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

Abstract

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...

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

“…This sugar was presumably taken up by the cells as F-6-P (Fig. 1), similar to what occurs in other Bacillus species (36). Both strains grew well on this sugar, suggesting that pBM19 genes are not critical for mannitol consumption in B. methanolicus.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…150) B. subtilis MtlR is the regulator of the mtlAFD operon encoding the PTS for mannitol, 151) and it specifically interacts with DNA regions containing the mtl promoter. 152) Disruption of the mtlR gene prevented the utilization of mannitol, indicating that MtlR functions as a transcription activator of the mtlAFD operon. However, the role of PTSmediated regulation has not been studied yet.…”

The lmo0501 gene coding for a putative transcription activator protein in Listeria monocytogenes promotes growth under cold, osmotic and acid stress conditions

“…Beside these three domains, the Lmo0501 protein also possesses PTS enzyme II motifs that resemble the EIIB bgl-like and EIIA fructose/mannitol subunits. While B. subtilis and B. stearothermophilus MtlRs are known transcription regulators activating the expression of mannitol assimilation and catabolism genes, the Lmo0501 functions in L. monocytogenes are presently unknown (Henstra et al, 2000;Henstra et al, 1999;Watanabe et al, 2003).…”

“…stearothermophilus (Henstra et al, 2000;Henstra et al, 1999;Watanabe et al, 2003). In addition to conserved DNA binding winged helix -turn-helix (HTH), MtlRs possess two PTS regulation domains, PRDI and PRDII, that are phosphorylation regulated by PTS components (van Tilbeurgh and Declerck, 2001).…”