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. 2003 Aug;185(16):4816-24.
doi: 10.1128/jb.185.16.4816-4824.2003.

Mannitol-1-phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in Bacillus Subtilis: Possible Cooperation of Mtl and Gut Operons

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Free PMC article

Mannitol-1-phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in Bacillus Subtilis: Possible Cooperation of Mtl and Gut Operons

Shouji Watanabe et al. J Bacteriol. .
Free PMC article

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-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.

Figures

FIG. 1.
FIG. 1.
(A) Organization of genes relating to mannitol-specific PTS consisting of mtlA, mtlD, and mtlR and the gene structure of mtlA and mtlD in the disrupted mutants MTLAd and MTLDd and the insertion mutants ΔmtlA and ΔmtlD. (B) Gene organization of gutR, gutB, and gutP for the glucitol transport system. A “P” with an arrowhead and stem-loop structure indicates a putative promoter for σA showing the direction of transcription and of the ρ-independent transcriptional terminator. Numbers in parentheses are predicted amino acid lengths of each gene product. “Kb” indicates the distance from the replication origin of the B. subtilis chromosome. Inserted DNA regions in ΔmtlA and ΔmtlD mutants are also indicated (▪).
FIG. 2.
FIG. 2.
Northern hybridization of transcripts from mtlA, mtlD, and mtlR genes of B. subtilis 168 grown on glucose (Glc), mannitol (Mtl), or glucitol (Gut). Total RNA (10 μg) from cells grown on each sole carbon source were dissolved by agarose gel electrophoresis, and transcripts were detected by using mtlA probe for mtlA (A), mtlD probe for mtlD (B), and mtlR probe for mtlR (C), which are indicated by black bars in the lower portions of the schematic drawings of mtlA, mtlD, and mtlR. We prepared 1,103-, 1,072-, and 1,020-bp probes for mtlA, mtlD, and mtlR, respectively, by amplifying regions near the NH2-terminal coding regions of each gene by PCR with the following specific primer sets: MTLA-F2 (5′-ATGAAAGTGAAAGTGCAACGCTTT-3′) and MTLA-R2 (5′-TGCTTTGGCTTGTTCCGCCTCTAA-3′), MTLD-F2 (5′-GCCTTACATTTCGGTGCGGGAAAT-3′) and MTLD-R2 (5′-GTTCATGGGACTGAATGCCGCACA-3′), and MTLR-F2 (5′-TTAAAGCACTTATTATTACAAAAC-3′) and MTLR-R2 (5′-TATGCGGCTGACGGCCGGCTCCAA-3′). Fragments were purified and labeled with [α-32P]dCTP by using a random labeling kit from Takara-Shuzo, Ltd. Total RNA was extracted from cells at the mid-log phase of growth on 1% glucose (lanes 1, 4, and 7), mannitol (lanes 2, 5, and 8), or glucitol (lanes 3, 6, and 9). Arrows under the genes indicate the direction and sizes of the transcripts in kilonucleotides.
FIG. 3.
FIG. 3.
Growth curves of B. subtilis 168 and the disrupted mutants MTLAd, MTLDd, and MTLRd, on glucose (A), mannitol (B), or glucitol (C). Overnight cultures of 168, MTLAd, MTLDd, and MTLRd in S6 minimal medium containing 1% glucose at 37°C were inoculated into fresh medium containing 1% glucose, 1% mannitol, or 1% glucitol with or without IPTG and then cultured at 37°C. Cell growth in each environment was monitored by absorbance at 660 nm. Symbols: •, strain 168 without IPTG; ○, MTLAd without IPTG; ▴, MTLAd with IPTG; ▵, MTLDd with or without IPTG; ▪, MTLRd without IPTG. The growth of each strain was tested more than four times, and the results were similar each time.
FIG. 4.
FIG. 4.
