Topological and deletion analysis of CorS, a Pseudomonas syringae sensor kinase

Angela V. Smirnova and Matthias S. Ullrich

International University Bremen, School of Engineering and Sciences, Research II, Campus Ring 1, 28759 Bremen, Germany

Correspondence
Matthias S. Ullrich
m.ullrich{at}iu-bremen.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180. COR is produced at the virulence-promoting temperature of 18 °C, but not at 28 °C, the optimal growth temperature of PG4180. Assuming that the highly hydrophobic N-terminus of CorS might be involved in temperature-signal perception, the membrane topology of CorS was determined using translational phoA and lacZ fusions, leading to a topological model for CorS with six transmembrane domains (TMDs). Interestingly, three PhoA fusions located downstream of the sixth TMD showed a thermoresponsive phenotype. Enzymic activity, immunoblot, and protease-sensitivity assays were performed to localize the CorS derivatives, to analyse the expression level of hybrid proteins and to examine the model. In-frame deletions of the last four, or all six TMDs gave rise to non-functional CorS. The results indicated that the transmembrane region is important for CorS to function as a temperature sensor, and that the membrane topology of CorS might be involved in signal perception.


Abbreviations: COR, coronatine; HPK, histidine protein kinase; TMD, transmembrane spanning domain


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plant pathologists have long been aware that the development of bacterial disease symptoms is most severe during periods of cold and humid weather. Thus, low temperature might favour virulence-gene expression in some plant-pathogenic bacteria. Although numerous examples of the thermoregulation of virulence in plant-pathogenic bacteria have been described (Fullner and Nester, 1996; Hugouvieux-Cotte-Pattat et al., 1992; Palmer & Bender, 1993; Rowley et al., 1993; Van Dijk et al., 1999), little is known about the detailed molecular mechanisms of temperature-sensing and temperature-signal transduction. This is especially intriguing, since thermoregulation of gene expression in some plant pathogens is the reverse of the observed types of thermoregulation in human and animal pathogens, where high temperature usually fosters virulence (Eriksson et al., 2002).

The thermoresponsive production of the phytotoxin coronatine (COR) has been investigated in detail in the plant pathogen Pseudomonas syringae pv. glycinea PG4180, which causes bacterial blight of soybeans (Budde et al., 1998; Palmer & Bender, 1993; Ullrich et al., 1995). Structurally, COR resembles a polyketide, and consists of two distinct moieties, coronafacic acid (CFA) and coronamic acid (CMA), which function as intermediates in the biosynthetic pathway to COR and are fused together by an amide bond (Mitchell et al., 1994; Parry et al., 1994). PG4180 produces COR predominantly at 18 °C, whereas there is no detectable COR production at 28 °C. Biosynthesis of COR in PG4180 is regulated at the transcriptional level by a modified two-component regulatory system composed of CorS, CorR and CorP (Ullrich et al., 1995), and by RpoN ({sigma}54), an alternative sigma factor (Alarcón-Chaidez et al., 2003).

A classical two-component system consists of a histidine protein kinase (HPK) and a response regulator (RR), both of which are characterized by receiver and transmitter domains. The first reaction in the signalling cascade is autophosphorylation of a highly conserved histidine residue of the HPK transmitter module. This reaction is under the control of the HPK sensory or receiver domain, which responds to environmental signals. The phosphate group is subsequently transferred from the HPK to an aspartyl residue of the conserved N-terminal receiver module of the RR. This induces activation of the transmitter domain, which often contains a conserved helix–turn–helix (H-T-H) DNA-binding motif. The transmitter domain subsequently binds specifically to target DNA regions in order to activate signal-dependent gene expression (Hoch, 2000).

Interestingly, the CorRSP system consists of two response regulators, CorR and CorP, and an HPK, CorS. CorR is a classical response regulator of the FixJ family of regulatory proteins (Grebe & Stock, 1999), and has conserved receiver and transmitter domains. The transmitter domain comprises a typical H-T-H DNA-binding motif. However, CorP lacks the H-T-H DNA-binding motif.

Moreover, it has been shown that CorR, but not CorP, is able to bind specifically to DNA upstream of COR biosynthetic promoters (Peñaloza-Vázquez & Bender, 1998; Wang et al., 1999). Evidence for the DNA binding of CorR was achieved through protein overproduction in PG4180 at 18 °C. The overproduction of CorR in PG4180 at 28 °C and in the corS-mutant background resulted in an inactive protein in DNA-binding assays (Wang et al., 1999). This fact highlighted the importance of the functional HPK CorS, which initially might be autophosphorylated at 18 °C and then activates CorR by phosphorylation. Rangaswamy & Bender (2000) demonstrated in vitro the phosphorylation of both CorR and an N-terminally truncated form of CorS, thereby confirming biochemically the function of these proteins in phosphotransfer.

