Journal of Bacteriology, March 2004, p . 1330-1336, Vol . 186, No . 5

Dimerization of the RamC Morphogenetic Protein of Streptomyces coelicolor

Michael E . Hudson and Justin R . Nodwell^*

Department of Biochemistry, Health Sciences Centre, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

Received 1 October 2003/ Accepted 21 November 2003

 
RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium Streptomyces coelicolor . This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanismof action . In this study we found that the presence of multiplecopies of a defective allele of ramC inhibits morphogenesisin S . coelicolor, consistent with either titration of a targetor formation of inactive RamC multimers . We identified a domainin RamC that is C terminal to the putative kinase domain andforms a dimer with a K_d of 0.1 µM . These data suggestthat RamC acts as a dimer in vivo.

 
Germination of Streptomyces coelicolor spores results in the propagation of filamentous substrate hyphae that grow by elongating and branching, which gives rise to a colony referred to as a substrate mycelium . After 24 to 36 h of growth, colonies producea second filamentous, nonbranching type of cells called aerialhyphae that project from the colony surface . These two celltypes have different fates; the substrate hyphae produce secondarymetabolites, including many compounds that have antibiotic activity[3], while the aerial hyphae produce spores [5] . It has been demonstrated previously that the ramC gene encodes a membrane-associatedprotein having an amino-terminal serine/threonine kinase-likedomain that is required for the production of aerial hyphae[12, 23] . RamC is produced in the substrate hyphae but is absentfrom the aerial hyphae, at least by the time that spore formationhas commenced [23], and our current hypothesis is that RamCphosphorylates an unknown target protein and that this helpsdrive the formation of aerial hyphae . There is a growing bodyof evidence that intercellular signaling triggers this developmentalstep in the S . coelicolor life cycle [6, 12, 15, 20-23, 31], and it is possible that RamC is part of this mechanism.

While genetic evidence suggests that RamC is a serine/threonine kinase, it is certainly a very unusual one . Numerous genes encoding this class of kinase have been identified in various bacteria, including, in particular, the myxococci, the mycobacteria, pseudomonads, and Streptomyces [1, 2, 17, 27, 32] . The active centers of mostof these kinases are highly conserved compared to each otherand their eukaryotic counterparts . In contrast, the degree ofsequence similarity of the RamC amino-terminal domain to the amino-terminal domains of the other kinases is rather limited, and the similar region includes a 120-amino-acid element insertedin the putative nucleotide binding region [12] that has notbeen found in any other kinase discovered so far . Indeed, atpresent, the C-terminal boundary of the putative kinase domainhas not been defined with certainty, and there has been no convincingdemonstration of RamC kinase activity in vitro [unpublishedobservations].

Aside from information concerning the amino terminus there is little information regarding the structural characteristicsor mode of action that can be derived from the primary sequence.There is a notable repeated sequence C terminal to the putativekinase domain consisting of six back-to-back repeats of theconsensus sequence VDETTR; however, this does not suggest anyknown structural motif . Furthermore, there are no RamC homologueswith known functions in the genome databases; the only clearhomologues are the products of the amfT genes of Streptomycesgriseus and Streptomyces avermitilis, and both of these geneslie in gene clusters that are obviously related to the ram genes[13, 26].

We are dissecting RamC to elucidate its mechanism of actionduring morphogenesis in S . coelicolor. We report here that the presence of a defective allele of ramC on a multicopy plasmid has a partial dominant negative effect on morphogenesis of S. coelicolor, which is consistent with the possibility that RamC might act as a dimer or other higher-order complex in vivo.While the putative kinase domain of RamC did not appear to oligomerize,a short sequence C terminal to it that includes the VDETTR repeatbrought about the formation of stable dimers . Our results suggestthat RamC acts as a dimer.

 
Bacterial strains and growth conditions. Strains used for this work are listed in Table 1 . S . coelicolor was grown on R2YE media [16] at 30°C . Escherichia coli wasgrown on Luria-Bertani medium at 37°C . For two-hybrid analysis, E . coli strain DHP-1 was grown on MacConkey minimal medium [Difco] supplemented with 1% maltose for 12 to 24 h at 30°C [9, 14] . Ampicillin and chloramphenicol were used at concentrationsof 100 and 25 µg/ml, respectively . Thiostrepton was usedat a concentration of 50 µg/ml.

