Laboratory of Physiology and Genetics of Actinomycetes, Institute of Microbiology, AS CR, Videnska 1083, 142 20, Prague-4, Czech Republic1
Author for correspondence: Pavel Tichy. Tel: +420 2 475 2356. Fax: +420 2 475 2347. e-mail:tichy{at}biomed.cas.cz
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ABSTRACT |
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Keywords: PkwA, WD-repeat protein, protein Ser/Thr kinase, phosphorylation, Thermomonospora curvata
Abbreviations: DIG, digoxigenin; MBP, maltose-binding protein; WD, Trp-Asp; WDA, WD domain of PkwA
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INTRODUCTION |
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The pkwA gene of Thermomonospora curvata CCM3352 (Janda et al., 1996 ) was the first reported example of a gene encoding a WD-repeat protein in prokaryotes. The DNA sequence indicated that it encodes a protein with seven tandem repeats of 31 amino acids, having the characteristic dipeptides GH (Gly-His) and WD (Trp-Asp). There is also a putative Ser/Thr-type kinase domain at its amino terminus, which represents the first case of a eukaryotic-type Ser/Thr kinase (for a review, see Zhang, 1996
) with a WD repeat in actinomycetes. Interestingly, Ser/Thr-type kinases found in Streptomyces spp., for instance AfsK from Streptomyces coelicolor A3(2) (Matsumoto et al., 1994
), and Pkg3, Pkg4 (Vomastek et al., 1998
) and Pkg2 (Nadvornik et al., 1999
) from Streptomyces granaticolor show a conserved region of seven tandem repeats of 1112 amino acids with similarity to the tryptophan-docking motif at their carboxy terminus, which could also fold into a propeller-like structure (Vomastek et al., 1998
; Nadvornik et al., 1999
).
In the last three years, several genes encoding WD-repeat proteins have been discovered in the genus Streptomyces, such as wdpA from S. coelicolor A3(2) (AJ131817) and wdlA from Streptomyces lincolnensis RIA 1246 (GenBank accession no. AF116463) (both cloned, sequenced and expressed in our laboratory; unpublished data). In addition, two genes (AL078635, AL021411) have been detected during the S. coelicolor A3(2) sequencing project. A recently found gene (AL136500) encodes a hypothetical protein very similar to PkwA. Putative WD-repeat-encoding genes in some other prokaryotes such as Synechocystis PCC6803 (Q55563), Thermotoga maritina (AE001822), Myxococcus xanthus (AF162663), Deinococcus radiodurans (AE000513) and Chlorobium tepidum have also been reported. However, all of these data are just nucleotide sequences; there is no direct evidence for the expression of WD-repeat proteins in any prokaryotes and hence their role in these organisms remains unknown. Nevertheless, the fact that a number of WD-repeat-protein-encoding genes is present in prokaryotes suggests that this ancient regulatory protein family can no longer be regarded as confined to eukaryotes. Since it is known that these proteins are involved in diverse functions, their very presence in prokaryotes may indicate novel regulatory systems.
In this paper we demonstrate the expression of PkwA, a WD-repeat protein, in the thermophilic actinomycete T. curvata CCM3352. We show that the expresssion of PkwA is limited to exponential phase of spore-derived growth and that the WD domain of PkwA can undergo phosphorylation in the presence of membrane fractions of T. curvata.
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METHODS |
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Preparing constructs for PkwA and WDA expression in E. coli.
A plasmid that expresses full-length PkwA (residues 1742) in E. coli with a hexa-His-tag at the carboxy terminus was constructed. An EcoRI site at the start of pkwA was generated in pMPT2 by PCR and the resulting construct was named pMPT2-E (Fig. 1). The suitable restriction sites XhoI and HindIII were also generated and the termination codon was removed from the 3' end of pkwA in pMPT2-E by PCR. The 2.2 kb EcoRIXhoI fragment carrying pkwA was then subcloned in pET24a (+) (Novagen) and named pMPT2-EX (Fig. 1
). In this construct there are an extra 14 codons from the vector at the N terminus of PkwA.