The C-terminal transmitter domain of CorS (residues 202–550) forms the kinase core. The kinase core includes a dimerization and histidine phosphotransfer domain (DHp, residues 244–309) and a conserved catalytic and ATP-binding domain (CA, residues 353–464). Within the DHp domain of CorS, the H-box with the invariant histidine residue (His-254), a presumed site of phosphorylation, is well defined. The CA domain of CorS contains four conserved motifs, the N, D, F and G boxes, which are characteristic of HPKs and are involved in ATP-binding, catalysis and phosphotransfer (Bilwes et al., 1999). The N-terminal part from residues 1 to 201 is highly hydrophobic, and is presumably embedded in the bacterial membrane. The hydrophobic N-terminus may function as the sensor domain of CorS in signal perception, given that the fatty-acid composition of the membrane changes with temperature in order to maintain membrane fluidity. Therefore, a precise determination of the structure of the CorS N-terminal region is crucial to an understanding of signal perception.

Initially, in-frame deletions of the predicted four and six transmembrane spanning domains (TMDs) were generated and analysed to determine the importance of TMDs for the function of CorS in signal perception. Based on computer analysis, CorS might possess six or even seven TMDs. To elucidate the structure of the N-terminal part of CorS, we used the method of translational fusion with alkaline phosphatase and {beta}-galactosidase (Manoil & Beckwith, 1986; Manoil et al., 1988).


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
Rabbit polyclonal anti-PhoA antibodies were kindly donated by R. Schuelein (Pharmacological Institute, Free University Berlin, Germany). Anti-rabbit Ig–alkaline phosphatase conjugates were purchased from Sigma.

Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli cells were grown in Luria broth (LB) at 37 °C. P. syringae cells were maintained for 2–5 days at 28 °C on mannitol-glutamate (MG) agar plates (Keane et al., 1970). Subsequently, P. syringae cells were grown at 18 °C and 28 °C shaken at 280 r.p.m. in Hoitink–Sinden minimal medium optimized for COR production (HSC) (Palmer & Bender, 1993). Antibiotics were used at the following concentrations: ampicillin, 100 µg ml–1; chloramphenicol, 25 µg ml–1; tetracycline 25 µg ml–1; streptomycin, 25 µg ml–1; kanamycin, 25 µg ml–1. {beta}-Glucuronidase, alkaline phosphatase and {beta}-galactosidase activities were detected by the blue colour formation of colonies on agar plates containing 20 µg ml–1 5-bromo-4-chloro-3-indolyl-{beta}-glucuronic acid (X-Gluc), 160 µg ml–1 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine salt (X-Phos), and 100 µg ml–1 5-bromo-4-chloro-3-indolyl-{beta}-galactoside (X-Gal), respectively. The plates were incubated for 4–6 days at 18 °C and 28 °C.


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Table 1. Bacterial strains and plasmids used in this study

Abbreviations: Apr, ampicillin resistant; Kmr, kanamycin resistant; Smr, streptomycin resistant; Spr, spectinomycin resistant; Tcr, tetracycline resistant; mob, mobilization function; tra, transfer function.

 
Molecular biology techniques.
Standard methods were performed, if not otherwise indicated, according to Sambrook & Russell (2001). Plasmid DNA was isolated by alkaline lysis and purified using QIAGEN-tips (Qiagen). DNA was treated with restriction enzymes and other nucleic-acid-modifying enzymes (Klenow fragment, alkaline phosphatase, T4 DNA polymerase and T4 DNA ligase) according to the manufacturer's instructions. DNA fragments were analysed on agarose gels, and different restriction fragments were purified using the QIAEX II gel extraction kit (Qiagen). PCR fragments were amplified using Taq DNA polymerase (Qiagen) and the proof-reading DNA polymerase PfuTurbo (Stratagene), or a mix of Pwo/Taq (Roche). PCR products were purified with the QIAquick PCR purification kit (Qiagen). Plasmid DNA was mobilized into P. syringae strains using the tri-parental mating technique described by Bender et al. (1991). DNA sequencing and all primer syntheses were performed by MWG Biotech (Ebersberg, Germany). The oligonucleotide primers used for PCR are listed in Table 2.


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Table 2. Oligonucleotide primers used in this study

Enzyme recognition sites are underlined. S, Sense strand; AS, antisense strand.

 
Computer analysis.
The sequence of CorS was subjected to hydropathy profile analysis based on the Kyte–Doolittle method (Kyte & Doolittle, 1982), using the program PROTEAN (DNA-STAR Software). To analyse and predict the membrane topology of CorS, TopPred II (Claros & von Heijne, 1994), TMHMM (Sonnhammer et al., 1998), HMMTOP (Tusnády & Simon, 1998), DAS (Cserzo et al., 1997), MEMSAT (Jones et al., 1994), SOSUI (Hirokawa et al., 1998), and Vector NTI (Informax Inc., USA) were used. Domain organization of CorS was analysed by the Pfam (Bateman et al., 2002) and SMART (Schultz et al., 2000) programs.

Construction of in-frame deletions.
Primers corSoutF, inRSer22, corSoutR, corSinF and corSinR (Table 2) were used to generate deletion PCR fragments. Plasmid pH34 was used as the template for PCR. For the deletion of all six TMDs, the first 1·5 kb PCR product (I) started at the EcoRI site of plasmid pH34, at which the primer generated a SacII site, and terminated at the codon for Ser-22, at which the primer generated an EcoRI site (Fig. 1A). For the deletion of the last four TMDs, the second 1·7 kb PCR product (II) had the same 5' start as PCR product I, but terminated at the codon for Thr-103, at which the primer generated an EcoRI site. The third 1·3 kb PCR product (III) was used for both deletion constructs. It started at the codon for Ser-202, at which the primer generated an EcoRI site and terminated at the HindIII site of plasmid pH34, at which the primer generated a KpnI site. The third PCR fragment (III) was cloned into pBluescript II SK, yielding plasmid pASKE14. Subsequently, both SacII–EcoRI fragments (PCR products I and II) were separately cloned into pASKE14, resulting in constructs pASSK29S22 and pASSK31T103 (Fig. 1A). pASSK29S22 contained a deletion of a 0·5 kb DNA region from corS, and pASSK31T103 contained a 0·3 kb deletion. Both constructs were confirmed by nucleotide sequencing.



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Fig. 1. (A) Schematic representation of in-frame deletions in corS. Regions deleted from corS are shown by a hatched rectangle. Deletion regions are bordered by the indicated amino acid residues of CorS: Ser-22, Thr-103 and Ser-202. The primers used for PCR, the respective PCR products (I), (II) and (III), the size of deleted fragments and the plasmids containing the in-frame deletions are also indicated. Restriction sites destroyed in the course of subsequent cloning steps are shown in parentheses. (B) Schematic representation of translational fusions of CorS to alkaline phosphatase (PhoA) and {beta}-galactosidase (LacZ) reporter genes. The translational corS : : phoA fusions were expressed under the control of the corS promoter, indicated by an arrow. Restriction sites relevant for plasmid pH34 and for the construction of plasmids bearing in-frame deletions and translational fusions are indicated.

 
Subsequently, the two inserts of pASSK29S22 and pASSKT103 were subcloned into pRK415-EB, a derivative of pRK415 (Keen et al., 1988) lacking the multiple cloning site between EcoRI and BamHI, and which is able to replicate in P. syringae. For this, the 3·1 kb and 2·9 kb SacII–KpnI fragments derived from plasmids pASSK31T103 and pASSK29S22, respectively, were blunt-ended with T4 DNA polymerase, while pRK415-EB was cleaved with HindIII and blunt-ended with Klenow fragment DNA polymerase. Successful cloning resulted in plasmids pASH31 and pASH29. Both plasmids were used for the complementation analysis of mutant PG4180.D4 containing the gusA reporter plasmid pRGMU1 (Ullrich & Bender, 1994).

Construction of translational fusions.
Primers corSoutF and corSThr27 to corSGln281 (Table 2) were used to generate DNA fragments starting at the EcoRI site of plasmid pH34, at which the primer generated a SacII site, and terminating at codons for certain amino acid residues located either in the putative periplasmic or cytoplasmic loops of CorS, at which the primers generated KpnI sites (Fig. 1B). All fragments contained the native corS promoter and a ribosome-binding site. SacII–KpnI PCR fragments were cloned in pBluescript II SK, and subsequently were fused to a 2·6 kb KpnI fragment containing a promoterless phoA gene, which lacked both a ribosome-binding site and the signal-peptide sequence, and which was derived from plasmid pPHO7 (Guttierrez & Devedjian, 1989). This resulted in constructs bearing translational corS : : phoA fusions under the control of the corS promoter (Fig. 1B). Precise in-frame fusion of all fragments to the phoA gene was verified by DNA sequence analysis of the junction regions. Subsequently, SacII–PstI fragments containing corS : : phoA translational fusions were cloned into the broad-host-range vector pBBR1MCS (Kovach et al., 1994), which replicates in P. syringae.

To generate corS : : lacZ translational fusions, the 2·6 kb KpnI fragment containing phoA was substituted by a 3·0 kb KpnI fragment encoding the lacZ gene in all corS : : phoA fusions in pBBR1MCS. The DNA fragment encoding lacZ was PCR amplified using primers lacZF and lacZR (Table 2) and plasmid pMC-1871 (Amersham-Pharmacia Biotech), which contains an intact lacZ gene without its ribosome-binding site and without the first eight non-essential codons.

Estimation of specific alkaline phosphatase, {beta}-galactosidase and {beta}-glucuronidase activities.
Bacteria were harvested from 1·5 ml of bacterial culture for all enzymic measurements. Alkaline phosphatase (PhoA) and {beta}-galactosidase (LacZ) activities were determined as described by Rutz et al. (1999). To determine PhoA activity, the pellet was resuspended in 300 µl 1 M Tris/HCl (pH 8·0), and cells were permeabilized by the addition of 25 µl 0·1 % SDS and 2 drops chloroform. Samples were stirred for 40 min at 28 °C. A 200 µl volume of cell extract was transferred to a 96-well microtitre plate. A spectrophotometric assay was performed with p-nitrophenyl phosphate as substrate (5 mg p-nitrophenyl phosphate ml–1 in 1 M Tris/HCl, pH 8·0, buffer containing 5 mM MgCl2). Plates were incubated for 180 min at 28 °C, and A405 was measured in an MRX microplate reader (Dynatech). Specific alkaline phosphatase activity was defined as units per mg of cellular protein. One unit of PhoA activity corresponded to 1 µmol of p-nitrophenol released per minute at 28 °C. The concentrations of p-nitrophenol used for the calibration curve ranged from 2 to 120 µmol. Protein concentrations were determined by the Bradford assay (Bradford, 1976).

