Characterization of the Gene for a Protein Kinase Which Phosphorylates the Sporulation-Regulatory Proteins SpoOA and

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1989, p /89/ $02.00/0 Copyright C) 1989, American Society for Microbiology Vol. 171, No. 11 Characterization of the Gene for a Protein Kinase Which Phosphorylates the Sporulation-Regulatory Proteins SpoOA and SpoOF of Bacillus subtilist MARTA PEREGO, SHERILYNN P. COLE, DAVID BURBULYS, KATHLEEN TRACH, AND JAMES A. HOCH* Division of Cellular Biology, Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, North Torrey Pines Road, La Jolla, California Received 24 April 1989/Accepted 28 July 1989 The kina (spoiij) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the SpoOA and SPOOF sporulation regulatory proteins. SpoOF protein was a much better phosphoreceptor for this kinase than SpoOA protein in vitro. Mutants with deletion mutations in the kina gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrb gene or on the timing of subtilisin expression and therefore did not impair the repression function of the SpoOA protein. Placement of the kina locus on a multicopy vector suppressed the sporulation-defective phenotype of spoob, spooe, and spoof mutations but not of spooa mutations. The results suggest that the spoob-, spooe-, and spoof-dependent pathway of activation (phosphorylation) of the SpoOA regulator may be by-passed through the kina gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the SpoOA protein. Sporulation in Bacillus subtilis is regulated by a complex program of developmental gene expression coupled with a precise series of intracellular structural events that together result in an environmentally impervious dormant body. Sporulation generally begins under cultural conditions that are not conducive to vegetative growth. The initiation of the sporulation process depends on the successful action of the products of the spoo genes (12). The spoo gene products are responsible for transduction to the transcription machinery of the metabolic signals that induce sporulation. One major role of the spooa gene product is to release repression of stationary-phase gene expression brought about by the AbrB protein (26, 29). This is accomplished by activation of the SpoOA protein, which becomes a repressor of the synthesis of the AbrB repressor (Strauch, Spiegelman, and Hoch, unpublished data). Studies of the primary structure of the SpoOA and SpoOF proteins revealed that the SpoOF protein was homologous to the amino-terminal half of the SpoOA protein (7, 31). These closely related regions are characteristic of the homology between receiver proteins in two-component transmitterreceiver regulatory systems (15, 24). It follows then that the SpoOA and SpoOF proteins may be the recipients of signals from transmitter proteins. Ninfa and Magasanik (22) discovered that the mechanism of signal transmission from the nitrogen-regulatory transmitter NtrB to the receiver protein NtrC was phosphorylation of the latter by the former in the presence of ATP. Similarly, the activation of chemotaxis proteins CheB and CheY by the CheA transmitter protein is a phosphorylation event (10). The transmitter protein NtrB can phosphorylate the chemotaxis receiver proteins, and the * Corresponding author. t Publication no BCR from the Department of Molecular and Experimental Medicine chemotaxis transmitter protein CheA can phosphorylate the NtrC receiver (23). These enzymatic results suggest that all two-component regulatory systems identified by homology to transmitters and receivers may function by a phosphorylation mechanism and that the observed homologies are associated with the kinase and phosphoacceptor functions. Although the potential receiver proteins SpoOA and SpoOF had been identified among the spoo genes, no spoo gene product has been found that had the characteristic sequence of a transmitter protein. In this report we show that the spoiij gene (renamed kina, kinase) codes for a protein with homology to transmitter kinases and that the kina gene product can phosphorylate both the SpoOA and SpoOF proteins. While this work was in its early stages, Stragier and co-workers (International Spores Conference, Woods Hole, Mass. April 1988) first showed that the spoiij gene coded for a protein which was homologous to the