 
Dominant negative mutant. Wild-type ramC and the inactive mutant ramCD369A gene were excisedfrom plasmids pTO8 and pTO8-D369A by using NheI and HindIII.The fragments were cloned into plasmid pIJ922 [Table 2] at the XbaI and HindIII sites . The resulting constructs were designated pRamC and pRamCD369A . These plasmids were passed through the nonmethylating strain Er^2-1 and transformed into S . coelicolor[16].

 
Bacterial two-hybrid system. Oligonucleotides [Table 3] were used to amplify segments oframC and introduce BamHI restriction sites on either end forcloning into pT18Bam and pT25 . The resulting constructs, pT18-N,pT18-R, and pT18-C, fused ramC segments to the 5' end of theT18 portion of cyaA; constructs pT25-N, pT25-R, and pT25-C fusedthe same segments to the 3' end of the T25 portion of cyaA. Combinations of these constructs were introduced into the cyaE . coli strain DHP-1 and were analyzed by using the MacConkey indicator medium [as described by Eccleston et al . [9] and Karimovaet al . [14]].

 
Repeat domain fusion protein. The segment of ramC encoding the repeat [rep] domain was amplifiedby using oligonucleotides MBP-rep-top and MBP-rep-bot [Table3], which introduced an EcoRI restriction site and a PstI restriction site at the 5' and 3' ends of the DNA fragment, respectively. This construct was then introduced into pMAL-c2X to generate pMAL-rep.

Cultures of E . coli strain ER2508 carrying pMAL-rep were grown at 37°C to an optical density at 600 nm of 0.6 . Expressionof maltose binding protein [MBP]-rep was induced with 1 mM isopropyl-ß-D-galactopyranoside at 37°C for 3 h . Cells were harvested by centrifugation,washed in 100 mM Tris [pH 8.0], and resuspended in buffer A[100 mM HEPES [pH 7.4], 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol]containing 1 mM phenylmethylsulfonyl fluoride . Cells were lysedby three passes through a French press, and cell debris wasremoved by centrifugation at 20,000 x g for 30 min . The cleared lysate was applied to a 10-ml amylose resin column [New England Biolabs] by using an Akta Prime fast protein liquid chromatograph [Amersham Biosciences] and a flow rate of 1.0 ml/min . The columnwas washed with 11 column volumes of buffer A, and bound proteinswere eluted by using buffer B [buffer A containing 10 mM maltose]at a flow rate of 1.5 ml/min . The presence of MBP-rep in eachfraction was determined by sodium dodecyl sulfate [SDS]-polyacrylamidegel electrophoresis [PAGE] . Fractions containing the fusionprotein were pooled and concentrated by using Ultrafree-15 centrifugationfilters [Millipore] . The concentrated protein was applied toa Superdex-200 gel filtration column [Amersham Biosciences]equilibrated with buffer A at a rate of 0.2 ml/min . Fractionscontaining MBP-rep were pooled, dialyzed against buffer C [25mM HEPES [pH 7.4], 200 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA]containing 20% glycerol, and stored at -20°C until theywere used . Protein concentrations were determined by the Bradfordassay [Bio-Rad] by using bovine serum albumin as the standard.MBP was purchased from New England Biolabs . The protein was dialyzed against buffer C with glycerol and stored at -20°C until it was used.

Gel filtration. A Superdex-200 analytical gel filtration column [Amersham Biosciences]was calibrated with ferritin [440 kDa; Stokes radius [ r^S] =6.1 nm], aldolase [158 kDa; r^S = 4.8 nm], albumin [67 kDa; r^S = 3.5 nm], ovalbumin [43 kDa; r^S = 3.0 nm], chymotrypsinogen A [25 kDa; r^S = 2.1 nm], and RNase A [13.7 kDa; r^S = 1.6 nm]in buffer A at a flow rate of 0.2 ml/min by using a Beckman high-performance liquid chromatograph . The void volume was determined by using blue dextran 2000 . Protein elution was monitored at 280 nm, and a standard curve of the elution volume parameter[K_av] versus r^S was determined by using the equation K_av= [V_e- V_o]/[V_t - V_o], where V_e is the elution volume, V_t is the totalcolumn volume, and V_o is the void volume . To determine the r^Sof the fusion protein, 90 µl of a solution containing0.5 mg of MBP per ml and/or 0.5 mg of MBP-rep per ml was appliedto the column and developed as described above . The K_av andapparent r^S of MBP and MBP-rep were determined from the standardcurve.