To determine LacZ activity, the pellet was resuspended in 300 µl buffer Z (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM {beta}-mercaptoethanol, pH 7·0), and cells were permeabilized by the addition of 25 µl 0·1 % SDS and 2 drops chloroform. Samples were stirred for 40 min at 28 °C. A 200 µl volume of cell extract was transferred to a 96-well microtitre plate. ONPG (4 mg ml–1) in buffer Z (pH 7·0) was used as the substrate solution. Plates were incubated for 90 min at 28 °C, and A405 was measured in an MRX microplate reader. Specific {beta}-galactosidase activity was defined as units per mg of cellular protein. One unit of LacZ activity corresponded to 1 µmole of o-nitrophenol released per minute at room temperature. The concentrations of o-nitrophenol used for the calibration curve ranged from 10 to 1000 µmol.

{beta}-Glucuronidase activity (GUS) was quantified by a fluorescence assay, as described previously (Xiao et al., 1992). The pellet was lysed in 500 ml GUS extraction buffer (50 mM Na2HPO4, adjusted to pH 7·0, 10 mM EDTA disodium salt, 0·1 % N-laurolylsarcosyl sodium salt, 0·1 % Triton X-100, 0·07 % {beta}-mercaptoethanol), and incubated on ice for 30 min. Subsequently, cells were disrupted by 3x15 s ultrasonic treatment and transferred to precooled 96-well microtitre plates. The substrate solution used was 2 mM 4-methylumbelliferyl-{beta}-D-glucuronide in GUS extraction buffer. Plates were incubated for 10 min at 37 °C, and fluorescence emission was measured at 450 nm after excitation at 390 nm in a Fluorolite fluorometer (Dynatech). Specific {beta}-glucuronidase activity was defined as units GUS per mg of cellular protein. One unit of GUS activity corresponded to 1 µmol of 4-methylumbelliferol released per minute at 37 °C.

Immunodetection of CorS–PhoA proteins.
Equal amounts of protein for Western blot analysis were separated by SDS-PAGE on two gels running in the same chamber. Subsequently, proteins from one gel were electrotransferred to a Hybond-C nitrocellulose membrane (Amersham-Pharmacia Biotech). The second gel was stained with GelCode Blue stain reagent (Perbio Science, Bonn, Germany). CorS–PhoA fusion proteins were detected with PhoA polyclonal antibody (dilution 1 : 1000) and with goat anti-rabbit Ig conjugated to alkaline phosphatase as the secondary antibody (dilution 1 : 7500). The chromogenic reaction was initiated by adding nitrotetrazolium blue and 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine salt (Blake et al., 1984).

Subcellular cell fractionation of P. syringae and trypsin treatment of spheroplasts.
Subcellular fractionation was done according to the method described by Boyd et al. (1987). Cells were permeabilized in SP buffer (0·1 M Tris/HCl at pH 7·5, 0·5 mM EDTA-Na, 0·5 M sucrose), osmotically shocked by addition of 20 mM MgCl2 (1 : 20 dilution) and treated with 1 mg lysozyme ml–1 on ice to generate spheroplasts. Spheroplasts were lysed in 200 µl lysis buffer (50 mM Tris/HCl, pH 8·0, 150 mM NaCl, 1 % Triton X-100, 0·1 % SDS). A 80 µl volume of the lysate was used directly for Western blotting. A 120 µl volume of the lysate was treated with trypsin (1 µg ml–1 final concentration). Protease digestion was stopped by the addition of 12 µl AEBSF protease inhibitor solution (10 mM). Proteins were precipitated by cold 10 % TCA, resuspended in 40 µl Tris/HCl (pH 8·0), and used for Western blotting.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Predictions of CorS membrane topology
According to a hydrophobicity plot generated by the program PROTEAN, the N-terminal region of CorS (approximately 200 amino-acid residues) contained multiple hydrophobic zones (Fig. 2A). This indicated a potential membrane localization for this portion of the protein. We employed different topology prediction programs to generate a topological model for CorS. According to the TopPred II prediction, CorS might possess 5, 6, or even 7 TMDs. Interestingly, the seventh TMD was predicted to occur downstream of the conserved H-box, where the site of autophosphorylation, residue His-254, is located (Fig. 2B). This prediction seemed unlikely, because in order to be phosphorylated, the histidine residue must be located in the cytoplasm. The second, sixth and seventh TMDs were predicted to be putative and showed the lowest hydrophobicity scores among all TMDs. Vector NTI and MEMSAT yielded similar results: they predicted six TMDs, among which the second and sixth TMDs had the lowest hydrophobicity scores (Fig. 2B). The low hydrophobicity scores resulted from a number of non-hydrophobic amino-acid residues present in both TMDs. However, this is not unusual in proteins with many TMDs that form a bundle in which non-hydrophobic residues may make contacts between helices (Sonnhammer et al., 1998). The summarized results obtained from analysis with the programs TopPred II, TMHMM, HMMTOP, DAS, MEMSAT, SOSUI, and Vector NTI preferentially supported a topological model for CorS with six TMDs, and with both the N- and the C-terminus located in the cytoplasm.