A plasmid that expresses a His-tag derivative of the WD domain of PkwA (WDA) was also constructed. The EcoRI site was generated at the 3300 position in pkwA (GenBank accession no. AF115313) by PCR using pMPT2-E (Fig. 1) as template. The amplified fragment (
1.0 kb) was digested with EcoRI/HindIII, subcloned in pET28b (+) (Novagen) and named pMPT2-E2 (Fig. 1
). This construct contains an extra 36 codons from the vector at the N terminus of the WD domain, which includes a hexa-His-tag and a T7-tag.
Expression and purification of PkwA and WDA.
Both pMPT2-EX and pMPT2-E2 constructs were individually transformed into E. coli strain BL21 (DE3) (Novagen). Transformants were grown at 37 °C in LB medium containing 50 µg kanamycin ml-1 until the optical density at 600 nm reached 0.6. Cells were induced with 1 mM (final concentration) IPTG for 3 h and then placed on ice for 5 min; they were then harvested by centrifugation at 13000 g (BiofugeA; Heraeus Sepatech). Cells were disrupted by sonication and native protein purification of both PkwA and WDA His-tag derivatives was performed on a metal affinity matrix according to the standard protocols supplied with the Talon metal affinity resin (Clontech).
Maintenance and growth curve of T. curvata CCM3352.
T. curvata strain CCM3352 was obtained from the Czechoslovak Culture Collection (Brno, Czech Republic) and maintained on SPTC agar medium as described by Janda et al. (1997) . Cultivation and growth measurements of T. curvata in mineral salt-vitamin minimal medium (MM-medium) was performed as described by Petricek et al. (1989)
.
Detection of PkwA in T. curvata CCM3352.
To examine PkwA expression in T. curvata, spores stored in 20% (v/v) glycerol at -70 °C were inoculated (108 spores in 100 ml medium) into MM-medium (Petricek et al., 1989 ) and incubated on an orbital shaker (Gallenkamp) (200 r.p.m.) at 52 °C in a 500 ml Erlenmeyer flask. Samples were collected at 6 h intervals. Pellicles were harvested by centrifugation (35000 g, 15 min) (SS34, RT5C Sorvall; Dupont), washed once with ice-cold X-buffer (20 mM Tris/HCl pH 7.5 containing 1 mM DTT, 1 mM EDTA and 0.1 mM PMSF) and stored frozen at -20 °C. The samples were resuspended in ice-cold X-buffer (
50 mg in 0.5 ml) and disrupted by ultrasonic treatment (Labsonic 2000; USA). Each cycle consisted of sonication for 1 min followed by cooling for 2 min. These two steps were repeated until the pellicles were completely disrupted. The whole sonication process was carried out on ice. The lysate was centrifuged and supernatant was separated. The crude extract was boiled for 3 min with SDS-PAGE loading buffer and the proteins were separated on 0.1% SDS/10% acrylamide PAGE gels (Laemmli, 1970
). The gel was electroblotted onto Hybond C nitrocellulose membrane (Amersham) using Tris/glycine/methanol buffer (pH 8.0) and 150 mA constant current for 2.5 h. PkwA was probed using anti-PkwA mAb 3G2 (1:100) and the signal was developed by horse-radish-peroxidase-labelled anti-mouse antibody (1:1000) (Sevac) as described in the standard ECL (enhanced chemiluminescence) protocol manual (Amersham).
Total RNA extraction from T. curvata.
Total RNA from 18, 24, 30, 36 and 48 h spore-derived cultures of T. curvata was prepared as described by Petricek et al. (1992) . The integrity of RNA was checked on agarose electrophoresis containing 2.2 M formaldehyde.
Preparation of digoxigenin (DIG)-labelled RNA probe by in vitro transcription.
The 1.4 kb EcoRIBamHI fragment (pMPT2-E, Fig. 1) from pkwA was subcloned in pGEM-4Z (Promega) and the resulting plasmid was named pGEMEB. pGEMEB was linearized by SmaI, separated by electrophoresis, isolated from the agarose gel and used for in vitro transcription. The reaction was performed according to the standard procedure supplied with the DIG RNA Labelling Kit (SP6/T7) (Roche Molecular Biochemicals) using 1 µg linearized pGEMEB. The 145 bp BamHISmaI fragment of pkwA was transcribed in the opposite direction using the T7 promoter and T7 RNA polymerase.
