POST-TRANSLATIONAL REGULATION OF THE rpoS AND pSrA GENES IN pSeudomonAS putidA WCS358: THE ROLE OF CLPXP PROTEASE

The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degradеd by ClpXP in the early exponential phase of growth. The оbtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp.


INTRODUCTION
Gram-negative bacteria have evolved sophisticated stress response mechanisms that allow them to adapt to a broad range of stressful conditions.The central regulator during the stationary phase in Pseudomonas spp., as in other Gram-negative bacteria, is the stationary phase RpoS alternative sigma factor (H e n g g e -A r o n i s , 2002; J o r g e n s e n et al., 1999; S u h et al., 1999).In Escherichia coli, RpoS regulates more than 100 genes involved in cell survival, cross-protection against various stresses, and virulence (I s h i h a m a , 2000).Similarly, in pseudomonads RpoS regulates a large set of genes, as recently demonstrated using microarray transcriptome analysis (S c h u s t e r et al., 2004).The expression of RpoS in E. coli is carefully controlled at the levels of transcription, translation, and posttranslation, and its synthesis considerably increases at the onset of the stationary phase.Regulation has been studied to a lesser extent in fluorescent pseudomonads, where -contrary to the case of E. coli -transcriptional regulation plays a major role (Ve n t u r i , 2003).It has been shown that the TetR family regulator designated PsrA plays a major role in positively regulating rpoS transcription at the entry of pseudomonads into the stationary phase (K o j i c and Ve n t u r i , 2001; K o j i c et al., 2002).
ClpXP is a bipartite protease responsible for the degradation of RpoS in E. coli (H e n g g e -A r o n i s , 2002).The ClpX component of ClpXP is a hexameric ring ATPase belonging to the Clp/Hsp100 subfamily of the AAA+ ATPases (S c h i r m e r et al., 1996).ClpX by itself has the capacity to recognize specific substrates and to denature and/or remodel the tertiary structures of these proteins in an ATP-dependent reaction (J o n e s et al., 1998).The ClpP component of ClpXP is a serine peptidase with broad sequence specificity.ClpP consists of two stacked heptameric rings, which enclose a central chamber containing the enzyme's 14 active sites.In E. coli, ClpP also associates with ClpA, an ATPase related to ClpX, to form the ClpAP protease (K a t a y a m a et al., 1988).
In this paper, the role of ClpXP protease in posttranslational regulation of the RpoS and PsrA proteins in Pseudomonas putida WCS358 is described for the first time.

Bacterial strains, media, and growth conditions
The bacterial strains used in this study were Pseudomonas putida WCS358 (G e e l s and S c h i p p e r s , 1983), P. putida WCS358 clpX::Km (B e r t a n i et al., 2003), P. putida WCS358 rpoS:: Km (K o j i c and Ve n t u r i , 2001) and P. putida WCS358 psrA::Km (K o j i c and Ve n t u r i , 2001).Bacteria were grown in lB medium (1% tripton, 0.5% NaCl, and 0.5% yeast extract) with addition of kanamycin (100 μg/ml) for P. putida WCS358 clpX::Km, P. putida WCS358 rpoS::Km and P. putida WCS358 psrA::Km.Agar plates were prepared by the addition of agar (1.5%, w/v) (Torlak, Belgrade, Serbia).Bacteria were grown at 30°C.

Preparation of cellular protein extracts
lB medium (200 ml) was inoculated with over night cultures (1% finally) of P. putida WCS358, P. putida WCS358 clpX::Km, P. putida WCS358 rpoS:: Km and P. putida WCS358 psrA::Km.Bacterial cells were obtained by centrifugation (5000 rpm, 15 min, 4°C) in a microfuge from eppendorf (Hamburg, Germany) at optical density (OD) of 0.7, 1.5, and 2.5 at 600 nm for all four strains.Pellets were resuspended in 1 ml of the reaction buffer [40 mM HePeS-KOH (pH 7.5), 5 mM MgCl 2 , 60 mM KCl, 0.5 mM DTT, 0.4 mM eDTA (pH 8), and 3.4% glycerol in water], transferred to microtubes, and kept on ice.Cell suspensions were sonicated on ice five times for 10 sec at 10 mA with a 30-sec pause between each sonication.After sonication, cell suspensions were centrifuged for 1 h at 13 000 rpm and 4°C in the above-indicated microfuge.The resulting supernatant (500 μl) were transferred to Ti50 ultracentrifuge vials and centrifuged in a model l7-55 ultracentrifuge (Ti50 rotor) from Beckman Coulter (Fullerton, USA) for 2.5 h at 25 000 rpm and 4°C.Finally, 300 μl of supernatant was transferred to clean cold microtubes and mixed with the sample-loading buffer [125 mM Tris-HCl (pH 6.8) 10 mM eDTA (pH 8) 4% SDS, 25% glycerol, 5% 2-mercaptoethanol and 0.07% bromphenolblue] in a 1:1 volume ratio.Prior to loading, samples were heated at 100°C for 5 min and analyzed by SDS-PAGe on 15% gels.Cellular protein extracts from rpoS and psrA mutants of Pseudomonas putida WCS358 were used as a negative control for analysis of rpoS and psrA expression in further experiments.