transmitter class of proteins. MATERIALS AND METHODS Bacterial strains and growth conditions. The Bacillus subtilis strains used in this study are shown in Table 1. Transformations were performed by the method of Anagnostopoulos and Spizizen (1) with 1 to 2 jig of plasmid DNA or 50 to 100 ng of chromosomal DNA. Transformants were selected on Schaeffer agar plates supplemented with chloramphenicol (5,ug/ml). Selection for the macrolide-lincosamide-streptogramin B (MLS) resistance marker conferred by transposon Tn917 was done as described by Youngman et al. (32). The efficiency of sporulation was tested by inoculating a single colony in 5 ml of Schaeffer sporulation medium supplemented with chloramphenicol (5 jig/ml) in the case of strains carrying replicative plasmids; cultures were grown for 24 h

2 6188 PEREGO ET AL. TABLE 1. Bacterial strains used in this study Strain Relevant genotype Source or reference W168 Wild type This laboratory JH642 trpc2 phe-j This laboratory JH646 trpc2 phe-l spooa12 This laboratory JH647 trpc2 phe-1 spoell This laboratory JH648 trpc2 phe-1 spoob136 This laboratory JH649 trpc2 phe-j spoof221 This laboratory JH651 trpc2 phe-1 spooh81 This laboratory KS19 spoiij::tn917fihu19 27 JH12638 trpc2 phe-j spoiij::tn9j7fihu19 This laboratory JH12315 trpc2 phe-j amye::[apre'sg lacz pc194 cat] JH12604 trpc2 phe-j amye::[abrb'pjm5155- Strauch et al.a lacz pc194 cat] JH12678 trpc2 phe-j spoiij::tn917qihu19 This laboratory amye::[apre'sg35.1-lacz pc194 cat] JH12679 trpc2 phe-j spoiij::tn917fqhu19 This laboratory amye::[abrb'pjm5155-lacz pc194 cat] a Strauch et al., submitted for publication. at 37 C. Serial dilutions were plated before and after treatment with CHC13 in order to obtain the viable cell count and the spore count. Escherichia coli DH5a competent cells were purchased from Bethesda Research Laboratories and used for plasmid constructions. Transformants were selected on LB supplemented with ampicillin at 100 p.g/ml or J. BACTERIOL. chloramphenicol at 10,ug/ml. The vectors used were ptv20 and ptv21a2 (32), the integrative vectors pjh101 (6) and pjm103 (26), and the shuttle vector pbs19 (G. Gray, unpublished). DNA techniques. Plasmid DNA preparation from E. coli, chromosomal DNA isolation from B. subtilis, and DNA manipulations were done as described previously (26). Cloning of chromosomal DNA fragments flanking the Tn917 insertion junctions in plasmids pjmecoptv20 and pjmecoptv21a2 was done as described by Youngman et al. (32) and Perego et al. (26). Plasmids pjm8100 and pjm8110 were cloned by the colony hybridization technique as described by Maniatis et al. (19). To prepare pjm8100 and pjm8110, 20,ug of chromosomal DNA was digested to completion with BclI and ClaI, respectively. Digests were run on a 0.75% agarose gel. BclI fragments of approximately 3 kilobases (kb) were electroeluted, phenol extracted, ethanol precipitated, resuspended in TE buffer, and then ligated into BamHI-cut pjm103. ClaI fragments of 1.7 kb were subjected to the same procedure and then cloned in AccI-cut pjm103. The fragments carried by plasmids pjm20/2 and pjm8104 were used as nick-translated probes. Plasmid pjm8115 was constructed by cloning the purified fragments from plasmids pjm8104 and pjm8103 upstream and downstream, respectively, of the cat gene from plasmid pjm1o5c, a derivative of Bluescript vector (Stratagene) carrying the Cmr marker from pc194 (13) in the EcoRV site of the multilinker (Perego and Hoch, manuscript in preparation). Sequence analysis. The supercoil sequencing method of Chen and Seeburg (3) was used in order to sequence the Downloaded from -o -.2 m I I I.. orf X U - ~~~~t:4 - < ~~~~ o ci co o Q E -a X 0cn3 L LU UI r( I4 I- 1-4 = ffi s ffi r pjm 8110 pjm 8113 pjm 8112 pjm 8122 pjm pjm 8116 AI kin A pjm 8111 pjm81 15 cat,- I 0 _3 1 Oz Cn CnE X~ = xco) I I I V I a I orf Y pjm Eco PTV20 ii pjm 20/2 pjm 8100 pjm 8102 pjm pjm 8106 I -i l pjm 8105 pjm 8101 l 1- -~ w a 0 a a a n m A m w I I Al I Ia ai I i---- II a I %.L- I 500 bp on January 26, 2019 by guest FIG. 1. Restriction map, plasmids, and sequencing strategy for kina. The arrows at the top show the extent of each sequencing reaction used to obtain the final sequence. The approximate location of the original transposon mutation is shown. The inserts in the various plasmid vectors are shown at the bottom.