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Fig. 2. CorS hydrophobicity profile, CorS surface probability profile and CorS predicted membrane topology. (A) Hydrophobicity was plotted according to the algorithm of Kyte & Doolittle, (1982), and revealed the hydrophobic N-terminal part of CorS. (B) Schematic representation of the topological structure of CorS, derived from prediction analyses. The table summarizes results of prediction analyses by the TopPred II, Vector NTI and MEMSAT programs. Open rectangles represent the predicted certain and putative TMDs of CorS. Circles, black arrows and numbers indicate residues of CorS fused to either PhoA or LacZ. Charged residues are indicated with ‘+’ and ‘–’. The conserved H-box is indicated by a dotted rectangle, and the putative site of phosphorylation, His-254, is indicated by a grey rectangle.

 
Deletion analysis of the membrane-spanning region of CorS
To test whether the apparent TMDs are important for CorS to function as a temperature sensor, the region of the gene encoding either all six or the last four TMDs was subjected to in-frame deletion analysis. The truncated corS gene in pASH31 encodes a protein with two TMDs, whereas in pASH29 it presumably encodes a soluble protein (Fig. 3A). Neither plasmid containing the truncated versions of corS complemented PG4180.D4 (pRGMU1) with respect to cmaABT promoter activity. GUS activities measured in transconjugant PG4180.D4 (pRGMU1; pASH29) and in the mutant PG4180.D4 (pRGMU1) were similarly negligible. The residual promoter activity for PG4180.D4 (pRGMU1; pASH31) at 18 °C comprised only 8 % of the cmaABT promoter activity of the wild-type (Fig. 3B), and was only slightly higher than in the mutant, whereas complete loss of promoter activity occurred at 28 °C. In contrast, the control plasmid pMUH34, containing intact corS, was able to completely complement the mutant phenotype, and consequently restored temperature-dependent transcriptional activation of cmaABT (Fig. 3B). These results suggested that CorS lacking TMDs is a non-functional protein.



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Fig. 3. Complementation analysis with truncated corS derivatives. (A) Schematic representation of truncated CorS proteins resulting from the in-frame deletions of corS. (B) Complementation analysis of mutant PG4180.D4 (corS corR) with plasmid pMUH34 and plasmids containing in-frame deletions of corS. cmaABT : : uidA expression was determined in PG4180 (pRGMU1), PG4180.D4 (pRGMU1), and PG4180.D4 (pRGMU1) derivatives harbouring plasmids pMUH34, pASH29, and pASH31. The H, N, D, F and G boxes indicate the conserved sequence motifs identified in various HPKs. Values represent the averages of two experiments with three replicates.

 
Phenotypic and enzymic analyses of CorS–PhoA and CorS–lacZ translational fusions
To confirm the computer-based topology predictions biochemically, a series of translational fusions of hybrid proteins of CorS with either alkaline phosphatase (PhoA) or {beta}-galactosidase (LacZ) were constructed. The design of the hybrid proteins was such that at least one fusion was placed in each predicted loop facing either the periplasm or the cytoplasm. Five additional CorS–PhoA fusions were generated upstream and downstream of the conserved H-box and the putative seventh TMD. Plasmids carrying hybrid constructs were mobilized into P. syringae PG4180 and tested for PhoA and LacZ activity.

MG medium agar plates containing the substrates for either alkaline phosphatase (X-Phos) or {beta}-galactosidase (X-Gal) were used for the visual estimation of the enzymic activity of CorS–PhoA and CorS–LacZ fusion proteins, respectively. PhoA fusions to amino-acid residues Ala-51, Leu-125 and Val-177 exhibited a clear PhoA+ phenotype (blue colonies), whereas PhoA fusions to amino-acid residues Thr-27, Tyr-85 and Leu-151 gave rise to a PhoA phenotype (white colonies). The PhoA+ phenotype of PhoA fusions to Ala-51, Leu-125 and Val-177 indicated that fusions to these amino-acid residues are located in periplasmic loops of CorS, whereas the PhoA phenotype of the other fusions implied that amino-acid residues Thr-27, Tyr-85 and Leu-151 are located in the cytoplasm.

Results obtained with the respective CorS–LacZ fusions were in agreement with results for the PhoA fusions. LacZ fusions to amino-acid residues Thr-27, Tyr-85 and Leu-151 exhibited a clear LacZ+ phenotype (blue colonies), thus supporting the assumption of their cytoplasmic location. LacZ fusions to Ala-51 and Leu-125 showed a LacZ phenotype (white colonies), which verified their periplasmic location. However, the LacZ fusion to amino-acid residue Val-177 showed a LacZ+ phenotype, contradicting the result obtained for the Val-177–PhoA fusion, which exhibited a PhoA+ phenotype. An additional downstream CorS–LacZ fusion to residue Arg-204 also exhibited a LacZ+ phenotype.

The translational corS : : phoA fusions that were expressed under control of the corS promoter were not generally affected by temperature. Most cells harbouring CorS–PhoA fusions with PhoA+ phenotypes and cells carrying CorS–LacZ fusions with LacZ+ phenotypes formed blue colonies at both temperatures and, consequently, cells harbouring CorS–PhoA fusions with PhoA phenotypes and cells containing CorS–LacZ fusions with LacZ phenotypes formed white colonies at both temperatures. However, fusions of PhoA to the amino acid residues Arg-204, Gln-214 and Asp-227, which are located downstream of the sixth predicted TMD and upstream of the conserved H-box of CorS, showed a temperature-dependent phenotype. Cells harbouring these fusions formed white colonies at 18 °C and blue colonies at 28 °C. PhoA fusions were also constructed to two residues near the C-terminus: Leu-249, which is located in proximity to residue His-254, the autophosphorylation site of CorS, and residue Gln-281, which is located downstream of the H-box and of the predicted putative seventh TMD. Cells containing these fusions remained white at both temperatures, demonstrating that the C-terminus of CorS is located in the cytoplasm, regardless of temperature.

Subsequently, reporter-enzyme activities were quantitatively estimated for cells harbouring either CorS–PhoA or CorS–LacZ fusions. For this, cultures of the respective cells were grown in minimal HSC medium and harvested at an OD600 of 3·0. As a control for enzymic measurements, wild-type cells lacking recombinant PhoA and LacZ fusions were used. The enzymic activity of none of the fusion proteins analysed was temperature-dependent. Approximately equal levels of specific activity were measured at 18 °C and 28 °C for CorS–PhoA and CorS–LacZ fusion proteins (data not shown).

PhoA fusions to amino-acid residues Ala-51, Leu-125 and Val-177, which exhibited PhoA+ phenotypes on MG agar plates, showed high specific activities (2·3–3·8 units mg–1 protein) compared to cytoplasmic fusion proteins (Table 3). Specific activities of PhoA in 28 °C samples for fusions Arg-204–PhoA, Gln-214–PhoA and Asp-227–PhoA, which had exhibited a temperature-dependent phenotype on solid medium, were only slightly elevated compared to activities of cytoplasmic PhoA fusions (Table 3), suggesting that the interesting temperature-dependent phenotype observed on agar plates could not be reproduced in liquid medium.


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Table 3. Specific alkaline phosphatase and {beta}-galactosidase activities for CorS–PhoA and CorS–LacZ translational fusions in PG4180

Cells for enzymic measurements were grown at 28 °C in minimal HSC medium.

 
Quantitative estimation of the specific activities of LacZ in 28 °C samples for CorS–LacZ fusions demonstrated that the LacZ+ phenotypes of fusions to the amino-acid residues Thr-27, Tyr-85, Leu-151 and Arg-204 on MG plates corresponded to high levels of specific activities for these fusion proteins (34–160 units mg protein–1; Table 3). An elevated level of LacZ activity for the Val-177–LacZ fusion was in conflict with a high level of PhoA activity for the corresponding PhoA fusion. Orientation of the Val-177 residue with respect to the membrane was verified in immunoblot analyses and in a protease-sensitivity assay.

Immunoblot analyses and protease-sensitivity assay
To demonstrate that cellular location-based differences in the enzymic activities of various hybrid proteins were not the result of different levels of protein expression, immunoblot analyses for CorS–PhoA hybrid proteins with antibodies raised against PhoA were performed. Protein samples from the same P. syringae cultures that were grown at 28 °C and subsequently used for measurement of enzymic activities were subjected to Western blot analysis. For this, PG4180 cells expressing CorS–PhoA fusions were fractionated to generate spheroplasts. The spheroplasts were lysed in the presence of SDS and Triton X-100. The lysates containing solubilized proteins were separated by 10 % SDS-PAGE and blotted on nitrocellulose membranes. Representative results are shown in Fig. 4(A). Western blot analysis of all CorS–PhoA fusions demonstrated that hybrid proteins of the expected molecular size were produced. The actual expression levels for the hybrid proteins did not account for the differences observed for the respective PhoA activities, thus confirming that the observed differences in enzymic activities were not due to differential expression of the fusion proteins. However, some degree of protein instability was observed. For all chimeras, a band of about 48 kDa was detected, which corresponded to the size of the PhoA moiety.



