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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
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 helixturnhelix (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 202550) forms the kinase core. The kinase core includes a dimerization and histidine phosphotransfer domain (DHp, residues 244309) and a conserved catalytic and ATP-binding domain (CA, residues 353464). 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 -galactosidase (Manoil & Beckwith, 1986
; Manoil et al., 1988
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 25 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 HoitinkSinden minimal medium optimized for COR production (HSC) (Palmer & Bender, 1993
). Antibiotics were used at the following concentrations: ampicillin, 100 µg ml1; chloramphenicol, 25 µg ml1; tetracycline 25 µg ml1; streptomycin, 25 µg ml1; kanamycin, 25 µg ml1.
-Glucuronidase, alkaline phosphatase and
-galactosidase activities were detected by the blue colour formation of colonies on agar plates containing 20 µg ml1 5-bromo-4-chloro-3-indolyl-
-glucuronic acid (X-Gluc), 160 µg ml1 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine salt (X-Phos), and 100 µg ml1 5-bromo-4-chloro-3-indolyl-
-galactoside (X-Gal), respectively. The plates were incubated for 46 days at 18 °C and 28 °C.
|
|
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 SacIIEcoRI 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.
|
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. SacIIKpnI 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, SacIIPstI 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, -galactosidase and
-glucuronidase activities.
Bacteria were harvested from 1·5 ml of bacterial culture for all enzymic measurements. Alkaline phosphatase (PhoA) and -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 ml1 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 -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 ml1) 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
-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.
-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 %
-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-
-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
-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 CorSPhoA 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). CorSPhoA 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 ml1 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 ml1 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
MG medium agar plates containing the substrates for either alkaline phosphatase (X-Phos) or -galactosidase (X-Gal) were used for the visual estimation of the enzymic activity of CorSPhoA and CorSLacZ 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 CorSLacZ 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-177PhoA fusion, which exhibited a PhoA+ phenotype. An additional downstream CorSLacZ 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 CorSPhoA fusions with PhoA+ phenotypes and cells carrying CorSLacZ fusions with LacZ+ phenotypes formed blue colonies at both temperatures and, consequently, cells harbouring CorSPhoA fusions with PhoA phenotypes and cells containing CorSLacZ 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 CorSPhoA or CorSLacZ 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 CorSPhoA and CorSLacZ 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 (2·33·8 units mg1 protein) compared to cytoplasmic fusion proteins (Table 3). Specific activities of PhoA in 28 °C samples for fusions Arg-204PhoA, Gln-214PhoA and Asp-227PhoA, 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.
|
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 CorSPhoA 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 CorSPhoA 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 CorSPhoA 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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generally, HPKs are intrinsic membrane proteins with two or more N-terminal transmembrane -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
-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 12 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alarcón-Chaidez, F. J., Keith, L., Zhao, Y. & Bender, C. L. (2003). RpoN (54) is required for plasmid-encoded coronatine biosynthesis in Pseudomonas syringae. Plasmid 49, 106117.[CrossRef][Medline]
Aravind, L. & Ponting, C. P. (1997). The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 22, 458459.[CrossRef][Medline]
Aravind, L. & Ponting, C. P. (1999). The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins. FEMS Microbiol Lett 176, 111116.[CrossRef][Medline]
Bartsevich, V. V. & Pakrasi, H. B. (1999). Membrane topology of MntB, the transmembrane protein component of an ABC transporter system for manganese in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 181, 35913593.
Bateman, A., Birney, E., Cerruti, L. & 7 other authors (2002). The Pfam protein families database. Nucleic Acids Res 30, 276280.
Bender, C. L., Young, S. A. & Mitchell, R. E. (1991). Conservation of plasmid DNA sequences in coronatine-producing pathovars of Pseudomonas syringae. Appl Environ Microbiol 57, 993999.