3 VOL. 171, 1989 PHOSPHORYLATION OF B. SUBTILIS SpoOA AND SpoOF 6189 chromosomal region carried by plasmid pjm8111. Sequenase (U.S. Biochemical Corp.) or T7 polymerase (Pharmacia) was used in the dideoxy chain elongation reactions. The sequencing strategy used is shown in Fig. 1. I-Galactosidase assay.,-galactosidase activity in strains harboring the abrb or the apre transcription fusions to lacz was assayed as previously described (5). The specific activity was expressed in Miller units (21). KinA purification. A KinA expression vector, pjm8118, was constructed by placing the chromosomal region carried by plasmid pjm8111 (Fig. 1) under control of the inducible tac promoter contained in plasmid pkqv4 (29). E. coli JM109 cells bearing plasmid pjm8118 were grown in Luria broth containing ampicillin (100 jig/ml) at 37 C with shaking. When the cells reached an OD540 of 1.0, IPTG (isopropylthio-p-d-galactoside; Stratagene) was added to a final concentration of 2 mm, and the incubation was continued for 2 h. The bacterial cells were pelleted by centifugation at 10,000 x g in a Sorvall GS-3 rotor for 15 min. The cell pellet was washed twice in 1/10 culture volume of buffer A (25 mm Tris hydrochloride [Tris-HCl, ph 8.0], 0.1 mm EDTA, 1 mm MgCl2, 10 mm KCl, 1mM CaCl2, 7 mm P-mercaptoethanol, 20,g of phenylmethylsulfonyl fluoride [PMSF] per ml) and resuspended in 20 ml of the same buffer. Lysozyme (Sigma) was then added to the resuspended cell pellet to a final concentration of 100,ug/ml, and the mixture was incubated on ice for 30 min. The resulting protoplasts were then sonicated in an ice-water bath for 10 cycles of 30-s bursts separated by 1-min rests. After centrifugation at 43,500 x g for 1 h in a Sorvall SS34 rotor, the supernatant was removed and diluted with buffer A to a final protein concentration of 20 mg/ml as determined by the Warburg-Christian method (18). Solid AmSO4 was slowly added to this crude extract to a final concentration of 10%. After gentle stirring at 4 C for 15 min, this suspension was allowed to sit on ice without stirring for an additional 15 min before being centrifuged at 43,500 x g for 15 min. The supernatant was removed, once again solid AmSO4 was slowly added to a final concentration of 40%, and the suspension was treated as above. The resulting 10 to 40%o AmSO4 cut was resuspended in 2 ml of buffer A and dialyzed overnight against 1 liter of buffer A with one change after 4 h. The desalted protein sample was applied to an AffiGel Blue column (3 by 14 cm; Bio-Rad Laboratories) that had been equilibrated with 5 volumes of buffer A. The column was washed with 4 volumes of buffer A plus 250 mm KCI, followed by 3 volumes of buffer A plus 2 M KCl. The protein eluted in a very wide peak during the 2 M KCI wash. The KinA-containing fractions (approximately 200 ml), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were pooled, dialyzed against 4 liters of buffer A overnight, and applied to a DEAE-Trisacryl M (3 by 16 cm; IBF-Biotechnics) column which had been equilibrated with buffer A. The column was washed with 3 volumes of buffer A plus 100 mm KCl and eluted with a 6-column-volume shallow gradient from 100 to 300 mm KCl in buffer A. The KinA-enriched fractions as determined by SDS-PAGE were pooled and concentrated to a