Western blot analysis
Transfer of proteins from SDS-PAGe gels to a PVDF membrane (Millipore, Bedford, USA) was done with a semi-dry fast blot (Fastblot B43, Biometra, Goettingen, Germany) according to the manufacturer's protocol.PVDF membranes were air-dried after protein transfer, then incubated for 1 h in PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 , pH 7.4, in 1 l H 2 O) containing 0.1% Tween 20 and 5% milk powder.After saturation, membranes were incubated in PBS-0.1% Tween 20 with rabbit rpoS and psrA polyclonal antibodies in a volume ratio of 1:2000 for 1 h.The membranes were washed in PBS-0.1% Tween 20 and then incubated in PBS-0.1% Tween 20 with rabbit immunoglobulins conjugated with HRP (DAKO A/S, Glostrup, Denmark) in a volume ratio of 1:4000 for 1 h, after which they were washed with PBS-0.1% Tween 20.The signal was detected with SIGMA FAST TM 3,3'diaminobenzidine tablets (Sigma Aldrich Chemie, Germany).

ReSUlTS AND DISCUSSION
In order to determine the role of ClpXP protease in post-translational regulation of the rpoS and psrA genes of Pseudomonas putida WCS358 (wild type), cellular protein extracts were isolated from this strain and its clpX, rpoS, and psrA mutants.Cellular levels of RpoS and PsrA in protein extracts of the early exponential, late exponential, and stationary phases of bacterial culture growth were then investigated using Western blot analysis with RpoS and PsrA polyclonal antibodies.
Western blot analysis of wild type strain and clpX mutant strain protein extracts revealed that in the early exponential phase of growth (culture OD 600 nm =0.7) both RpoS and PsrA protein levels were significantly higher in the clpX mutant strain than in the wild type (for RpoS, see Fig. 1, lanes 1 and 2; for PsrA, see Fig. 2, lanes 1 and 2).This result supports our presumption that ClpXP protease degrades RpoS and PsrA when these proteins are not needed for cell survival in non-stress conditions (such as the exponential phase of growth).Thus, regulation of rpoS and psrA gene expression at the level of protein stability in Pseudomonas is preventive.The response to stress renders cells broadly stress-resistant in such a way that damage is avoided rather than needing to be repaired.Surprisingly, in the late exponential phase of growth (culture OD 600 nm =1.5), the presence of RpoS and PsrA was shown in wild type but not in clpX mutant strain protein extracts (for RpoS, see Fig. 1, lanes 4 and 5; for PsrA, see Fig. 2, lanes 4 and 5).The experiments were performed in triplicate with the same outcome, confirming almost complete degradation of RpoS and PsrA in the late exponential phase of growth.The absence of detectable amounts of these proteins in late exponential phase of growth of P. putida WCS358 clpX::Km could be explained in the light of the associating potential of ClpP in E. coli.It is known that in addition to ClpX, ClpP interacts with other ATP-dependent molecular chaperones of ClpA in E. coli (K a t a y a m a et al., 1988).ClpA serves as a substrate-specifying adapter for ClpP peptidase in the ClpAP protease complex.Taking into account this possibility, we analyzed the sequenced genome of Pseudomonas putida KT2440 in silico looking for the presence of a gene coding for a ClpA-like protein and found a gene that codes for a protein showing 65% amino acid identity with the E. coli ClpA protein.The ClpA levels in E. coli increase during the late exponential and early stationary phases resulting in an increase of ClpAP activity (K a t a y a m a et al., 1990).Knowing that P. putida harbors ClpA ATPase, we could speculate that in the late exponential phase of growth, in the absence of ClpX, ClpP forms a complex with ClpA and degrades RpoS and PsrA.This degradation results in the absence of RpoS and PsrA in P. putida WCS358 clpX::Km during the late exponential phase.When cells enter the stationary phase of growth, PsrA activates transcription of the rpoS gene and the RpoS synthesized enables the cell to cope with starvation stress.Thus, equal levels of RpoS and PsrA were expected in both P. putida WCS358 and P. putida WCS358 clpX::Km in the stationary phase of growth (culture OD 600 nm =2.5) (for RpoS, see Fig. 1, lanes 7 and 8; for PsrA, see Fig. 2, lanes 7 and 8) and corresponded with previous reports for the RpoS protein in E. coli (H e n g g e -A r o n i s , 2002).
In summary, we found that rpoS and psrA expression is regulated at the post-translational level by ClpXP protease during the early exponential phase of growth in Pseudomonas putida WCS358.In view of the fact that RpoS and PsrA proteins are very important global transcriptional regulators involved in the response to the stationary phase, production of extracellular molecules, and quorum sensing (K o j i c and Ve n t u r i , 2001; S c h u s t e r Fig. 1.Western blot analysis of protein extracts from P. putida WCS358, P. putida WCS358 clpX::Km, and P. putida WCS358 rpoS::Km with antibodies against RpoS.Lanes 1, 4, and 7: RpoS levels in protein extract of P. putida WCS358 grown to OD 600 nm = 0.7, 1.5, and 2.5, respectively; Lanes 2, 5, and 8: RpoS levels in protein extracts of P. putida WCS358 clpX::Km grown to OD 600 nm = 0.7, 1.5, and 2.5, respectively; Lanes 3, 6, and 9: RpoS levels in protein extracts of P. putida WCS358 rpoS::Km grown to OD 600 nm = 0.7, 1.5, and 2.5, respectively -negative control.

rpoS AND pSrA GENES IN pSeudomonAS putidA WCS358: THE ROLE OF CLPXP PROTEASE
Abstract -The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads.This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358.Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degradеd by ClpXP in the early exponential phase of growth.The оbtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp.