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.

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.

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A novel ␣-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60°C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H 2 O 2 (1.8 M).Starch, a main component of our daily diet, is frequently found not only in food residues on dishes but also in food stains on clothes (36). Enzymatic hydrolysis of starch is catalyzed by ␣-amylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.1), ␤-amylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.2), glucoamylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.3), ␣-glucosidase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.20), and debranching enzymes such as pullulanase (pullulan 6-glucanohydrolase; EC 3.2.1.41) and isoamylase (glycogen 6-glucanohydrolase; EC 3.2.1.68). These amylolytic enzymes, especially ␣-amylase and pullulanase, are very important, particularly in the food and detergent industries (1, 28). We have found and characterized some unique debranching enzymes, such as a highalkaline pullulanase (4), an alkali-resistant neopullulanase (16), an alkaline isoamylase (6), and an alkaline amylopullulanase (5) from cultures of alkaliphilic Bacillus strains. These alkaline amylolytic enzymes can be used as effective additives in laundry and automatic dishwashing detergents operating under high alkalinity, as we also reported alkaline cellulases and a highly alkaline protease from alkaliphilic Bacillus strains (21). In particular, the alkaline amylopullulanase is unique in that it hydrolyzes ␣-1,6 and ␣-1,4 linkages in various carbohydrates at different active sites (3, 12).␣-Amylases are used widely in technical applications, such as in bread making, production of glucose and/or fructose syrups and fuel ethanol from starches, and desizing of textiles and paper. The demand for ␣-amylase in laundry and automatic dishwashing detergents has also been growing for several years (36). However, most of the Bacillus ␣-amylases, such as the enzymes from Bacillus licheniformis (BLA) (29), Bacillus amyloliquefaciens (BAA) (38), and Bacillus stearothermophilus (BSA) (23), are acid or neutral enzymes having pH optima at around 6.5. These neutral enzyme...

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, alpha-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.