volume of 2 ml by repeated centrifugation at 15,000 x g in an Amicon Centriprep 10 concentrator. Glycerol was added to 25% final concentration, and the protein was stored at -70 C. The KinA protein was determined to be approximately 80 to 85% pure by Coomassie staining of an SDS-polyacrylamide gel (see Fig. 5). SpoOA and SpoOF proteins were purified from E. coli cells containing pkqv4 derivatives by procedures to be published (K. Trach, D. Burbulys, and J. Hoch, manuscript in preparation). Phosphorylation assay. The in vitro phosphorylation assay procedure used was that of Keener and Kustu (14) with some modifications. The phosphorylation reaction mixtures contained 175 nm KinA and 3.5 pum SpoOA or SpoOF protein in 130 mm Tris-HCl (ph 8.0)-40 mm KCI-4.5 mm MgCl2-4.0 mm CaCl2-3.5 mm,-mercaptoethanol-0.5 mm dithiothreitol-0.4 mm EDTA-12.5% (vol/vol) glycerol-10,ug of PMSF per ml. The reactions were initiated by the addition of 30,uCi of [,y32p]atp (7,000 Ci/mmol; ICN Biomedicals, Inc.) diluted in an equimolar mixture of ATP and MgCl2 to give a final ATP concentration in the assays of 85,uM. The reactions were incubated at 37 C for 1 h, mixed with 0.2 volume of a 5x protein gel buffer (17), and immediately frozen on dry ice until just prior to loading on a 15% polyacrylamide gel (17). The samples were not boiled prior to SDS-PAGE. The undried gel was exposed to Kodak XRP-5 film for 2 h at -70 C with one intensifying screen. RESULTS Cloning and characterization of the Tn917 insertion region. The Tn917 transposon mutation in B. subtilis KS19 was first characterized as an insertion either in the stage 0 sporulation gene spooe or in the stage II gene spoiif at position 130 on the chromosomal genetic map (27). We first used this transposon insertion mutant in an attempt to clone the spooe gene. Chromosomal DNA from strain KS19 transformed with linearized plasmids ptv20 and ptv21a2 was used to recover DNA fragments adjacent to both Tn917 insertion junctions as described previously (26, 32). Two plasmids, pjmecoptv20 (Fig. 1) and pjmecoptv21a2 (not shown), carrying a 2.2-kb and a 4.5-kb fragment, respectively, were obtained. These plasmids were used in transformation studies in order to determine the presence or absence of the spooe locus. Neither plasmid pjmecoptv20 nor plasmid pjmecoptv21a2 was able to transform the B. subtilis spooe mutant strains (25) to a sporulation-proficient phenotype, indicating that neither of the plasmids contained this locus. A genetic linkage map was made by integrating plasmid pjm20/2 (a derivative of pjmecoptv20 in the vector pjh101, Fig. 1) in the wild-type strain W168. With this chromosomal DNA as a donor in transformation, the linkage between the spooe marker and the chloramphenicol resistance marker conferred by the integrated plasmid was checked. We observed 97% recombination between the Cmr and the spooe markers. These results supported the conclusion that pjmecoptv20 and pjmecoptv21a2 were not carrying the spooe locus. Cloning the chromosomal region carrying the kina gene. Southern blot analysis on W168 chromosomal DNA with the fragment carried by pjm20/2 as the probe was performed (data not shown) in order to find a restriction fragment that would overlap the site of insertion of the Tn917fHU19 transposon. A