Chemical cross-linking. MBP and MBP-rep were subjected to chemical cross-linking byusing the homobifunctional cross-linking agent dimethyl suberimidate[DMS] [Pierce] . MBP or MBP-rep at a concentration of 5 µMwas mixed with DMS at a concentration of 10 to 1,000 µMin 20-µl reaction mixtures on ice for 30 min . MBP and MBP-rep at a concentration of 5 µM were also reacted with100 µM DMS on ice for 0 to 120 min . Each reaction wasstopped by addition of Tris buffer [pH 8.0] to a concentrationof 50 mM, and the mixture was analyzed by SDS-PAGE.

Analytical ultracentrifugation. Sedimentation equilibrium analysis was performed with a Beckman-CoulterXL-A analytical ultracentrifuge, a four-cell An-60 Ti rotor,and six-channel Epon-charcoal cells with sapphire windows at4°C; 110-µl samples of MBP-rep in buffer C were analyzedat concentrations corresponding to A₂₈₀ values of 0.27, 0.2,and 0.1 [which corresponded to 1.45, 1.1, and 0.55 µM]and rotor speeds of 5,000, 10,000 and 15,000 rpm . For reference,125 µl of buffer C was used in each reference cell . Concentrationgradients were observed at 280 nm by using a radial step sizeof 0.001 and five scan repetitions . Centrifugation was carriedout for 16 h, and the equilibrium state was confirmed by comparingabsorbance scans obtained at 15 and 16 h . Data for all rotorspeeds and protein concentrations were analyzed by using Beckmananalysis software based on the Origin 6.0 package [Microcal].To model the experimental data as either a single ideally interactingspecies or as an equilibrium of a monomer and higher-order complexes,a self-association model was used [29] . The protein partialspecific volume [0.725 ml/g] and solvent density [1.008 g/ml]were estimated by using the program SEDNTERP.

To determine the molar dissociation constant [K_d] of the MBP-repcomplex, the concentration-dependent association constant was derived by using the following equation: K_a[conc] = K_a[abs][l/2], where K_a[conc] is the per molar association constant, K_a[abs]is the absorbance-based association constant derived by usingthe Beckman software package, is the calculated molar extinctioncoefficient [70,410 cm^-1 M^-1], and l is the path length of thesample cell [1.2 cm] . K_d was calculated by taking the inverse of K_a[conc] [29].

 
Previously, it was found that the presence of a single copyof a defective allele of ramC in a morphologically wild-typestrain of S . coelicolor had no effect on morphogenesis [12], suggesting that balanced levels of active and inactive RamC variants permitted normal function . To determine whether thiswas true if there was an excess of the inactive polypeptide,we introduced ramC or a ramC allele containing the D369A mutation in the putative kinase domain into a variant of the vector pIJ922 to produce plasmids pramC and pramCD369A . Both alleles were expressed from the ramC promoter . pIJ922 contains the SCP2* origin of replication and has a copy number of approximately five relative to the S . coelicolor chromosome [16] . When introducedinto the ramC null strain N373 [23], pramC, but not a controlplasmid, complemented the developmental defect, as expected[data not shown] . In the morphologically wild-type parent strainM145, pIJ922 and pramC had no discernible effect on morphogenesis;however, the presence of pramCD369A caused a reproducible delayin the formation of aerial hyphae [Fig . 1] . This could be consistent either with titration of a target protein by inactive RamC or,if RamC formed a dimer or other higher-order complex, with titrationof functional RamC into complexes with the defective RamCD369A polypeptide . Any such heteromeric complexes of RamC and RamCD369A must have retained some activity, however, because after incubation for a longer time the cells growing on the plate shown in Fig. 1c formed aerial hyphae.

 
RamC [Fig . 2] can be divided into possible functional domains based on sequence motifs and results of previous work [12]. To determine whether any of these domains had the capacity to assemble into a higher-order complex, we fused segments of the ramC gene encoding the 420 amino acid residues at the amino terminus [N fragment], 64 residues containing the VDETTRrepeat sequence [R fragment], and the 399 residues at the carboxy terminus [C fragment] in frame to the two vectors of a two-hybrid system[14] . This two-hybrid system was based on the fact that expressionof the maltose utilization genes [mal] depends on the presenceof cAMP in E . coli and the fact that the Bordetella pertussis cyaA gene can complement an E . coli cya mutant . The B . pertussis adenylate cyclase enzyme can be split into two nonfunctional fragments [T18 and T25] that, when expressed in vivo as fusionsto polypeptides that interact with one another, can be broughttogether to restore enzymatic activity . This can be readilydetected by a pink colony phenotype on MacConkey medium containingmaltose [14].