Northern hybridization.
Samples of total RNA (25 µg) were denatured, separated by 2.2 M formaldehyde-agarose gel electrophoresis and vacuum-blotted onto Amersham Hybond N+ membranes following the procedure of Bormann et al. (1992)
. RNARNA hybridization and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD)-based chemiluminescent detection were performed with the DIG Luminescent Detection Kit for Nucleic Acids (Boehringer Mannheim), following the standard procedure recommended by the suppliers.
cDNA synthesis and PCR amplification.
cDNA was synthesized in 20 µl of a reaction mixture using a sequence-specific primer WD-Rev2 (5' AGG GTT GTG TGT TCT TC 3') annealing at nucleotides 41504166 of pkwA (GenBank accession no. AF115313) and MMLV reverse transcriptase (Amersham Pharmacia Biotech) according to the standard procedure described in the PCR Application Manual (Boehringer Mannheim). A 5 µl aliquot from the first-strand cDNA synthesis mixture was subjected to 30 cycles of amplification by PCR. In addition to amplification buffer and template cDNA, the amplification mixture contained (in a final volume of 100 µl) 2.5 U Taq polymerase (Promega), 1.5 mM MgCl2, 250 µM of each deoxynucleotide triphosphate and 50 pmol forward primer WD-For1 (5' AAC ACG CCG TCC TCA AA 3') annealing at nucleotides 37793795 of pkwA. Reverse primer WD-Rev2 was added to a final concentration in the reaction mixture of 50 pmol.
Assessing the effect of the WD domain on Pkg2.
E. coli strain BL21 (DE3) harbouring pEX2, which encodes Pkg2, was grown and induced by a method similar to that described for PkwA and WDA. The induced E. coli cells from 1 ml culture were harvested, washed three times in ice-cold phosphorylation buffer (50 mM Tris/HCl pH 7.5 containing 50 mM NaCl, 10 mM MgCl2, 1 mM EDTA and 10 mM ß-mercaptoethanol), resuspended in 150 µl of the same buffer and disrupted by sonication. The lysate was centrifuged (18000 g) at 4 °C for 10 min. The supernatant was collected and used as the source of Pkg2. Partially purified His-tag derivatives of both PkwA and WDA were transferred in ice-cold phosphorylation buffer using microcon-30 (Amicon). PkwA, WDA and Pkg2 were subjected to autophosphorylation as controls. The reaction was carried out at 37 °C for 10 min after mixing 5 µCi (1.85x105 Bq) [-32P]ATP (Amersham Pharmacia Biotech). To assess the effect of the WD domain, PkwA (
1.0 µg) and WDA (
3.0 and
5.0 µg) were separately mixed with Pkg2 and subjected to phosphorylation. All the above reactions were terminated by adding SDS-PAGE loading buffer. After electrophoresis, gels were soaked in boiling 16% trichloroacetic acid (Mannai & Cozzone, 1982
), dried under vacuum and exposed to an image processor (Fuji film). The signal was developed with Fuji-BAS-5000 (Fuji film).
WDA phosphorylation in T. curvata.
Twenty-four-hour spore-derived pellicles of T. curvata (50 mg) were harvested and washed three times in ice-cold phosphorylation buffer (500 µl each time), resuspended in 500 µl of the same buffer and disrupted by sonication as described above. The lysate was ultracentrifuged (100000 g) at 4 °C for 60 min. The supernatant (cytoplasmic fraction) was collected separately. The sediments containing membrane fractions were washed three times in ice-cold phosphorylation buffer (500 µl each time) and resuspended in 100 µl of the same buffer. Both cytoplasmic fraction and membrane fractions were subjected to autophosphorylation as negative controls. To assess WDA phosphorylation,
2.0 µg purified WDA was mixed separately with 5 µl cytoplasmic fraction and 5 µl membrane fractions of T. curvata, and phosphorylation was carried out by mixing with 5 µCi (1.85x105 Bq) [
-32P]ATP (Amersham Pharmacia Biotech). Various conditions were used for both control and test samples, such as different cations, different temperatures and different times of incubation. Termination of reaction, electrophoresis, gel treatment, drying and signal development were performed as described above.