BclI-BclI fragment of approximately 3 kb was identified and subsequently cloned (see Materials and Methods) in the BamHI site of the vector pjm103, giving plasmid pjm8100. Transformation analysis showed that 50% of the Cmr colonies obtained after integration of pjm8100 via Campbell-type recombination in strain JH12638 containing the original transposon mutation were sporulation proficient. This result shows that the transposon insertion-inactivated gene, which has a sporulation-deficient phenotype, was not

4 6190 PEREGO ET AL. entirely present on this plasmid. If the entire transcription unit for this gene had been present on the donor plasmid, 100% of the Cmr transformants should have been Spo+ by complementation. After further genetic analysis with plasmid subclones of pjm8100, we observed that integration of plasmids pjm8103 and pjm8104 in strain JH642 resulted in a Spo- phenotype, indicating that the fragments carried by these plasmids were internal to the gene of interest. On the other hand, plasmids pjm8100, pjm8102, and pjm8106 did not affect the sporulation ability of the same strain, implying that one end of the gene (or its transcription unit) had to be located between the Dral site and the XmnI sites of plasmid pjm8106. These results were obtained by microscopic analysis of bacterial colonies grown on Schaeffer sporulation agar medium. No endospores were observed in strain JH642 carrying plasmids pjm8103 and pjm8104, and approximately 40 to 60% endospores were observed in the parental strain JH642 and in strains JH642(pJM8102), JH642(pJM8106), and JH642 (pjm8100) after 18 h of growth. Further observation after 42 h of growth on solid medium revealed the presence of approximately 10% endospores in the pjm8103- and pjm8104-harboring strains and 70 to 80% spores in the remaining strains. All strains gave 70 to 80% spores after 72 h of growth, implying a delayed-sporulation phenotype for the mutant strains. We were unable to obtain Cmr integrants of plasmids pjm8101 and pjm8105, suggesting the presence of a gene which is essential for cell growth. Since plasmid pjm8104 was internal to the sporulationassociated gene, this chromosomal region was mapped by Southern blot analysis (data not shown) in order to obtain a flanking-region clone. The adjacent region was cloned on a 1.7-kb ClaI-ClaI fragment in the AccI site of pjm103, giving plasmid pjm8110. Neither this plasmid nor its derivatives pjm8112 and pjm8113 gave an Spo- phenotype after integration in strain JH642, which implied that the transcription unit for the unknown sporulation gene had to start somewhere inside the fragment carried by pjm8112. Plasmid pjm8111, which was constructed by fusing pjm8100 and pjm8110 at the AvaI site, restored sporulation to 100% of the Cmr transformants obtained after integration of this plasmid in strain JH12638, supporting the proposed location of the transcription unit. Sequence analysis. Sequence analysis of the chromosomal region carried by plasmid pjm8111 was performed as described in Materials and Methods (Fig. 2). A major open reading frame (ORF) was identified in the region that was shown by genetic analysis to contain the unknown sporulation-associated gene. This ORF coded for a protein of 606 amino acids with a total molecular mass of 69,170 