 
When plasmids pT18Bam and pT25 [which encode carboxy- and amino-terminal fragments of the B . pertussis adenylate cyclase] were introduced into the E . coli cya mutant DHP-1 [Table 1] and the resultingstrain was plated on MacConkey medium containing maltose, therewas no evidence of maltose fermentation, as expected . We introducedall possible combinations of pT18-N, pT18-R, and pT18-C withpT25-N, pT25-R, and pT25-C into DHP-1 and determined the capacityof the resulting strains to metabolize maltose . Combinations of the N and C fragments with each other or themselves did not restore a Mal⁺ phenotype to DHP-1, suggesting that none of the resulting fusion proteins had the capacity to interact with each other . However, when pT18-R and pT25-R were combined inDHP-1, the resulting colonies exhibited a weak but reproducibleMal⁺ phenotype . Combinations of either pT18-R or pT25-R withthe N or C fusions did not result in a Mal⁺ phenotype, suggesting that this was a specific property of the R fusions . Finally, combinations of pT18-R and pT25-R with pT18Bam or pT25 did notallow maltose utilization, indicating that the interaction didnot involve either fragment of adenylate cyclase but was againspecific for the R fusions . These data [summarized in Table4] suggested that the R fragment of RamC was able to form ahigher-order complex with itself.

 
To examine this effect in vitro, we created a gene fusion ofa ramC segment encoding residues 441 to 555 [the rep fragment, which was larger than the R fragment used in the two-hybridanalysis [see Fig . 2 and 6]] to the E . coli gene malE in thecontext of plasmid pMAL-c2X to create an expression vector for the fusion protein MBP-rep [Fig . 2] . The product of malE, MBP,is a monomer in solution, so we determined whether the rep fragmentcaused it to form a higher-order complex . We therefore determinedthe r^S values of purified MBP and MBP-rep by gel filtrationchromatography . As shown in Fig. 3 [upper panel], MBP elutedfrom a Superdex-200 column at 80 min, at a position betweenovalbumin [43 kDa; r^S = 3.0] and chymotrypsinogen [25 kDa; r^S= 2.1], which is consistent with its known molecular mass [43kDa], and gave a calculated r^S of 3.0 nm . These data are consistent with the fact that MBP is a monomer . In contrast, the MBP-repfusion, which has a calculated molecular mass of 55 kDa, elutedfrom a Superdex-200 column at 62 min, between ferritin [440kDa; r^S = 6.1 nm] and aldolase [158 kDa; r^S = 4.8 nm] [Fig. 3, middle panel] . The derived r^S value, 5.8 nm, is much largerthan the value expected for a monomeric 55-kDa protein, suggestingeither that the fusion protein had formed a nonspecific aggregate,that it was unfolded, or that it had formed a specific higher-ordercomplex . We believe that the shoulder on the MBP-rep peak [Fig.3, middle and lower panels] contained partially degraded protein,which could not be completely eliminated during purification.

 
The apparent oligomerization of MBP-rep could have been dueeither to a specific interaction of rep with itself or to anonspecific interaction with MBP . To distinguish between thesepossibilities, we carried out an experiment in which a 1:1 mixtureof MBP and MBP-rep was analyzed by gel filtration . The two polypeptideseluted from the column in discrete peaks at 80 and 62 min, respectively[Fig. 3, lower panel]; no intermediate peak was observed, and SDS-PAGE analysis confirmed that each peak contained exclusively MBP or MBP-rep [data not shown], suggesting that no heterooligomers of MBP and MBP-rep had formed . Therefore, the apparent oligomerization of MBP-rep was likely due to specific interactions of the RamC rep fragment with itself . The behavior of proteins and protein complexes during gel filtration chromatography is sensitiveto the shape of the protein or complex; hence, the results shownin Fig. 3 did not accurately reveal either the molecular weight or stoichiometry of the apparent MBP-rep complexes . Indeed, while the data were consistent with the formation of a higher-order complex by MBP-rep, they could also have suggested that thefusion was simply a very asymmetric molecule, a property thatwould result in excessively large r^S values [4].

