, there has been a great deal of research performed regarding protein production in B. subtilis (Simonen et al., 1993; Westers et al., 2004). Nevertheless, the yields of heterologous protein obtained from this organism are often insufficient (Harwood, 1992). Several bottlenecks in the B. subtilis secretion pathway have been reported, including poor targeting to the translocase, degradation of the secretory protein, and incorrect folding (Westers et al., 2004). One of the major bottlenecks involves the degradation of the produced protein by extracellular proteases; therefore, inactivation of extracellular proteases is essential for improvement of protein production with B. subtilis as the host.

The Gram-positive bacterium, Bacillus subtilis and related species are widely used as hosts for the extracellular production of industrially worthy enzymes, such as amylases, proteases, xylanase, and lipases (Braun et al., 1999; Tjalsma et al., 2000; Westers et al., 2004). These species possess a very high capacity for secreting a variety of exoenzymes into the growth medium, thereby reducing downstream purification processes. In addition, many of these are generally regarded as safe (GRAS) microorganisms, and do not produce endotoxins. Therefore, the secretion system of these species presents many advantages in terms of production capacity, structural authenticity, product purification, and safety. Nevertheless, the secretion of heterologous proteins from eukaryotes by these species is frequently inefficient (Table1). Hence, these species are never selected as the best cell factory for pharmaceutical proteins (Westers et al., 2004).

The organization of tandemly repeated sequences of ribosomal DNA (rDNA) in rice mutants derived from gamma-irradiated tetraploids was analyzed. Southern hybridization analysis of nuclear DNA revealed that most of the intergenic spacers (IGSs) in mutant rDNA are replaced concertedly by new molecular species. The new IGSs are produced by the amplification of a subrepeat of about 250 bp. Results obtained from sequence analyses indicate that various intermediate molecular species of the subrepeat were formed during structuring of the IGS region and that many rearrangements occurred between them. These findings demonstrate the effectiveness of recurrent irradiation of tetraploids for inducing artificial genome rearrangement, and also indicate the extreme plasticity and variability of genome structure in plants.

Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

A Mutation causing the abnormal development of grains was induced in rlce (Oryza sativa L.) by means of the tetraploid-irradiation technique. The grain was about half of the size of a normal brown rice and consisted entirely of the embryonic tissue. Microscopic observations revealed that the endosperm cells stopped dividing at early developmental stage, while the embryo continued growing into a large cellular clump in which a plumule, a radicle and a scutellum differentiated. We termed these grains endospermless grains. On the basis of the segregation ratios of endospermless grains, it is concluded that a recessive allele on a single locus or a deletion of small chromosomal segment is resposible for the abnormal development of grains.

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.

Background Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system. Results Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874. Conclusions We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.

Populations of artificial autotetraploids of rice (Oryza sativa L, cvs. Nipponbare and Fukunishiki) were repeatedly irradiated with gamma-rays through several generations. Plants which did not differ in appearance from the original diploid plants occurred occasionally in the populations. These plants were designated as diploid-like plants. Eight diploid-like plants were obtained so far, and their generations were advanced without irradiation in order to examine the mode of segregation of visible characters in their progeny. The results indicated that plants showing several kinds of mutant characters could be obtained and were fully fertile. Usefulness of the tetraploid-irradiation technique for developing fertile rice plants which exhibit gross phenotypic changes is discussed.