daltons (Da). Comparison of the sequence of the protein with those of other known proteins in the GenBank data base revealed a high level of homology in the C-terminal region with proteins such as PhoR, NtrB, and EnvZ, which are known to be part of two-component regulatory systems that respond to environmental stimuli (24). This observation suggested that the major ORF identified in plasmid pjm8111 could also code for a protein kinase. This gene was designated kina based on the results described below. The sequence of pjm8111 also showed the presence of a protein-coding sequence upstream of kina which did not have significant homology with proteins in the GenBank data base. Furthermore, its inactivation by integration of plasmid pjm8113 via Campbell-type recombination did not affect sporulation. This gene was followed closely by a strong stem-loop structure with a possible role as a terminator. The J. BACTERIOL. terminator recognition program of Brendel and Trifonov (2) did not flag this sequence as a terminator, however. Nevertheless, it is unlikely that kina is cotranscribed with this gene, based on the plasmid integration studies described above. A third ORF was identified downstream of the kina gene on the opposite strand. This protein of 393 amino acids (43,549 Da) had some homology (Fig. 3) with the hisc gene of E. coli (8). Its inactivation by integration of plasmid pjm8101 or pjm8105 was lethal, suggesting that it is an essential protein for cell growth even in complex medium. A potential stem-loop structure with strong similarity to rhoindependent terminators (2) was identified immediately downstream of the kina gene and appeared to function bidirectionally. Homology of kina to transmitter proteins. In two-component regulatory systems, the receiver proteins are homologous over approximately 120 amino acids located at their amino termini, while the transmitter proteins share homology at their carboxy termini (24). The KinA protein was most homologous overall to the B. subtilis phor gene product (28). These two proteins, aligned by the CLUSTAL4 program of Higgins and Sharp (11), are shown in Fig. 4. If the protein is arbitrarily divided in half at residue 303, 41 residues are identical in the amino-terminal half and 92 residues are identical in the carboxyl-terminal half of the protein. Included in this latter half were two highly conserved motifs among kinases (Fig. 4), A/S-H-E-I-K/R-T/ N-P-L, beginning at residue 402, and G-T-G-L-G-L, beginning at residue 569 (24). The carboxyl half of these proteins presumably interacts with the conserved amino terminus of receiver proteins (23). The amino terminus of transmitter kinases is unique from kinase to kinase and may be the site of interaction with whatever effecter molecules activate the various kinases. Phosphorylation of SpoOA and SpoOF by KinA. Although the sequence homology of KinA with that of other transmitter kinases was very suggestive of a kinase role for KinA, in vitro phosphorylation reactions were required to prove that KinA had this enzymatic function. To this end, the SpoOA and SpoOF proteins were purified to homogeneity, and the KinA protein was purified to an estimated 80% purity from E. coli extracts of strains producing high levels of each of these proteins from expression