Fig 1.
Mannitol, arabitol and glucitol utilization by Pseudomonas fluorescens DSM10506.
(A) Structure of the mannitol operon. In the presence of mannitol, arabitol, or glucitol, transcription of mtlE-Z is activated by MtlR. mtlR is located apart from the other genes in the genome of P. fluorescens DSM10506. (B) Mannitol, arabitol and glucitol are translocated into the periplasm likely by outer membrane porin OprB. MtlE (periplasmatic binding protein), MtlFG (transmembrane domains) and MtlK (ATP binding cassette domain) mediate specific transport of the polyols into the cytoplasm where they are oxidized by MtlD (mannitol 1-dehydrogenase). The products fructose (produced from mannitol and glucitol) and xylulose (produced from arabitol) are phosphorylated by MtlZ (fructose kinase) and MtlY (xylulose kinase) and thus trapped inside the cell. OM = outer membrane, IM = inner membrane.
Table 1.
Plasmids used in this study.
Fig 2.
Physical map of pJOE7771.1, a pBBR1MCS-2 derivative with low copy number, and nucleotide sequence of the mtlR/6-his-eGFP intergenic region.
Restriction sites used for the construction of pJOE7771.1, pJH189.1 and the PmtlE mutant plasmids pJH210.1-pJH258.1 are shown. Mannitol, arabitol, or glucitol-inducible expression of the reporter gene eGFP is mediated by PmtlE and mtlR. The -10 and -35 boxes of PmtlE are indicated in the nucleotide sequence. The transcription start site of PmtlE was determined by a modified 5’-RACE protocol (see materials and methods).
Table 2.
Relative fluorescence of plasmid-carrying E. coli HB101 or P. putida GN146 strains measured 6 h after inducer addition.
Unless stated otherwise, cultures were induced with mannitol.
Fig 3.
eGFP reporter gene expression with different regulator/promoter systems and inducers in P. putida GN146.
(A) Fluorescence of P. putida GN146 pJOE7771.1 (MtlR/PmtlE) induced with mannitol, arabitol, or glucitol (B) Fluorescence of P. putida GN146 pJOE7771.1 (MtlR/PmtlE, inducer: mannitol) and pJH257.2 (optimized MtlR/PmtlE with altered -35 sequence “TTGTCg”, inducer: mannitol) compared to P. putida GN146 pJOE7784.1 (RhaR-RhaS/PrhaBAD, inducer: rhamnose) and P. putida GN146 pJOE7801.1 (TetR/PtetA, inducer: anydrotetracycline). Fluorescence was measured 6 h after inducer addition.
Fig 4.
In vivo analysis of the MtlR binding site by fluorescence measurement of P. putida GN146 strains carrying pJOE7771.1-derived mutant plasmids.
The nucleotide sequences of the wild type (pJOE7771.1) and the mutants are shown. Mutated nucleotides are typed in lowercase. Perfect direct repeats are indicated by solid arrows. Similar direct repeats are indicated by dashed arrows. Fluorescence was measured 6 h after addition of mannitol. (A) Mutants with truncated 5’ sequences. (B) Mutants with blocks of base substitutions. (C) Mutants with doubled or shifted 15 bp sequence stretch -72 to -58.
Fig 5.
EMSA of 2 nM Cy5-labelled (or 8 nM FITC-labelled) DNA fragments incubated (+) with 445 nM (or 1,780 nM for FITC-labelled fragments) or (-) without MtlR.
(A) PmtlE operator mutants with truncated 5’ sequences (fragments Cy5-labelled). (B) PmtlE operator mutants with blocks of base substitutions (fragments Cy5-labelled). (C) PmtlE operator mutants with doubled or shifted 15 bp sequence stretch -72 to -58 (fragments FITC-labelled). The numbers of the DNA fragments equal the numbers of the plasmids in Fig 4.
Fig 6.
DNase I footprinting analysis of the MtlR binding site.
One representative experiment is shown. The sequencing reaction (ACGT) of pJOE7771.1 is shown on the left. Footprinting reactions were performed with 2.28 nM Cy5-labelled operator DNA (-) without or (+) with MtlR (66, 132 or 264 nM). The protected nucleotides are indicated by empty rectangles on the right and the bases that mark the borders of the protected region are indicated on the left. (A) Coding strand. (B) Non-coding strand. (C) Presentation of the nucleotides protected by MtlR in the sequence 5’ to PmtlE by black lines above (coding strand) and below (noncoding strand) the sequence.
Fig 7.
Determination of the equilibrium dissociation constant (KD), dissociation rate (kdiss) and half life time (t½) of the MtlR monomer binding to its upstream binding site.
(A) Representative EMSA and determination of KD. Lanes: (1) 2 nM Cy5-labelled operator DNA, (2–9) 2 nM Cy5-labelled operator DNA + 2, 11, 56, 111, 167, 223, 445, or 667 nM MtlR. The average KD values of at least three independent experiments were 30.8 ± 4.8 nM with mannitol and 32.6 ± 5.0 nM without mannitol. (B) Representative EMSA and determination of kdiss and t½. Lanes: (1) 2 nM Cy5-labelled operator DNA, (2) 2 nM Cy5-labelled operator DNA + 445 nM MtlR, (3–9) 2 nM Cy5-labelled operator DNA + 445 nM MtlR + 100 nM non-labelled competitor DNA loaded onto the gel 0, 15, 30, 45, 60, 75, and 90 min after addition of the competitor. The average kdiss and t½ values of at least three independent experiments were 1.1×10−4 ± 2.8×10−5 s-1 and 112 ± 24 min with mannitol and 1.2×10−4 ± 2.8×10−5 s-1 and of 99 ± 23 min without mannitol. The second upper band in some of the lanes on gel B is considered as an electrophoresis artefact (compare Fig 5C).