The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida

Abstract

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

Fig 1. Scheme of the lapA promoter region, promoter region fragments cloned into pBLKT (or p9TT_BlacZ) plasmid and the names of yielding constructs.

The first nucleotides of the mRNA 5ʹ ends determined by 5ʹ RACE are written in bold and designated as A-I to A-VIII. Potential promoters P_lapA1 to P_lapA8 are highlighted as white boxes in the black DNA. The black arrows show the beginnings of lapA and lapB genes.

Fig 2. Mapped mRNA 5ʹ ends, promoters and Fis binding sites at the lapA promoter region.

(A) Sequence of the lapA promoter region and downstream DNA. The start codons of the lapA and lapB genes are shown in bold. The first nucleotides of the mRNA 5ʹ ends determined by 5ʹ RACE are written in bold and designated as A-I to A-VIII. The potential –10 and –35 elements of the lapA promoters are shown in grey boxes with solid and dotted outlines, respectively. The Fis binding sites Fis-A1, Fis-A2 and Fis-A4 to Fis-A7 are shown in black brackets. The E. coli Fis binding consensus according to Finkel and Johnson (1992) and Shao et al. (2008) is shown above every Fis binding sequence [46,47]. The most important nucleotides in the E. coli Fis binding consensus are shown in bold. The point mutations in the Fis binding sequences are indicated by arrows on the antisense strand. The substituted nucleotides in the -10 boxes of potential promoters are shown in bold on the sense strand. In silico predicted FleQ binding sequences [48] are indicated by grey boxes. (B) Agarose gel electrophoresis of cDNA amplified by the RACE method for the identification of the lapA mRNA 5ʹ ends. The arrows point to the PCR products used to determine the mRNA 5ʹ ends. The primer used is shown under the gel images.

Fig 3. The effect of mutating P_lapA3 on the transcription from the proximal upstream DNA of lapA.

B-galactosidase (β-Gal) activity expressed from the lapA promoter-lacZ reporter constructs containing upstream DNA at position -27 to -200 was measured in P. putida wild-type strain PSm grown in LB medium to optical density of 0.5 (exponential phase) and for 18 hours (stationary phase). Schemes of lapA proximal upstream DNA carrying the functional P_lapA3 promoter and predicted promoters P_lapA1 and P_lapA2 are shown above the diagrams. The Dotted box denotes the mutated promoter P_lapA3, lacZ reporter gene is shown as a black arrow and location of sequences previously described as P_lapA1 and P_lapA2 in white boxes. Vertical bars denote 95% confidence intervals of means. Data of at least 9 independent measurements is shown. Letters a–c depict homogeneity groups according to ANOVA post hoc Bonferroni test. Identical letters denote non-significant differences (P>0.05) between averages of β-galactosidase activity.

Fig 4. Fis binding to Fis-A1 and Fis-A2 sites upstream of the lapA gene.

(A) Protection of the lapA upstream DNA against DNase I cleavage by Fis binding on the sense and the antisense strands. Lines at the right side of the panels indicate the regions protected by Fis from DNase I cleavage at the positions -110 to -85 on the sense strand and -109 to -84 on the antisense strand corresponding to Fis-A1; and -158 to -132 on the sense strand and -159 to -138 on the antisense strand corresponding to Fis-A2. (B) Gel shift assay of the Fis binding to the lapA promoter DNA containing the wild-type Fis binding site Fis-A1 and Fis-A2 and one or the other mutated site. 2 × 10¹⁰ molecules of radioactively labelled PCR products containing both Fis binding sites Fis-A1 and FisA2 or one mutated binding site FisA1mut-Fis-A2, and Fis-A1-FisA2mut were used in Fis binding assay. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) or a PCR product without Fis binding site (RF1). Arrows point to different dissociation of Fis from radioactively labelled DNA in favour of binding unlabelled Fis-specific DNA. Added unlabelled DNA was calculated in molecules. 0.46 μM Fis was used in each reaction mixture except mixtures without Fis in lanes 2, 12 and 22.

Fig 5. Fis binding to Fis-A4 site upstream of the lapA gene.

(A) Protection of the lapA upstream DNA against DNase I cleavage by Fis binding on the sense and the antisense strands. Lines at the right side of the panels indicate the regions protected by Fis from DNase I cleavage at the positions -417 to -381 on the sense strand and -409 to -385 on the antisense strand corresponding to Fis-A4. (B) Gel shift assay of the Fis binding to the lapA promoter DNA containing the wild-type Fis binding site Fis-A4 and mutated site Fis-A4mut. 2 × 10¹⁰ molecules of radioactively labelled PCR products containing Fis-A4 and FisA4-mut sites were used for Fis binding. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) or a PCR product without Fis binding site (RF1). Arrows point to different dissociation of Fis from radioactively labelled DNA in favour of binding unlabelled Fis-specific DNA. Added unlabelled DNA was calculated in molecules. 0.46 μM Fis was used in each reaction mixture except mixtures without Fis in lanes 2 and 12.

Fig 6. Fis binding to Fis-A5 and Fis-A6 sites upstream of the lapA gene.

(A) Protection of the lapA upstream DNA against DNase I cleavage by Fis binding on the sense and the antisense strands. Lines at the right side of the panels indicate the regions protected by Fis from DNase I cleavage at the positions -586 to -566 on the sense strand and -586 to -564 on the antisense strand corresponding to Fis-A5; and -627 to -603 on the sense strand and -626 to -604 on the antisnse strand corresponding to Fis-A6. (B) Gel shift assay of the Fis binding to the lapA promoter DNA containing the wild-type Fis binding site Fis-A5 and Fis-A6 and one or other mutated site. 2 × 10¹⁰ molecules of radioactively labelled PCR products containing both Fis binding sites Fis-A5 and FisA6 or one mutated binding site FisA5mut-Fis-A6, and Fis-A5-FisA6mut were used in the assay. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) or a PCR product without Fis binding site (RF1). Arrows point to different dissociation of Fis from radioactively labelled DNA in favour of binding unlabelled Fis-specific DNA. Added unlabelled DNA was calculated in molecules. 0.46 μM Fis was used in each reaction mixture except mixtures without Fis in lanes 2, 12 and 22.

Fig 7. Fis binding to Fis-A7 site upstream of the lapA gene.

(A) Protection of the lapA upstream DNA against DNase I cleavage by Fis binding on the sense and the antisense strands. Lines at the right side of the panels indicate the regions protected by Fis from DNase I cleavage at the positions -744 to -768 on the sense strand and -745 to -769 on the antisense strand corresponding to Fis-A7. (B) Gel shift assay of the Fis binding to the lapA promoter DNA containing the wild-type Fis binding site Fis-A7 and mutated site Fis-A7mut. 2 × 10¹⁰ molecules of radioactively labelled PCR products containing Fis-A7 and Fis-A7mut sites were used for Fis binding. Fis was outcompeted from Fis-DNA complex with unlabelled PCR product containing the Fis binding site (LF2) or a PCR product without Fis binding site (RF1). Arrows point to different dissociation of Fis from radioactively labelled DNA in favour of binding unlabelled Fis-specific DNA. Added unlabelled DNA was calculated in molecules. 0.46 μM Fis was used in each reaction mixture except mixtures without Fis in lanes 2 and 12.

Fig 8. The effect of mutated Fis binding sites in the lapA promoter region on the level of reporter gene lacZ expression.

B-galactosidase (β-Gal) activity expressed from the lapA promoter-lacZ reporter constructs was measured in P. putida wild-type strain PSm and fis overexpression strain F15 grown in LB medium with or without 1 mM IPTG for 18 hours. Schemes of Fis binding sites (shown as grey boxes) are shown below the diagrams. Dotted lines denote mutated Fis binding sites and the lacZ reporter gene is shown as a black arrow. The scheme is not to scale. Vertical bars denote 95% confidence intervals of means. Data of at least 5 independent measurements is shown. Letters a–c depict homogeneity groups according to ANOVA post hoc Bonferroni test. Within subfigures, identical letters denote non-significant differences (P>0.05) between averages of β-galactosidase activity.

Fig 9. The effect of fis overexpression to the transcription of lapA promoter region in exponential phase P. putida.

Β-galactosidase (β-Gal) activity expressed from the lapA promoter-lacZ reporter constructs was measured in P. putida wild-type strain PSm and fis overexpression strain F15 grown in LB medium with or without 1 mM IPTG to the optical density of 0.5. Vertical bars denote 95% confidence intervals of means. Data of at least 10 independent measurements is shown. Letters a–b depict homogeneity groups according to ANOVA post hoc Bonferroni test. Identical letters denote non-significant differences (P>0.05) between averages of β-galactosidase activity.

Fig 10. The effect of mutated Fis binding sites Fis-A6 and Fis-A7 to P_lapA8 promoter.

B-galactosidase (β-Gal) activity expressed from the lapA promoter-lacZ reporter constructs p9_P_lapA8B, p9_P_lapA8B_F6mut, p9_P_lapA8B_F7mut and p9_P_lapA8B_F6,7mut were measured in P. putida wild-type strain PSm and fis overexpression strain F15 grown in LB medium with or without 1 mM IPTG for 18 hours. Schemes of Fis binding sites (shown as grey boxes) are shown below the diagrams. Dotted lines denote mutated Fis binding sites, lacZ reporter gene is shown as a black arrow and promoter P_lapA8 in a small white box. The scheme is not to scale. Vertical bars denote 95% confidence intervals of means. Data of at least 9 independent measurements is shown. Letters a–i depict homogeneity groups according to ANOVA post hoc Bonferroni test. Identical letters denote non-significant differences (P>0.05) between averages of β-galactosidase activity.

Fig 11. The effect of mutated Fis binding sites to individual promoters P_lapA6 and P_lapA7.

B-galactosidase (β-Gal) activity expressed from the lapA promoter-lacZ reporter constructs. (A) P_lapA6 promoter construct with or without mutated Fis-A4 binding site was cloned into medium-copy plasmid pBLKT and low-copy plasmid p9TT_BlacZ. p9_P_lapA6B, p9_P_lapA6B_F4mut, (B) pB_P_lapA6B, pB_P_lapA6B_F4mut, (C) p9_P_lapA7 and p9_P_lapA7_F5mut were measured in P. putida wild-type strain PSm and fis overexpression strain F15 grown in LB medium with or without 1 mM IPTG for 18 hours. Schemes of Fis binding sites (shown as grey boxes) are shown below the diagrams. Dotted lines denote mutated Fis binding sites and mutated promoter P_lapA6, lacZ reporter gene is shown as a black arrow and promoters P_lapA6 and P_lapA7 as white boxes. The scheme is not to scale. Vertical bars denote 95% confidence intervals of means. Data of at least 9 independent measurements is shown. Letters a–e depict homogeneity groups according to ANOVA post hoc Bonferroni test. Within subfigures, identical letters denote non-significant differences (P>0.05) between averages of β-galactosidase activity.