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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 activity of the promoter region is lowered in P. aeruginosa rpoH mutant strain. Growth in microtiter plates at 37°C and transcriptional activity of P. aeruginosa PAO1 and rpoH mutant containing pHK-TSPO-157.
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Figure 6: The activity of the promoter region is lowered in P. aeruginosa rpoH mutant strain. Growth in microtiter plates at 37°C and transcriptional activity of P. aeruginosa PAO1 and rpoH mutant containing pHK-TSPO-157.

Mentions: A bioinformatic analysis of the 227 bp region lying upstream of Pfl01_2810-tspo using the MEME software (Brown et al., 2013), failed to identify an algU consensus binding site (data not shown), suggesting that the effect of AlgU on the expression of this promoter region may be indirect. However, a sequence that showed 90% of identity with the previously proposed binding consensus sequence for the E. coli RpoH sigma factor (σ32) was found 95 bp upstream the putative +1 translational start site of Pfl01_2810 (Cowing et al., 1985; Figure 4A). This consensus sequence was also detected upstream P. fluorescens Pf0-1 dnaK, groEL, or grpE genes (Figure 4A) encoding established direct RpoH targets in P. aeruginosa (Potvin et al., 2008) and/or in E. coli (Arsene et al., 2000). In these latter bacterial species, RpoH is responsible for the heat-shock response during the upshift of temperature from 30 to 42°C in the early exponential growth phase (Arsene et al., 2000). P. fluorescens Pf0-1 is a psychrotrophic strain that is able to grow at temperatures up to 32°C (Silby et al., 2009; Loper et al., 2012). We therefore tested the effect of growth at 32°C on the expression of the operonic structure Pfl01_2810-tspo. The promoter activity of the operonic structure was about fivefold increased during the exponential growth phase when bacteria were grown at 32°C compared to 28°C (Figure 4B). By contrast, no temperature-related modification could be observed during the stationary growth phase (Figure 4B). Pfl01_2810 mRNA expression level was further measured by qRT-PCR experiments on RNAs extracted from bacteria grown at 28 or 32°C. As shown on Figure 4C, an increase of the growth temperature resulted in significantly higher levels of expression (about 1.8- and 1.4-fold Pfl01_2810 expression after 1 and 2 h of growth, respectively). Taken together, our data show that the activity of the promoter region laying upstream Pfl01_2810 – tspo is increased in response to an elevation of the growth temperature. The identification of an RpoH consensus binding site upstream of Pfl01_2810 is therefore in line with the increased activity of this promoter region at elevated growth temperature. To get further insights into the involvement of RpoH in the temperature-dependent increase of the promoter activity, the transcriptional fusions pHK-TSPO-227, pHK-TSPO-157, pHK-TSPO-88 were constructed, in which three fragments of the studied promoter region were fused to the luxCDABE reporter system (Figure 5A). Among these three fusions, only pHK-TSPO-157 contained the RpoH putative binding site (Figure 5A, black star). The promoter activity of each construct was then assayed when bacteria were grown at 28° (Figure 5B) and 32°C (Figure 5C). The fusion containing the RpoH-dependent promoter was active at both growth temperatures (Figures 5B,C, full triangles), at levels similar to the ones obtained with the large promoter region fusion construct pHK-TSPO (compare with Figure 4B), suggesting that the main promoter activity of Pfl01_2810-tspo lies in this region. By contrast, no activity was detected at each temperature for the two other constructs during the exponential growth phase. To get further insight into the role of RpoH in Pfl01_2810 – tspo transcription, the pHK-TSPO-157 reporter fusion and the pAB133 empty vector were transferred into P. aeruginosa PAO1 and in its rpoH isogenic mutant strain (Jacobs et al., 2003). P. aeruginosa wild type and rpoH mutant growth at 37°C was similar. As in the case of P. fluorescens Pf0-1, the promoter region activity increased during the growth phase of wild-type P. aeruginosa and then decreased. As shown on Figure 6, the activity of this promoter region was reduced by about 25% in P. aeruginosa rpoH mutant strain, suggesting that RpoH may be at least partly involved in this phenotype.


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 activity of the promoter region is lowered in P. aeruginosa rpoH mutant strain. Growth in microtiter plates at 37°C and transcriptional activity of P. aeruginosa PAO1 and rpoH mutant containing pHK-TSPO-157.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: The activity of the promoter region is lowered in P. aeruginosa rpoH mutant strain. Growth in microtiter plates at 37°C and transcriptional activity of P. aeruginosa PAO1 and rpoH mutant containing pHK-TSPO-157.
Mentions: A bioinformatic analysis of the 227 bp region lying upstream of Pfl01_2810-tspo using the MEME software (Brown et al., 2013), failed to identify an algU consensus binding site (data not shown), suggesting that the effect of AlgU on the expression of this promoter region may be indirect. However, a sequence that showed 90% of identity with the previously proposed binding consensus sequence for the E. coli RpoH sigma factor (σ32) was found 95 bp upstream the putative +1 translational start site of Pfl01_2810 (Cowing et al., 1985; Figure 4A). This consensus sequence was also detected upstream P. fluorescens Pf0-1 dnaK, groEL, or grpE genes (Figure 4A) encoding established direct RpoH targets in P. aeruginosa (Potvin et al., 2008) and/or in E. coli (Arsene et al., 2000). In these latter bacterial species, RpoH is responsible for the heat-shock response during the upshift of temperature from 30 to 42°C in the early exponential growth phase (Arsene et al., 2000). P. fluorescens Pf0-1 is a psychrotrophic strain that is able to grow at temperatures up to 32°C (Silby et al., 2009; Loper et al., 2012). We therefore tested the effect of growth at 32°C on the expression of the operonic structure Pfl01_2810-tspo. The promoter activity of the operonic structure was about fivefold increased during the exponential growth phase when bacteria were grown at 32°C compared to 28°C (Figure 4B). By contrast, no temperature-related modification could be observed during the stationary growth phase (Figure 4B). Pfl01_2810 mRNA expression level was further measured by qRT-PCR experiments on RNAs extracted from bacteria grown at 28 or 32°C. As shown on Figure 4C, an increase of the growth temperature resulted in significantly higher levels of expression (about 1.8- and 1.4-fold Pfl01_2810 expression after 1 and 2 h of growth, respectively). Taken together, our data show that the activity of the promoter region laying upstream Pfl01_2810 – tspo is increased in response to an elevation of the growth temperature. The identification of an RpoH consensus binding site upstream of Pfl01_2810 is therefore in line with the increased activity of this promoter region at elevated growth temperature. To get further insights into the involvement of RpoH in the temperature-dependent increase of the promoter activity, the transcriptional fusions pHK-TSPO-227, pHK-TSPO-157, pHK-TSPO-88 were constructed, in which three fragments of the studied promoter region were fused to the luxCDABE reporter system (Figure 5A). Among these three fusions, only pHK-TSPO-157 contained the RpoH putative binding site (Figure 5A, black star). The promoter activity of each construct was then assayed when bacteria were grown at 28° (Figure 5B) and 32°C (Figure 5C). The fusion containing the RpoH-dependent promoter was active at both growth temperatures (Figures 5B,C, full triangles), at levels similar to the ones obtained with the large promoter region fusion construct pHK-TSPO (compare with Figure 4B), suggesting that the main promoter activity of Pfl01_2810-tspo lies in this region. By contrast, no activity was detected at each temperature for the two other constructs during the exponential growth phase. To get further insight into the role of RpoH in Pfl01_2810 – tspo transcription, the pHK-TSPO-157 reporter fusion and the pAB133 empty vector were transferred into P. aeruginosa PAO1 and in its rpoH isogenic mutant strain (Jacobs et al., 2003). P. aeruginosa wild type and rpoH mutant growth at 37°C was similar. As in the case of P. fluorescens Pf0-1, the promoter region activity increased during the growth phase of wild-type P. aeruginosa and then decreased. As shown on Figure 6, the activity of this promoter region was reduced by about 25% in P. aeruginosa rpoH mutant strain, suggesting that RpoH may be at least partly involved in this phenotype.

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