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RESULTS |
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Northern analysis and RT-PCR further confirm PkwA expression
To further analyse pkwA expression, a pkwA-specific DIG-labelled RNA probe, generated by in vitro transcription, was used in Northern analysis. Prior to hybridization, the specificity of the probe for pkwA was determined against linearized pGEMEB by DNARNA hybridization (Fig. 4a, lane 1). After 18 and 24 h growth, RNA from T. curvata showed a strong signal at approximately 2.4 kb, decreasing after 30 h growth (Fig. 4b
, lanes 13). These data confirm that pkwA was expressed 18, 24 and 30 h after inoculation, but that there was little or no observed expression after 36 and 48 h growth (Fig. 4b
, lanes 4 and 5).
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Pkg2 phosphorylates WDA
To examine the effect of the WD domain of PkwA on protein Ser/Thr kinase, the Pkg2 from S. granaticolor (Nadvornik et al., 1999 ) was used as a positive control. WDA, the WD domain of PkwA, was overexpressed as a His-tag derivative in E. coli BL21 (DE3), verified on Western blot probed by mAb 3G2 (Fig. 5a
, lanes 2 and 4) and partially purified using a Talon metal affinity matrix. In a control phosphorylation reaction, PkwA and WDA did not undergo autophosphorylation (Fig. 5b
, lanes 7 and 8) whereas the active Pkg2 was positively phosphorylated (Fig. 5b
, lane 3). When Pkg2 was subjected to phosphorylation in the presence of partially purified PkwA or WDA, a decrease in the autophosphorylation ability of Pkg2 was reproducibly observed (Fig. 5b
, lanes 46). At the same time, Pkg2 phosphorylated WDA (Fig. 5b
, lanes 5 and 6). To rule out the possibility that this phosphorylation of WDA was due to an E. coli endogenous kinase, WDA was incubated under the same conditions with E. coli BL21 (DE3) crude extract. However, the WDA was not phosphorylated (lane 2), thus proving that it is Pkg2 that specifically phosphorylates WDA. Fig. 5(c)
, showing a Western blot of lanes 3, 4 and 5 from Fig. 5(b)
run in parallel in the SDS-PAGE gels, demonstrates the presence of corresponding proteins in the reaction mixtures.
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Phosphorylation reactions were also performed in the presence of Ca2+ (10 mM) or Mg2+ (10 mM), alone or in combination; however, the results were negative (not shown).
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DISCUSSION |
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During the examination of the expression of PkwA on Western blots, it was found that 3G2 reacted with the exponential-growth-phase extracts of T. curvata (Fig. 3b, lanes 37) derived from spores but interestingly, no PkwA could be detected at any stage of growth initiated from vegetative mycelium (Fig. 3b
, lanes 911). This indicates possible involvement of PkwA in some crucial spore-derived developmental stage of the growth of T. curvata. The pattern of PkwA expression in spore-derived growth was further confirmed by Northern hybridization. Northern analysis showed the presence of pkwA transcript (Fig. 4b
) during early exponential growth, which was in good agreement with the results obtained by Western blot analysis. It also suggested that the transcription of pkwA could be over by the end of the early exponential phase. However, the results obtained by the more sensitive RT-PCR method (Fig. 4c
) enabled us to detect the presence of transcript during up to 36 h of growth (to late exponential phase).
The apparent molecular mass of PkwA in SDS-PAGE gels was approximately 100 kDa instead of the 80 kDa predicted from the amino acid sequence. However, when PkwA was expressed as a His-tag derivative in E. coli, it also yielded a band of about 100 kDa on Western blots (Fig. 3a, b
). Such unexpectedly slow migration of protein on SDS-PAGE gels has also been reported in other cases such as Pkg2 (Nadvornik et al., 1999
). Expression of recombinant PkwA could not be seen on a stained gel, but immunodetection using mAb 3G2 on Western blots confirmed expression (Fig. 3a
) similar to other reported cases (Matsumoto et al., 1994
; Urabe & Ogawara, 1995
).
