Expression of the translocator protein (TSPO) from Pseudomonas
fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and
RpoH.
Charlène Leneveu-Jenvrin, Emeline Bouffartigues, Olivier Maillot, Marc G. J. Feuilloley, Nicole Orange, Pierre
Cornelis, Nathalie Connil and Sylvie Chevalier
Laboratory of Microbiology Signals and Microenvironment, LMSM EA4312, University of Rouen, France
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 membrane
biogenesis, signaling and stress response. A tspo homologue 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.
(B) Co-transcription of Pfl01_2810 and tspo by 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®),
(C) Growth curves in microtitre wells in LB medium at 28 C and relative luminescence (RL) activity of
pTSPO and pHK-TSPO in P. fluorescens Pf0-1.
The tspo gene (Pfl01_2811) of P. fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 and is expressed transiently during the bacterial growth.
(A) Schematic representation of the genomic environment of tspo
and of transcriptional fusions pTSPO and pHK-TSPO. Grey 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.
Pfl01_ 2809 lux CDABE pHK-TSPO -253 bp lux CDABE pTSPO -227 bp P. fluorescens Pf0-1 chromosome
lux transcriptional fusions
Pfl01_2810 tspo A HKF HKRTSPOFTSPOR HKTSPOF HKTSPOR primers B 0,01 0,1 1 10 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 1012141618202224 O D60 0 R LU x 1 0 6/0 .5 se c x O D60 0 C 10 1 0.1 0.01 pHK-TSPO pTSPO
Growth RL activity Vector
Conclusions
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. 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, we detect a putative binding site for the
heat shock response RpoH sigma factor. 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.
Expression of tspo is increased in response to hyperosmolarity at least partly via the extracytoplasmic function sigma factor AlgU activity.
Growth curves (A, C) and transcriptional activity (B, D) of P. fluorescens Pf0-1 in microtiter wells in LB medium supplemented or not with 171mM or 342 mM NaCl (A, B) or with 342 mM or 684 mM sucrose (C, D).
(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 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.
A putative RpoH-consensus binding site is located in Pfl01_2810-tspo promoter region: effect of temperature on growth and TSPO expression.
(A) Sequence alignment of the putative RpoH
binding sites identified by MEME in the regions located upstream of dnaK in E. coli and groEL, dnaK, grpE and Pfl01_2810 in P.
fluorescens Pf0-1, relatively to the E. coli
RpoH consensus sequence. Localization of this sequence is given relatively to the +1 translational start site.
(B) Growth and transcriptional activity of P.
fluorescens Pf0-1 containing pHK-TSPO
reporter fusion in microtiter plates cultured at 28 C or 32 C.
(A) Schematic representation of the 4 transcriptional
fusions. RpoH binding sequence (black star).
(B, C) Growth in microtiter plates at 28 C (B) or 32 C
(C), and transcriptional activity of P. fluorescens Pf0-1 containing pHK-TSPO-88, pHK-TSPO-157 and pHK-TSPO-227 plasmids.
Growth in microtiter plates at 37 C and transcriptional activity of P. aeruginosa PAO1 and rpoH mutant containing pHK-TSPO-157. 0,01 1 0 2 4 6 Time (h) NaCl (mM) 0,01 0,1 1 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) Sucrose (mM) O D60 0 A C Growth Medium LB LB + 171 mM NaCl or 342 mM Sucrose LB + 342 mM NaCl or 684 mM Sucrose 0 15 20 25 30 0 2 4 6 8 10 12 14 16 18 20 22 24 R LU x 1 0 6/0 .5 se c x O D600 Time (h) 0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) B D
pHK-TSPO RL activityMedium
LB LB + 171 mM NaCl or 342 mM Sucrose LB + 342 mM NaCl or 684 mM Sucrose 0,01 0,1 1 10 024 68 10 12 14 16 18 20 22 24 R LU x 1 0 6/0 .5 se c x O D600 Time (h) A 10 1 0.1 0.01 O D600
GrowthpHK-TSPO RL activityMedium
LB LB + D-cycloserine P. fluorescens Pf0-1 Strain 0,01 0,1 1 10 0 1 2 3 4 5 6 7 8 9 10 024 68 10 12 14 16 18 20 22 24 O D600 Time (h) B 10 1 0.1 0.01 R LU x 1 0 6/0 .5 se c x O D600
GrowthpHK-TSPO RL activity
P. aeruginosa PAO1 P. aeruginosa algU mutant Strain
Pfl01_ 2809
pHK-TSPO lux CDABE
P. fluorescens Pf0-1 chromosome
lux transcriptional fusions
Pfl01_2810 tspo lux CDABE lux CDABE lux CDABE pHK-TSPO-88 pHK-TSPO-157 pHK-TSPO-227 A
Designation Sequence Position/+1 translational site E. coli dnaK TGAA N14 CCCTA 12 Pfl01_2810 TTGAA N12 CCGAT 95 P. fluorescens groEL TGAT N18 CCGCT 27 P. fluorescens dnaK TTGAA N15 CCCAT 81 P. fluorescens grpE TTGAA N15 CCCAT 55
Consensus TTGAA N12/18 CCCAT
A B 0,01 0,1 1 10 0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 1012141618202224 O D60 0 R LU x 1 0 6/0 .5 se c x O D60 0 Time (h) 10 1 0.1 0.0 1
Growth pHK-TSPO RL activity Temperature
28 C 32 C 0,01 0,1 1 0 10 20 30 40 0 24 68 10 12 14 16 18 20 22 24 O D600 Time (h) 0,01 0,1 1 0 2 4 6 8 10 12 0 2 46 810 12 14 16 18 20 22 24 R LU x 1 0 6/0 .5 se c x O D600 Time (h) B C O D60 0 R LU x 1 0 6/0 .5 se c x O D60 0 pHK-TSPO-88 pHK-TSPO-157
Growth RL activity Vector
pHK-TSPO-227 O D60 0 0,1 1 0 2 4 6 810 12 14 16 18 20 22 24 0 1 2 3 4 5
GrowthpHK-TSPO RL activity
P. aeruginosa PAO1 P. aeruginosa rpoH mutant Strain