plasmids (Fig. 5). Reaction mixtures were prepared with [_y-32p]atp under conditions similar to those for the NtrB-NtrC reactions described by Keener and Kustu (14). KinA was autophosphorylated in the absence of receiver proteins (Fig. 6, lane A). This enzymatic reaction is characteristic of this class of kinases. Addition of SpoOA or SpoOF protein (Fig. 6, lanes B and C) resulted in transfer of the phosphate to these receiver proteins. The enzyme appeared to be much more active with SpoOF as a receiver than with SpoOA. We estimated from shorter exposures of this autoradiograph that the level of radioactivity in SpoOF was from 25- to 50-fold higher than that observed in SpoOA. On a molar basis, the level of label in KinA and SpoOF was about equal. This was the result under the conditions employed and does not necessarily reflect the relative activities in vivo. The major point is that both SpoOF and SpoOA were phosphorylated and that KinA actively phosphorylated both proteins. Efficiency of sporulation of kina mutants. The Tn9J7QHU19 insertion mutation in strain KS19 was first described as causing an oligosporogenous stage II phenotype (27). Since the kina gene was the site of transposon insertion, we carried out a quantitative analysis of the sporulation effi-

5 VOL. 171, 1989 PHOSPHORYLATION OF B. SUBTILIS SpoOA AND SpoOF 6191 ACATTTTACTAC GSCACGAMC;CATCGTGGG GGTTTTTTTCATAC ATCTGACTTTGTGT TTACGTCGAAAACAAAGTGTTTAGTGTTGTTGTTCCTCAGTA T F Y Y G T N R N L T F V G Y Y P S K K P V A F S V V V P S V GTAGATGATAGATCAATAAAATATTGCCAAAMAGACTATCCACGCCT DTCACTAAAAGATTGGC D D D D K I N K I I A K R A I H A Y A E L E K K L S K K * >>>>>>>>>>> <<cs CTTTCTTTTATTCAAAAATTGACGTTCACCATAAGAATAGAAGGAGAATACTCATTTTCTAGCGAATCATACTAGGTAAAAGTCAATC -c-c GAGGGATTCTGTGGAAC GGATACGCAGCATGTTAA ACCACTTT ATTCATGAGTCTTGCTCTGG TCATTTATATAATCCTGCA M E Q D T Q H V K P L Q T K T D I H A V L A S N G R I I Y I S A N S K L H TTTGGGCTATCTCCAAGAG GATGATC GGATCATTCCTCAACTTCGATAGAGCCATTTTTGGTGACTATTTTTATAGAACTTGATGCCGTGCACCTT L G Y L Q G E M I G S F L K T F L H E E D Q F L V E S Y F Y N E HR L M P C T F TCGTTTTATTAAAAAGATCATACGATTGTG,TGGGTGGCTTAATGTTGAAGAGTTCTTGCGAG R F I K K D H T I V W V E A A V E I V T T R A E R T E R E I I L K M K V L E E E AACAGSCCATCAA TCCCTAACGGAAAAGATCGACTAGC0ATGCAAAAAACOGAT GATTGAGTTGGTTGMTCTCCCGAGTCCGCTATG T G H Q S L N C E K H E I E P A S P E S T T Y I T D D Y E R L V E N L P S P L C CATCAGTGTCAAAGGCAGTCGTCTATGTAAC GCGCGA TGCTTTCAACTGGCAACAGATGCTATTATTGGTAAATCGTCCTATGAA TTTATTGAAATTC I S V K G K I V Y V N S A M L S M L G A K S K D A I I G K S S Y E F I E E E Y H TGATATCGTHTEATGAC GAAT ATPAGTGATGTTCATTCCCCGAC 1080 D I V K N R I I R M Q K G M E V G M I E Q T W K R L D G T P V H L E V K A S P T CGTCTACAAAAMACCAGACTGAGCTGCTGCTGCTGATCGATATCTCTTCAGAAAAATTCCAACATCCTGCAAAAAACCTGAACGATATCAGCTGCTGATTCAAATTCCAT V Y K N Q Q A E L L L L I D I S S R K K F Q T I L Q K S R E R Y Q L L I Q N S I TGATACCATTGCGGTGATTCACAATGATGTT TTATGAATTATCAGATTTCCCTGTTTGACTATTAGTTATGAAAAATACGTCGTCA 1320 D T I A V I H N G K W V F M N E S G I S L F E A A T Y E D L I G K N I Y D Q L H TCCTTGCGATCACGU;3GATGTAAA GAGAGAATCCAA AACATTGCGCAAAATCTGAAATTGTCAMATCCTGGTTCACCTTTCAACAMTCATCTATACGGAGAT P C D H E D V K E R I Q N I A E Q K T E S E I V K Q S W F T F Q N R V I Y T E M