Bender, C. L., Liyanage, H., Palmer, D., Ullrich, M., Young, S. & Mitchell, R. (1993). Characterization of the genes controlling biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid. Gene 133, 3138.[CrossRef][Medline]
Bilwes, A. M., Alex, L. A., Crane, B. R. & Simon, M. I. (1999). Structure of CheA, a signal-transducing histidine kinase. Cell 96, 131141.[Medline]
Blake, M. S., Johnston, K. H., Russel-Jones, G. J. & Gotschlich, E. C. (1984). A rapid, sensitive method for detection of alkaline phosphatase-conjugated antibody on Western blots. Anal Biochem 136, 175179.[Medline]
Bogdanov, M., Heacock, P. N. & Dowhan, W. (2002). A polytropic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J 21, 21072116.
Boyd, D., Manoil, C. & Beckwith, J. (1987). Determinants of membrane protein topology. Proc Natl Acad Sci U S A 84, 85258529.[Abstract]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Budde, I. P., Rohde, B. H., Bender, C. & Ullrich, M. S. (1998). Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine. J Bacteriol 180, 13601367.
Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685686.[Medline]
Cserzo, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. (1997). Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10, 673676.[CrossRef][Medline]
Eriksson, S., Hurme, R. & Rhen, M. (2002). Low-temperature sensors in bacteria. Philos Trans R Soc Lond B 357, 887893.[CrossRef][Medline]
Falke, J. J. & Hazelbauer, G. L. (2001). Transmembrane signalling in bacterial chemoreceptors. Trends Biochem Sci 26, 257265.[CrossRef][Medline]
Figurski, D. H. & Helsinki, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 16481659.[Abstract]
Franke, C. M., Tiemersma, J., Venema, G. & Kok, J. (1999). Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. J Biol Chem 274, 84848490.
Fullner, K. J. & Nester, E. W. (1996). Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178, 14981504.[Abstract]
Grebe, T. W. & Stock, J. B. (1999). The histidine protein kinase superfamily. Adv Microb Physiol 41, 139227.[Medline]
Guan, L., Ehrmann, M., Yoneyama, H. & Nakae, T. (1999). Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa. J Biol Chem 274, 1051710522.
Guttierrez, C. & Devedjian, J. C. (1989). A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res 17, 3999.[Medline]
Haardt, M. & Bremer, E. (1996). Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli. J Bacteriol 178, 53705381.[Abstract]
Heath, J. D., Charles, T. C. & Nester, E. W. (1995). Ti plasmid and chromosomally encoded two-component systems important in plant cell transformation by Agrobacterium species. In Two-Component Signal Transduction, pp. 367385. Edited by J. A. Hoch & T. J Silhavy. Washington, DC: American Society for Microbiology.
Hennessey, E. S. & Broome-Smith, J. K. (1993). Gene-fusion techniques for determining membrane-protein topology. Curr Opin Struct Biol 3, 524531.[CrossRef]
Hirokawa, T., Boon-Chieng, S. & Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378379.[Abstract]
Hoch, J. A. (2000). Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3, 165170.[CrossRef][Medline]
Hugouvieux-Cotte-Pattat, N., Dominguez, H. & Robert-Baudouy, J. (1992). Environmental conditions affect transcription of pectinase genes of Erwinia chrysanthemi 3937. J Bacteriol 174, 78077818.[Abstract]
Jones, D. T., Taylor, W. R. & Thornton, J. M. (1994). A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33, 30383049.[Medline]
Keane, P. J., Kerr, A. & New, P. B. (1970). Grown gall of stone fruit. II. Identification and nomenclature of Agrobacterium isolates. Aust J Biol Sci 23, 585595.
Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988). Improved broad-host-range plasmid for DNA cloning in Gram-negative bacteria. Gene 70, 191197.[CrossRef][Medline]
Kovach, M. E., Phillips, R. W., Elzer, P. H. & Roop, R. M. (1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800802.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Manoil, C. & Beckwith, J. (1986). A genetic approach to analyzing membrane protein topology. Science 233, 14031408.[Medline]
Manoil, C., Boyd, D. & Beckwith, J. (1988). Molecular genetic analysis of membrane protein topology. Trends Genet 4, 223226.[CrossRef][Medline]
Mitchell, R. E., Young, S. A. & Bender, C. L. (1994). Coronamic acid, an intermediate in coronatine biosynthesis by Pseudomonas syringae. Phytochemistry 35, 343348.[CrossRef]
Ouchane, S. & Kaplan, S. (1999). Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides. J Biol Chem 274, 1729017296.
