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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

AlgU is related to the operonic structure transcription. (A) The growth of P. fluorescens Pf0-1 containing pHK-TSPO and the relative bioluminescence levels generated by pHK-TSPO are shown when bacteria were grown in microtiter wells in LB with or without D-cycloserine. (B) Growth and relative bioluminescence levels of P. aeruginosa PAO1 and its isogenic algU mutant containing pHK-TSPO in LB in microtiter wells.
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Figure 3: AlgU is related to the operonic structure transcription. (A) The growth of P. fluorescens Pf0-1 containing pHK-TSPO and the relative bioluminescence levels generated by pHK-TSPO are shown when bacteria were grown in microtiter wells in LB with or without D-cycloserine. (B) Growth and relative bioluminescence levels of P. aeruginosa PAO1 and its isogenic algU mutant containing pHK-TSPO in LB in microtiter wells.

Mentions: In members of the Pseudomonas genus, the response to hyperosmolarity has been shown to be triggered by the master extracytoplasmic function (ECF) sigma factor AlgU (Schnider-Keel et al., 2001). In P. aeruginosa mucoid strains, AlgU has been extensively studied due to its involvement in the mucoid conversion, leading to increasing the severity of cystic fibrosis symptoms (Schurr et al., 1996). In non-mucoid strains, AlgU is the major cell wall stress response regulator (Wood et al., 2006). AlgU has been shown to be activated in response to sub-lethal concentrations of D-cycloserine antibiotic leading to peptidoglycan alterations in P. aeruginosa (Wood et al., 2006). In an attempt to give further insights into Pfl01_2810 – tspo expression, 50 μg.ml-1 of D-cycloserine was added to P. fluorescens Pf0-1 growth medium. At this sub-lethal concentration, the presence of the antibiotic slightly reduced the growth parameters of P. fluorescens, without affecting the final biomass (Figure 3A). The presence of D-cycloserine strongly increased the activity of pHK-TSPO during the exponential growth phase. However, the promoter region activity decreased rapidly, without showing an increase during the stationary growth phase, as it was observed after a hyperosmolar treatment. Taken together, these data suggest that the first peak could be linked to the activity of AlgU in our conditions, and that the increase observed during the stationary growth phase under hyperosmolar treatment could be AlgU-unrelated. To get further insight into the role of AlgU in the Pfl01_2810 – tspo transcription, the pHK-TSPO reporter fusion and the pAB133 empty vector were transferred into P. aeruginosa PAO1 and in its algU isogenic mutant strain (Bazire et al., 2010). As in the case of P. fluorescens Pf0-1, the promoter fusion activity increased during the early exponential growth phase of wild-type P. aeruginosa, but was clearly reduced by more than 2 fold in the algU mutant strain (Figure 3B). However, no further increase of the fusion activity was observed during the stationary phase in P. aeruginosa wild type and algU mutant strains, suggesting that this effect might be specific to P. fluorescens Pf0-1 and/or to the microtiter growth conditions, as discussed above. Taken together, these data suggest an involvement of AlgU in the operonic structure transcription.


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)

AlgU is related to the operonic structure transcription. (A) The growth of P. fluorescens Pf0-1 containing pHK-TSPO and the relative bioluminescence levels generated by pHK-TSPO are shown when bacteria were grown in microtiter wells in LB with or without D-cycloserine. (B) Growth and relative bioluminescence levels of P. aeruginosa PAO1 and its isogenic algU mutant containing pHK-TSPO in LB in microtiter wells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4585239&req=5

Figure 3: AlgU is related to the operonic structure transcription. (A) The growth of P. fluorescens Pf0-1 containing pHK-TSPO and the relative bioluminescence levels generated by pHK-TSPO are shown when bacteria were grown in microtiter wells in LB with or without D-cycloserine. (B) Growth and relative bioluminescence levels of P. aeruginosa PAO1 and its isogenic algU mutant containing pHK-TSPO in LB in microtiter wells.
Mentions: In members of the Pseudomonas genus, the response to hyperosmolarity has been shown to be triggered by the master extracytoplasmic function (ECF) sigma factor AlgU (Schnider-Keel et al., 2001). In P. aeruginosa mucoid strains, AlgU has been extensively studied due to its involvement in the mucoid conversion, leading to increasing the severity of cystic fibrosis symptoms (Schurr et al., 1996). In non-mucoid strains, AlgU is the major cell wall stress response regulator (Wood et al., 2006). AlgU has been shown to be activated in response to sub-lethal concentrations of D-cycloserine antibiotic leading to peptidoglycan alterations in P. aeruginosa (Wood et al., 2006). In an attempt to give further insights into Pfl01_2810 – tspo expression, 50 μg.ml-1 of D-cycloserine was added to P. fluorescens Pf0-1 growth medium. At this sub-lethal concentration, the presence of the antibiotic slightly reduced the growth parameters of P. fluorescens, without affecting the final biomass (Figure 3A). The presence of D-cycloserine strongly increased the activity of pHK-TSPO during the exponential growth phase. However, the promoter region activity decreased rapidly, without showing an increase during the stationary growth phase, as it was observed after a hyperosmolar treatment. Taken together, these data suggest that the first peak could be linked to the activity of AlgU in our conditions, and that the increase observed during the stationary growth phase under hyperosmolar treatment could be AlgU-unrelated. To get further insight into the role of AlgU in the Pfl01_2810 – tspo transcription, the pHK-TSPO reporter fusion and the pAB133 empty vector were transferred into P. aeruginosa PAO1 and in its algU isogenic mutant strain (Bazire et al., 2010). As in the case of P. fluorescens Pf0-1, the promoter fusion activity increased during the early exponential growth phase of wild-type P. aeruginosa, but was clearly reduced by more than 2 fold in the algU mutant strain (Figure 3B). However, no further increase of the fusion activity was observed during the stationary phase in P. aeruginosa wild type and algU mutant strains, suggesting that this effect might be specific to P. fluorescens Pf0-1 and/or to the microtiter growth conditions, as discussed above. Taken together, these data suggest an involvement of AlgU in the operonic structure transcription.

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