<i>In vivo</i> analysis of the MtlR binding site by fluorescence measurement of <i>P</i>. <i>putida</i> GN146 strains carrying pJOE7771.1-derived mutant plasmids.

<p>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.</p

DNase I footprinting analysis of the MtlR binding site.

<p>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 P_<i>mtlE</i> by black lines above (coding strand) and below (noncoding strand) the sequence.</p

Physical map of pJOE7771.1, a pBBR1MCS-2 derivative with low copy number, and nucleotide sequence of the <i>mtlR</i>/6-his-<i>eGFP</i> intergenic region.

<p>Restriction sites used for the construction of pJOE7771.1, pJH189.1 and the P_<i>mtlE</i> mutant plasmids pJH210.1-pJH258.1 are shown. Mannitol, arabitol, or glucitol-inducible expression of the reporter gene <i>eGFP</i> is mediated by P_<i>mtlE</i> and <i>mtlR</i>. The -10 and -35 boxes of P_<i>mtlE</i> are indicated in the nucleotide sequence. The transcription start site of P_<i>mtlE</i> was determined by a modified 5’-RACE protocol (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133248#sec002" target="_blank">materials and methods</a>).</p

Functional Characterization of the Mannitol Promoter of <i>Pseudomonas fluorescens</i> DSM 50106 and Its Application for a Mannitol-Inducible Expression System for <i>Pseudomonas putida</i> KT2440

<div><p>A new pBBR1MCS-2-derived vector containing the <i>Pseudomonas fluorescens</i> DSM10506 mannitol promoter P<i>_mtlE</i> and <i>mtlR</i> encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with <i>Pseudomonas putida</i> and <i>eGFP</i> as reporter gene. The new vector was applied for functional characterization of P<i>_mtlE</i>. Identification of the DNA binding site of MtlR was achieved by <i>in vivo</i> eGFP measurement with P<i>_mtlE</i> wild type and mutants thereof. Moreover, purified MtlR was applied for detailed <i>in vitro</i> investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to P<i>_mtlE</i> as a dimer. The proposed DNA binding site of MtlR is AGTGC-N₅-AGTAT-N₇-AGTGC-N₅-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of P<i>_mtlE</i>. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.</p></div

<i>eGFP</i> reporter gene expression with different regulator/promoter systems and inducers in <i>P</i>. <i>putida</i> GN146.

<p>(A) Fluorescence of <i>P</i>. <i>putida</i> GN146 pJOE7771.1 (MtlR/P_<i>mtlE</i>) induced with mannitol, arabitol, or glucitol (B) Fluorescence of <i>P</i>. <i>putida</i> GN146 pJOE7771.1 (MtlR/P_<i>mtlE</i>, inducer: mannitol) and pJH257.2 (optimized MtlR/P_<i>mtlE</i> with altered -35 sequence “TTGTCg”, inducer: mannitol) compared to <i>P</i>. <i>putida</i> GN146 pJOE7784.1 (RhaR-RhaS/P_{<i>rhaBAD</i>}, inducer: rhamnose) and <i>P</i>. <i>putida</i> GN146 pJOE7801.1 (TetR/P_<i>tetA</i>, inducer: anydrotetracycline). Fluorescence was measured 6 h after inducer addition.</p

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.

<p>(A) P_<i>mtlE</i> operator mutants with truncated 5’ sequences (fragments Cy5-labelled). (B) P_<i>mtlE</i> operator mutants with blocks of base substitutions (fragments Cy5-labelled). (C) P_<i>mtlE</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133248#pone.0133248.g004" target="_blank">Fig 4</a>.</p

Mannitol, arabitol and glucitol utilization by <i>Pseudomonas fluorescens</i> DSM10506.

<p>(A) Structure of the mannitol operon. In the presence of mannitol, arabitol, or glucitol, transcription of <i>mtlE-Z</i> is activated by MtlR. <i>mtlR</i> is located apart from the other genes in the genome of <i>P</i>. <i>fluorescens</i> 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.</p

Plasmids used in this study.

<p>^<i>a</i> Plasmids with mutations upstream of the -35 sequence of P_<i>mtlE</i>.</p><p>^<i>b</i> Plasmids with mutations in the -35 sequence of P_<i>mtlE</i>.</p><p>Plasmids used in this study.</p

Determination of the equilibrium dissociation constant (<i>K</i>_<i>D</i>), dissociation rate (<i>k</i>_<i>diss</i>) and half life time (t_<i>½</i>) of the MtlR monomer binding to its upstream binding site.

<p>(A) Representative EMSA and determination of <i>K</i>_<i>D</i>. 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 <i>K</i>_<i>D</i> 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 <i>k</i>_<i>diss</i> and t_<i>½</i>. 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 <i>k</i>_<i>diss</i> and t_<i>½</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133248#pone.0133248.g005" target="_blank">Fig 5C</a>).</p

Descriptive characteristics of observed MDTMs.

<p>Descriptive characteristics of observed MDTMs.</p