The deduced amino acid sequence of pkwA (Janda et al., 1996 ) suggests the presence of a putative Ser/Thr-type kinase domain at the amino terminus of PkwA. However, we failed to detect autophosphorylation under various conditions. Pkg3 reported from S. granaticolor represents another such case (Vomastek et al., 1998
). According to the primary structure, PkwA and Pkg3 belong to the family of so-called RD protein kinases, which requires phosphorylation of an activation loop between domains VII and VIII for their activation (Johnson et al., 1996
). It is very likely that the kinase domain of PkwA could be phosphorylated and activated by some other kinase that could be functioning together with PkwA in T. curvata.
The WD-repeat proteins are also known for their interaction with kinases (Ron et al., 1994 , 1995
; Kolman & Egelhoff, 1997
; Kwak et al., 1997
; Datta et al., 1998
) and some can even exert an inhibitory effect (Bhalerao et al., 1999
). A sequence alignment of the WD domain of PkwA showed significant sequence similarity with other such WD-repeat proteins. We therefore wanted to examine the possible effect of WDA on active Pkg2, a protein Ser/Thr kinase from S. granaticolor (Nadvornik et al., 1999
). The aim of an attempt to autophosphorylate Pkg2 with partially purified PkwA or WDA was to prove that whatever the effect may be, it is due to the WD domain. The results showed that Pkg2 autophosphorylation was lower in the presence of PkwA or its WD domain, WDA (Fig. 5b
, lanes 4, 5 and 6). However, at the same time Pkg2 phosphorylated WDA (Fig. 5b
, lanes 5 and 6). It is very likely that the WDA could exert a dual effect, influencing Pkg2 autophosphorylation and at the same time acting as a substrate for Pkg2. This scenario would be similar to Skp2, p21Cip1/WAF1 or p27Kip1 that inhibit CDK (cycline-dependent kinase) activity and at the same time act as substrates and undergo phosphorylation (Yam et al., 1999
; Zhang et al., 1994
; Sheaff et al., 1997
). However, the WDA phosphorylation by Pkg2 encouraged us to look for the putative kinase in T. curvata. The cytoplasmic fraction and the membrane fractions obtained from 24-h-old mycelia of T. curvata were used as a possible source. When testing under various conditions in the presence of partially purified WDA, the membrane fractions of T. curvata showed a phosphorylated band of the molecular size of WDA (Fig. 5d
, lane 2). However, the control reaction with the membrane fractions alone was negative (Fig. 5d
, lane 1), implying that the phosphorylated band in the test was indeed WDA. This result was obtained in the presence of Mn2+; the reactions carried out in the presence of other cations such as Ca2+ or Mg2+, alone or in combination, were negative. This implies that the putative kinase phosphorylating WDA prefers Mn2+, as in the case of Pkn2 protein kinase of Myxococcus xanthus or Mbk of Mycobacterium tuberculosis (Udo et al., 1997
; Peirs et al., 1997
). We also observed a faint band in the test reaction carried out with the cytoplasmic fraction and WDA (Fig. 5d
, lane 4). This could be due to contamination of the membrane fractions caused by disruption of the mycelia by sonication. According to these results, if the putative kinase phosphorylating WDA in T. curvata is membrane-bound then it would be strikingly similar to Pkg2, as the latter is also membrane-spanning (Nadvornik et al., 1999
). It is tempting to speculate that PkwA could be a member of a signalling pathway occurring in early exponential stages of growth derived from spores of T. curvata. The phosphorylation of WDA and the presence of a putative protein Ser/Thr kinase domain in PkwA further strengthen this hypothesis. It is also very likely that the WD domain of PkwA could serve as a molecular switch in a phospho-relay signal transduction mechanism in T. curvata.
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ACKNOWLEDGEMENTS |
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Received 28 June 2000;
revised 4 August 2000;
accepted 30 August 2000.