Palmer, D. A. & Bender, C. L. (1993). Effects of environmental and nutritional factors on production of the polyketide phytotoxin coronatine by Pseudomonas syringae pv. glycinea. Appl Environ Microbiol 59, 16191626.[Abstract]
Parkinson, J. S. (1993). Signal transduction schemes of bacteria. Cell 73, 857871.[Medline]
Parry, R. J., Mhaskar, S. V., Lin, M.-T., Walker, A. E. & Mafoti, R. (1994). Investigations of the biosynthesis of the phytotoxin coronatine. Can J Chem 72, 8699.
Peñaloza-Vázquez, A. & Bender, C. L. (1998). Characterization of CorR, a transcriptional activator which is required for biosynthesis of the phytotoxin coronatine. J Bacteriol 180, 62526259.
Rangaswamy, V. & Bender, C. L. (2000). Phosphorylation of CorR and CorR, regulatory proteins that modulate production of the phytotoxin coronatine in Pseudomonas syringae. FEMS Microbiol Lett 193, 1318.[CrossRef][Medline]
Rowley, K. B., Clements, D. E., Mandel, M., Humphreys, T. & Patil, S. S. (1993). Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production. Mol Microbiol 8, 625635.[Medline]
Rutz, C., Rosenthal, W. & Schuelein, R. (1999). A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain. J Biol Chem 274, 3375733763.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. & Bork, P. (2000). SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res 28, 231234.
Sonnhammer, E. L., von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of the 6th International Conference on Intelligent Systems for Molecular Biology, pp. 175182. Montreal, Canada: AAAI Press.
Suzuki, I., Los, A. D., Kanesaki, Y., Mikami, K. & Murata, N. (2000). The pathway for perception and transduction of low-temperature signals in Synechocystis. EMBO J 19, 13271334.
Taylor, B. L. & Zhulin, I. B. (1999). PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63, 479506.
Tusnády, G. E. & Simon, I. (1998). Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283, 489506.[CrossRef][Medline]
Ullrich, M. S. & Bender, C. L. (1994). The biosynthetic gene cluster for coromanic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases. J Bacteriol 176, 75747586.[Abstract]
Ullrich, M. S., Peñaloza-Vázquez, A., Bailey, A. M. & Bender, C. L. (1995). A modified two-component regulatory system is involved in temperature-dependent biosynthesis of the Pseudomonas syringae phytotoxin coronatine. J Bacteriol 177, 61606169.[Abstract]
Van den Eede, G., Deblaere, R., Goethals, K., Montagu, M. V. & Holsters, M. (1992). Broad host range and promoter selection vectors for bacteria that interact with plants. Mol PlantMicrobe Interact 5, 228234.[Medline]
Van Dijk, K., Fouts, D. E., Rehm, A. H., Hill, A. R., Collmer, A. & Alfano, J. R. (1999). The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner. J Bacteriol 181, 47904797.
Wang, L., Bender, C. L. & Ullrich, M. S. (1999). The transcriptional activator CorR is involved in biosynthesis of the phytotoxin coronatine and binds to the the cmaABT promoter region in a temperature-dependent manner. Mol Gen Genet 262, 250260.[CrossRef][Medline]
Xiao, Y., Lu, Y., Heu, S. & Hutcheson, S. W. (1992). Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster. J Bacteriol 174, 17341741.[Abstract]
Zhang, M., Wang, G., Shapiro, A. & Zhang, J. T. (1996). Topological folding and proteolysis profile of P-glycoprotein in membranes of mutidrug-resistant cells: implications for the drug-transport mechanism. Biochemistry 35, 97289736.[CrossRef][Medline]
Received 13 January 2004;
revised 23 April 2004;
accepted 30 April 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |