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Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH.

Leneveu-Jenvrin C, Bouffartigues E, Maillot O, Cornelis P, Feuilloley MG, Connil N, Chevalier S - Front Microbiol (2015)

Bottom Line: In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected.Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain.Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology Signals and Microenvironment, University of Rouen Evreux, France.

ABSTRACT
The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

No MeSH data available.


Related in: MedlinePlus

The tspo gene (Pfl01_2811) of Pseudomonas fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 that is expressed transiently in the bacterial growth course. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Gray bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. (B) Co-transcription of Pfl01_2810 and tspo by reverse transcription-PCR (RT-PCR) assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad®), primers sequences are given in Table 2. (C) Growth curves in microtitre wells in Luria-Bertani (LB) medium at 28°C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. (D) Growth curves in erlenmeyer in LB medium at 28°C and RL activity of pHK-TSPO in P. fluorescens Pf0-1.
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Figure 1: The tspo gene (Pfl01_2811) of Pseudomonas fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 that is expressed transiently in the bacterial growth course. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Gray bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. (B) Co-transcription of Pfl01_2810 and tspo by reverse transcription-PCR (RT-PCR) assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad®), primers sequences are given in Table 2. (C) Growth curves in microtitre wells in Luria-Bertani (LB) medium at 28°C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. (D) Growth curves in erlenmeyer in LB medium at 28°C and RL activity of pHK-TSPO in P. fluorescens Pf0-1.

Mentions: To monitor tspo transcription, tspo and Pfl01_2810-tspo promoter regions were fused to the promoter-less luxCDABE cassette in the replicative low-copy-number pAB133 vector (Gmr) (Bazire et al., 2005). The promoter regions of interest are located in Figures 1A and 5A, relatively to the tspo and Pfl01_2810 genes. The primer sequences, which were used for PCR amplifications, are shown in Table 2. The SacI-SpeI-digested PCR products were inserted into pAB133, yielding pTSPO, pHK-TSPO, pHK-TSPO-227, pHK-TSPO-157, and pHK-TSPO-88. The inserts were verified by DNA sequencing (Beckman Coulter Genomics).


Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH.

Leneveu-Jenvrin C, Bouffartigues E, Maillot O, Cornelis P, Feuilloley MG, Connil N, Chevalier S - Front Microbiol (2015)

The tspo gene (Pfl01_2811) of Pseudomonas fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 that is expressed transiently in the bacterial growth course. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Gray bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. (B) Co-transcription of Pfl01_2810 and tspo by reverse transcription-PCR (RT-PCR) assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad®), primers sequences are given in Table 2. (C) Growth curves in microtitre wells in Luria-Bertani (LB) medium at 28°C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. (D) Growth curves in erlenmeyer in LB medium at 28°C and RL activity of pHK-TSPO in P. fluorescens Pf0-1.
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Figure 1: The tspo gene (Pfl01_2811) of Pseudomonas fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 that is expressed transiently in the bacterial growth course. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Gray bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. (B) Co-transcription of Pfl01_2810 and tspo by reverse transcription-PCR (RT-PCR) assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad®), primers sequences are given in Table 2. (C) Growth curves in microtitre wells in Luria-Bertani (LB) medium at 28°C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. (D) Growth curves in erlenmeyer in LB medium at 28°C and RL activity of pHK-TSPO in P. fluorescens Pf0-1.
Mentions: To monitor tspo transcription, tspo and Pfl01_2810-tspo promoter regions were fused to the promoter-less luxCDABE cassette in the replicative low-copy-number pAB133 vector (Gmr) (Bazire et al., 2005). The promoter regions of interest are located in Figures 1A and 5A, relatively to the tspo and Pfl01_2810 genes. The primer sequences, which were used for PCR amplifications, are shown in Table 2. The SacI-SpeI-digested PCR products were inserted into pAB133, yielding pTSPO, pHK-TSPO, pHK-TSPO-227, pHK-TSPO-157, and pHK-TSPO-88. The inserts were verified by DNA sequencing (Beckman Coulter Genomics).

Bottom Line: In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected.Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain.Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology Signals and Microenvironment, University of Rouen Evreux, France.

ABSTRACT
The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

No MeSH data available.


Related in: MedlinePlus