Hiroshi Kakeshita

Essential Bacillus subtilis genes

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

The effect of Srb, a homologue of the mammalian SRP receptor α-subunit, on Bacillus subtilis growth and protein translocation

To determine the signal recognition particle (SRP)-SRP receptor (Srb) system in Bacillus subtilis (Bs), we cloned the Bs srb gene, which encodes a homologue of the mammalian SRP receptor alpha-subunit [Oguro et al., DNA Res. 2 (1995) 95-100]. We sequenced a 6098-bp DNA containing srb and analyzed the gene organization. Primer extension experiment and Northern blot analysis revealed that srb constitutes an operon with two additional ORFs. A database search of known proteins revealed that one encodes a homologue of Escherichia coli RNase III [36.0% identical amino acids (aa)] and the other encodes a homologue of yeast Smc1 (26.6% identical aa). We then constructed a Bs mutant in which srb expression was induced by IPTG. The depletion of Srb caused a defect in the cell growth and the cells became filamentous and twisted. Furthermore, pulse-chase experiments using this mutant revealed that the 17% of the beta-lactamase precursor accumulated in the cell after a 4-min chase in the absence of IPTG, although almost all of the precursors were converted into the mature from after a 1-min chase in the presence of IPTG.

Protein Traffic for Secretion and Related Machinery of Bacillus subtilis

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP–protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP–Sec pathway in protein localization to the cell membrane and spore was analyzed.

Mannitol-1-Phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in Bacillus subtilis: Possible Cooperation of mtl and gut Operons

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.

Expression of the ftsY gene, encoding a homologue of the alpha subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis.

Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A). During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.

Improvement of Heterologous Protein Secretion by Bacillus subtilis

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).