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Fig. 4. Western blot analysis of CorS–PhoA hybrid proteins and protease-sensitivity assay. Equal amounts of solubilized proteins from spheroplasts (A) and solubilized proteins treated with 1µg trypsin ml–1 (B) were resolved by 10 % SDS-PAGE and immunoblotted with anti-alkaline-phosphatase antiserum. Lanes: 1, no plasmid; 2, pAST27phoA; 3, pASA51phoA; 4, pASY85phoA; 5, pASL125phoA; 6, pASL151phoA; 7, pASV177phoA; 8, pASR204phoA; 9, pASD227phoA; 10, pASL249phoA; 11, pASQ281phoA. Molecular weight standards are shown on the left, and stars indicate the position of the hybrid proteins. The position of the 48 kDa band corresponding to the size of the PhoA protein is marked by an arrow.

 
For CorS–PhoA fusions, the periplamic or cytoplasmic localization of the respective hybrid proteins was additionally verified by protease-sensitivity assays. If a hybrid protein is translocated into the periplasm it becomes protease resistant due to intramolecular disulfide-bond formation. In contrast, in the cytoplasm, it remains protease sensitive (Rutz et al., 1999). Upon protease treatment, solubilized CorS–PhoA hybrid proteins should be cleaved to the molecular size of the PhoA protein (48 kDa) if the PhoA portion is located in the periplasm. In contrast, the hybrid protein should be completely digested if the PhoA portion is located in the cytoplasm. To test this, the lysates containing solubilized proteins were treated with trypsin. Upon trypsin treatment, a band of about 48 kDa was detected for hybrid proteins of Ala-51, Leu-125 and Val-177 fusions, which all showed high PhoA activities (Fig. 4B). The protease resistance of PhoA in these cases confirmed that PhoA was exposed to the periplasm. Thus, it was proven that Ala-51, Leu-125 and Val-177 residues indeed face the periplasm. When the hybrid proteins were located in the cytoplasm, they were cleaved by protease, and the 48 kDa signal could not be detected (Fig. 4B).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The N-terminal regions of various HPKs have been shown to sense, either directly or indirectly, diverse environmental stimuli. In response to environmental stimuli, the signal-modified N-terminal part of an HPK modulates conformational changes in the C-terminal part, which then transmits the signal intracellularly (Parkinson, 1993). A sensor kinase, VirA, from the plant pathogen Agrobacterium tumefaciens undergoes reversible conformational changes from an inactive state (OFF) to a standby mode, and subsequently to an active form (ON), in order to initiate virulence-gene expression. These conformational changes are mediated by indirect or direct effects of environmental stimuli: monosaccharides, phenolic compounds such as acetosyringone, pH, and temperature (Heath et al., 1995). Even though VirA seems to sense temperature changes, and the virulence of A. tumefaciens is promoted by activation of VirA at low temperature (Heath et al., 1995), the primary as well as secondary structures of VirA and CorS differ greatly (data not shown), making any comparison inconclusive.

Generally, HPKs are intrinsic membrane proteins with two or more N-terminal transmembrane {alpha}-helices. Therefore, the process of transmembrane signalling is fundamental to many sensory systems, but still poorly understood. Since temperature markedly affects the fatty-acid composition of the membrane, the physical state of the membrane (fluidity) might trigger a conformational change in one or more TMDs of a HPK like CorS. This mechanism was already hypothesized for other HPKs known to be involved in temperature sensing, such as DesK from Bacillus subtilis (Aguilar et al., 2001) and Hik33 from the cyanobacterium Synechocystis sp. PCC 6803 (Suzuki et al., 2000). However, just as for VirA, the secondary structure of Hik33 is only distantly related to the structure of CorS. Interestingly, Hik33 and VirA share structural features common to many other HPKs, with a periplasmic loop flanked by two transmembrane helices, whereas DesK resembles CorS with respect to N-terminal hydrophobic zones, which comprise four TMDs in DesK (Aguilar et al., 2001).

A number of conserved intracellular domains, such as the haem- and flavin-binding PAS domain (Taylor & Zhulin, 1999), the phytochrome- and cGMP-binding GAF (Aravind & Ponting, 1997), and the HAMP linker (Aravind & Ponting, 1999), have been implicated in playing a role in signal perception and transduction in HPKs. However, none of them was identified in CorS, which was analysed by the Pfam and SMART programs. However, all membrane-topology programs predicted multiple TMDs in the N-terminus of CorS. This fact allowed us to speculate that the transmembrane region of CorS is involved in signal perception.