To determine whether a complex was formed, we carried out a cross-linking experiment with the reagent DMS . Various amountsof DMS and 5 µM MBP were mixed together and allowed toreact . After 30 min the products were electrophoresed on anSDS-PAGE gel, and the gel was stained with silver . As shownin Fig . 4 [upper panel], addition of DMS to MBP had little orno effect on its subsequent migration on an SDS-PAGE gel evenat a molar ratio of DMS to protein of 200:1, which is consistentwith the monomeric nature of MBP . In contrast, when DMS wasadded to MBP-rep, a relatively modest molar ratio of DMS tothe polypeptide [2:1 to 5:1] induced the formation of a covalentcomplex that migrated more slowly on SDS-PAGE gels . The formationof this cross-linked species was relatively inefficient; increasingthe amount of DMS resulted in proportionate increases in thecross-linked product, but the preparation never reached saturationeven with a vast molar excess of DMS compared to the amountof MBP-rep . We also examined time course variation in this experiment[Fig . 4, lower panel] and observed the same cross-linked species.We presumed that the inefficiency of cross-linking reflectedthe scarcity or orientation of DMS-reactive residues in therep region of the fusion protein; DMS reacts with primary amines,and there is only one of these in the rep fragment . The fiveto seven minor cross-linked species surrounding the major bandmay have represented cross-links between full-length MBP-rep and partial proteolyzed protein . We do not believe that anyof the bands reflected trimers, tetramers, or higher-order complexes because if this were the case, we would have expected a progression from lower-molecular-weight species to higher-molecular-weight species as cross-linking proceeded . We suspect, therefore, thatall of the cross-linked species shown in Fig . 4 are dimers of MBP-rep.

 
Finally, to determine the stoichiometry of the MBP-rep complex,we carried out an equilibrium sedimentation experiment withpurified MBP-rep . This technique yields a precise mass measurementthat is independent of a protein's or protein complex's shapeand therefore allows precise assignment of stoichiometry [7]. Figure 5 shows sample data for an experiment carried out at 15,000 rpm in which MBP-rep at concentrations of 0.55, 1.1,and 1.45 µM were used . The data for MBP-rep were fittedto the expected curves for a monomer, a dimer, a trimer, a tetramer,and a pentamer of a 55-kDa protein . MBP-rep's behavior was anexcellent match for the behavior predicted for a dimer in allthree curves . The residual plot described above showed the positionof points relative to the origin, corresponding to the positionsrelative to the curve predicting the behavior of a dimer of55-kDa proteins . The random distributions of points above andbelow the origin reflected the strong correlation of these datawith the dimeric state . The results of experiments performedby using 5,000 and 10,000 rpm [data not shown] were virtuallyidentical to those shown in Fig . 5, demonstrating that therewas a rep fragment-induced dimer rather than any other oligomericstate or a monomer.

 
The equilibrium sedimentation data were used to derive an absorbance-based association constant [K_a[abs]] for the MBP-rep dimer of 206and therefore an association constant [K_a] of 8.7 x 10⁶ M^-1 and a dissociation constant [K_d] of 115 nM [see Materials and Methods] . We noted that these values are consistent with our observation that all of the MBP-rep behaved as a dimer duringthe gel filtration experiment [Fig . 3] as the protein was applied to the gel filtration column at a concentration of 9 µM.

RamC overexpressed in E . coli was irreversibly insoluble in our hands, and this prevented us from testing the full-lengthprotein for dimer formation . Nevertheless, taken together, ourdata demonstrate that the rep fragment of RamC is an efficient dimerization motif and therefore suggest that full-length RamCis also dimeric in nature . This in turn is consistent with amodel in which the delay in morphogenesis induced by the presenceof multiple copies of the ramCD369A allele is caused by thepresence of heterodimers of wild-type RamC with RamCD369A andhomodimers of RamCD369A . We concluded, therefore, that idealRamC function requires assembly of homodimers of the activeprotein.

Dimer formation and autophosphorylation are common themes inthe biochemistry of protein kinases [8, 10, 11, 18, 19, 24,25, 28, 30] . At present, we do not know the role of RamC dimerization;however, it is possible that the in vivo activity of RamC involvesautophosphorylation or phosphorylation of a dimeric target.A most intriguing question concerns the role of the C-terminalhalf of the protein . We presume that the activity of this portionalso depends on dimer formation.

 
We thank Tamara O'Connor for critical reading of the manuscriptand Huy Nguyen for technical assistance.

This work was supported by an Ontario graduate scholarship toM.H . J.N . was supported by a new investigator award and by operatinggrant MT-15108 from the Canadian Institutes for Health Research.

 
* Corresponding author . Mailing address: Department of Biochemistry, McMaster University, Health Sciences Centre, 1200 Main Street W., Hamilton, Ontario, Canada L8N 3Z5 . Phone: [905] 525-9140, ext . 27335 . Fax: [905] 522-9033 . E-mail: nodwellj@mcmaster.ca.

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