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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-␤-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.Bacteria express many phosphoenolpyruvate-dependent phosphotransferase systems (PTS) consisting of sugar-specific transport proteins and phosphorylation mechanisms to utilize various sugars. The PTS are primarily specific to each sugar, but they can transport other sugars to a lesser extent (4). Furthermore, their substrate specificity can be changed by the amino acid replacement(s) (1, 2). Genome sequence analysis of Bacillus subtilis (15) has identified 21 complete PTS, 17 of which were characterized or estimated via the corresponding sugars (21; see also the B. subtilis genetic databases BSORF [http://bacillus.genome.ad.jp/] and SubtiList [http://genolist .pasteur.fr/SubtiList/]). The operon for the mannitol-specific PTS is located at 38.5°of the B. subtilis chromosome. Mannitol-specific PTS have been investigated in Escherichia coli (16), in Bacillus stearothermophilus (8), and in other organisms (3, 10). The mtl operon in E. coli consists of the mtlA, mtlR, and mtlD genes that encode the mannitol transporter (enzyme IIC-BA mtl ), a transcriptional regulator, and mannitol-1-phosphate dehydrogenase, respectively, and that in B. stearothermophilus comprises four genes: mtlA (enzyme IICB mtl ), mtlR, mtlF (enzyme IIA mtl ), and mtlD. In contrast, the B. subtilis mtl operon consists of only the mtlA (enzyme IICBA mtl ) and mtlD genes, and the operon does not contain a gene for a transcriptional regulator. However, the gene ydaA, which was recently renamed mtlR and which is homologous to the mtlR genes of E. coli and B. stearothermophilus, is located 14.4 kb down stream from the mtl o...

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.