The topological analysis of CorS with different prediction programs indicated that six TMDs presumably span the membrane, and that the N- and C-termini of the protein are located in the cytoplasm. Uncertain topological information was derived by this analysis for a putative sixth TMD, which showed a relatively low hydrophobicity score, and a putative seventh TMD, predicted to be located downstream of the conserved H-box. An experimental approach based on the generation of translational fusions between C-terminally truncated CorS portions and either PhoA or LacZ was used to establish the topological assignments of all TMDs. The activities of hybrid fusion proteins were determined in qualitative and quantitative assays. Their expression levels in P. syringae were furthermore analysed by immunoblotting. High activities of PhoA fusions at amino-acid residues Ala-51, Leu-125 and Val-177 indicated that these residues were located in the periplasm (as indicated in Fig. 2B). LacZ fusions at these residues showed the expected lack of activity for Ala-51 and Leu-125, whereas an elevated LacZ activity level was observed for the fusion at amino-acid residue Val-177. According to previous topological studies of various membrane proteins, it is known that LacZ is a less-reliable reporter enzyme than PhoA (Bartsevich & Pakrasi, 1999; Hennessey & Broome-Smith, 1993). It has been suggested that {beta}-galactosidase fused to periplasmic domains sometimes exhibits high activity because of disruption of the membrane integration of the fusion protein by this large reporter enzyme (Bartsevich & Pakrasi, 1999). In contrast, a PhoA fusion requires active translocation of the reporter enzyme moiety through the cytoplasmic membrane for activity. Because the PhoA fusion at Val-177 showed high specific activity in a quantitative assay and, in addition, because Western blot analysis and a protease-sensitivity assay clearly demonstrated its periplasmic location, we concluded that Val-177 was indeed periplasmic. Some instability of most of the hybrid proteins was observed in Western blots. In most cases, the bands for cytoplasmic hybrid proteins showed weaker signals than the bands of periplasmic hybrid proteins. This result is probably attributable to a proteolytic degradation of the hybrid proteins, as reported earlier (Guan et al., 1999; Haardt & Bremer, 1996; Ouchane & Kaplan, 1999).

Negligible PhoA activities and elevated LacZ activities for fusions at the remaining amino-acid residues tested (Thr-27, Tyr-85, Leu-151 and Arg-204) indicated their cytoplasmic location and, in addition, proved that the N- and C-termini of CorS were located in the cytoplasm.

Our experimental data confirmed the predicted topology of CorS, with six TMDs. Additionally, deletion analysis indicated that CorS lost its function when its TMDs were removed. The very low, but measurable, promoter activity for PG4180.D4 (pRGMU1; pASH31T103) containing the truncated CorS derivative with two TMDs at 18 °C might be an experimental artifact, possibly due to differences in the stability of residual amounts of reporter gene mRNA at the two tested temperatures. Temperature has previously been shown to act on the secondary structure of RNAs, resulting in long-term changes in translation efficiency (Eriksson et al., 2002). Indeed, deletion analysis supported our assumption that the hydrophobic N-terminal part of CorS is important for signal perception.

Interestingly, three PhoA fusions, at amino-acid residues Arg-204, Gln-214 and Asp-227, which were located between the sixth TMD and the H-box, showed a temperature-dependent phenotype on indicator agar plates. Colonies harbouring these particular PhoA fusions showed a positive phenotype at 28 °C, but a negative phenotype at 18 °C. This implied that the PhoA portion of these three fusions might be translocated into the periplasm at 28 °C. The PhoA fusions at Leu-249 and Gln-281, located in close proximity upstream and downstream of the H-box, respectively, showed a PhoA phenotype, and were therefore clearly cytoplasmic at both temperatures. As yet, it is impossible to confirm the temperature-sensitive phenotype of these three fusions in a quantitative assay. Minimal HSC medium optimized for COR production was used for bacterial growth in liquid culture because P. syringae does not grow well in MG liquid medium. In HSC medium, a quantitative difference between PhoA activities for fusions at Arg-204, Gln-214 and Asp-227 with respect to temperature dependence was not observed. In addition to the presented data, measurements of PhoA activities were taken at various time points throughout bacterial growth in liquid broth (data not shown). Under no conditions could we detect a thermoresponsive PhoA activity in liquid medium. The reason for this remains obscure.

Nevertheless, the temperature-sensitive phenotypes of the three fusions located between the sixth TMD and the H-box led us to speculate that the CorS topology might be thermoresponsive in this particular protein region. The sixth TMD and the linker region between the sixth TMD and the H-box may be involved in a temperature-dependent conformational change affecting autophosphorylation of the conserved histidine residue.

Uncertain topological information derived from computer predictions, and experimental data which argued for a change in the topology of membrane proteins, had previously been reported for mammalian multidrug transporters, P-glycoproteins (Zhang et al., 1996), and for the lactococcal bacteriocin ABC transporter LcnC (Franke et al., 1999). Recently, it has been demonstrated that a polytopic membrane protein, such as the lactose permease (LacY) of E. coli, can change its membrane topology in a reversible manner in response to alterations in the phospholipid composition (Bogdanov et al., 2002). Moreover, even a subtle attractant-induced conformational change in transmembrane bacterial chemoreceptors of about 1–2 Å can affect the function of the cytoplasmic HPK CheA (Falke & Hazelbauer, 2001).

It remains unclear how the fluidity and/or the fatty-acid composition of the membrane might affect the function of CorS. It would be intriguing to answer this important question in the future, as well as to elucidate the precise mechanism of the conformational change in CorS which initiates the signal-transduction pathway and ultimately results in temperature-dependent gene expression in P. syringae.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Max-Planck-Gesellschaft. Anti-PhoA antibody was provided by Dr R. Schuelein. We thank Stephan Aufhammer for valuable technical help, and Erhard Bremer, Uwe Völker, Carol Bender and Helge Weingart for stimulating discussions.


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Received 13 January 2004; revised 23 April 2004; accepted 30 April 2004.



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