Growth curves of B. subtilis 168 (A) and its insertion mutants, ΔmtlA (B) and ΔmtlD (C), on 1% glucose (•), 1% mannitol (▴), and 1% glucitol (○) at 37°C. Overnight cultures of 168 and both mutants in synthetic S6 medium containing 1% glucose were inoculated into fresh S6 medium containing 1% glucose, 1% mannitol, or 1% glucitol, and growth was monitored by determining the A660.
FIG. 5.
FIG. 5.
Growth curves of B. subtilis 168 and the disrupted mutants GUTRd, GUTBd, and GUTPd on mannitol (A) and glucitol (B). Overnight cultures of 168, GUTBd, GUTPd, and GUTRd in synthetic S6 medium containing 1% glucose were inoculated into S6 medium containing 1% mannitol or 1% glucitol with or without IPTG and cultured at 37°C. Growth was monitored as the absorbance at 660 nm. Symbols: •, strain 168 without IPTG; ▵, GUTBd without IPTG; ▴, GUTBd with IPTG; ○, GUTPd without IPTG; ▪, GUTRd without IPTG.
FIG. 6.
FIG. 6.
Purification of B. subtilis MtlR from the cell lysate of E. coli transformant. MtlR tagged with His6 was purified from cell lysates of transformants (2-liter culture at 37°C) after 4 h of induction by 1 mM IPTG. Electrophoretic profiles of cell lysates after induction by IPTG each time and purified MtlR preparations are shown after being stained with Coomassie brilliant blue. MW, molecular size markers (given in kilodaltons).
FIG. 7.
FIG. 7.
Gel mobility shift assay with purified MtlR. (A) Binding between MtlR and 32P-labeled DNA fragment containing promoter and upstream region of mtlA and competition for the binding by nonlabeled fragment. A 101-bp DNA fragment containing the promoter and upstream region of mtlA was amplified by PCR with the primer pair MTLA-F3 (5′-AGGGACTGTAAGCGTTTTAACATAG-3′) and MTLA-R3 (5′-ATATAAACCCTCCCTGTTTTGTTTG-3′) and chromosomal DNA from 168 grown in LB medium. Lane 1 shows MtlR binding to a 101-bp DNA fragment without competitor. Lanes 2, 3, and 4 show competition for binding by 5, 50, and 500 nmol of nonlabeled competitor, respectively. (B) Binding between MtlR and a 32P-labeled mtlA DNA fragment was not inhibited by nonlabeled 225-bp DNA fragment containing the promoter and upstream region of gutB and gutR. The 225-bp DNA fragment was amplified by using the primer pair GUTB-F (5′-CAAGTTCAGCCATAAGCCTGCACCT-3′) and GUTB-R (5′-GTGTGAGTCATTTGGCAAGTTCCTT-3′) and chromosomal DNA. Lane 5 shows the absence of competitor DNA. Lanes 6, 7, and 8 show that 5, 50, and 500 nmol of 225-bp DNA did not compete for binding. (C to E) MtlR did not bind to 32P-labeled DNA fragments containing promoters and upstream regions of gut (lanes 9, 10, 11, and 12) (C), lev (lanes 13, 14, and 15) (D), and rbs (lanes 16, 17, and 18) (E) operons. Lanes 9, 13, and 16, absence of MtlR; lanes 10, 14, and 17, 5 nmol of MtlR; lane 11, 20 nmol of MtlR; and lanes 12, 15, and 18, 50 nmol of MtlR. Each reaction was mixed with 32P-labeled 225-bp (5 nmol) fragments of the gut promoter region or 32P-labeled 101-bp fragments (5 nmol each) of the promoters and upstream regions of lev or rbs. Two 101-bp fragments were amplified by using the primer set LEV-F (5′-ATCTATTGCTCCTTTCCTGT-3′) and LEV-R (5′-TGCTATTGGCTGAAATAACA-3′) or the primer set RBS-F (5′-CTGCTTTTGGGTATCATTAAAAAAC-3′) and RBS-R (5′-CTTAATCTTCCTTTCTTGTCGTCTT-3′), as well as chromosomal DNA. “C” and “DF” indicate the DNA-protein mobility shift complex and probe DNA, respectively.

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