GGTCTGCATTCCGACGACCTTTTTTGGGGTGCGTCGTCATTCTTCGTAACCGGAAATATGAGATAGTA ATCGGAATACACCG V C I P T T F F G E A A V Q V I L R D I S E R K Q T E E L M L K S E K L S I A G GCAGCTCGCGGCGGGATCGCCCATGAGATCCGCAACCC TCTTACAGCGATCAAAGGATTTTTACAGCCTA TTTGATATTGTGTTTTC Q L A A G I A H E I R N P L T A I K G F L Q L M K P T M E G N E H Y F D I V F S TGAACTCAGCCGTATCGGT TGTMTCTCGA TCAAACTGTCAATTGTT G GTGAGGTTCTGTT E L S R I E L I L S E L L M L A K P Q Q N A V K E Y L N L K K L I G E V S A L L AGACCMAGGGATAAGCTTTTAACATTATGAAAGCACATTTTTATTATM COGTAACATAACGTATTCATT AATTTTM AAATGC E T QA N L N G I F I R T S Y E K D S I Y I N G D Q N Q L K- Q V F I N L I K N A 1920 AGTTGAATCAATGCCTGATGGGGGAAAGTAGCTTATCATAACCGAGTGAGCATTCTGTTCATGTTACTGTCAGCAA_GAGTATA CCTGAAAAGGTACTAAACCOGAT V E S M P D G G T V D I I I T E D E H S V H V T V K D E G E G I P E K V L N R I 2040 TGGAGAGCCATTTTTTAAACAAAAAGAOCTGTATGGTAATMTGTATCTGAACTAGAGTTATTACATGTGGCACCACTAAAAGCA G E P F L T T K E K G T G L G L M V T F N I I E N H Q G V I H V D S H P E K G T AGFFATPKCAKTT A TTA TT A F K I S F P K K * >>>>>>>>> CCCCC FIG. 2. Nucleotide sequence of the kina gene. The sequence of the kina gene was determined as described in Materials and Methods. Stem-loop structures with potential as terminators are indicated

6 6192 PEREGO ET AL. J. BACTERIOL. BS OrfY MEHLLNPKAREIEISGIRKFSNLVAOHEDVISLTIGQPDFFTPHHVKAAAK-KAIDENVTSYTPNAGYLELRQAVQLYM4KKADFNYDAESEIIITTGASQAIDAAFRTILSPGDEVI EC HisC MSTV--- TITDLARENVRNL ---- TPYOSARRLGGNGDVWLNANEYPTAVEFOLTOOTLNRYPECOP- ----KAVIENY---- AQYAGVKPEQVLVSRGADEGIELLIRAFCEPGKDAI * * * * * * * ** * * ** * BS OrfY M-PGPIYPGYEPIINLCGAKPVIVDTTSHGFKLTARLIEDALTPNTKCCWLPYPSNPTGVTLSEEELKSIAALLKGRNVFVLSDEIYSELTYDRPHYSIATYLRD--QTIVINGLSKS EC HisC LYCPPTYGMYSVSAETIGVECRTV-PTPDNWQLDLQGISDKLD-GVKAVYVCSPNNPTGQLINPQDFRTLLELTRGKAI--WADEAYIEFC-- -PQASLAGWLAEYPHLAILRTLSKA * * * * * * * * ** * * * **** * * * ** ** * * * *** BS OrfY HTMTGURIGFLFAPKDIAKHILKV-HOYNVSCASSISQKAALEAVTNGFDDALINREQYKKRL ---DYVYDRLVSMGLDVVKPSGAFYIFPSIKSFGMTSFDFSMALLE--- DAGVAL EC HisC FALAGLRCGFTLANEEVINLLMKVIAPYPLSTPVADIAAQALSP-----OGIVANRERVAQIIAEREYLIAAL--KEIPCVEQ-----VFDSETNYILARFKASSAVFKSLWDQGIIL * * ** * ** * * ** *** * * * *.* * * * ** * BS OrfY VPGSSFSTYGEGYVRLSFACSMDTLREGLDRLELFVLKKREAMQTINNGV EC HisC RDQNKQPSLS-GCLRITVGT-----REESORVI DALRA--EQV * * ** * * * FIG. 3. Comparison of OrfY protein with HisC. The B. subtilis (BS) ORF downstream of kina and on the opposite strand, orfy, was compared with hisc of E. coli (EC) (8) by the CLUSTAL4 program of Higgins and Sharp (11). Asterisks indicate identical residues, and dots show related residues. ciency of several kina mutants. A deletion-cmr insertion kina mutant was constructed by integrating the linearized plasmid pjm8115 (Fig. 1) into the chromosome of B. subtilis JH642 via a double crossover event. This strain, together with other strains shown in Table 2, was subjected to growth in sporulation conditions as described in Materials and Methods. After treatment with chloroform, the number of surviving cells was compared with the number of total cells in the culture. As can be seen in Table 2, in strains JH642(pJM8104) (in which the kina ORF was interrupted by the insertion via single crossing-over of a plasmid that carries an internal fragment of the kina gene) and in strain JH642(pJM8115) (which was the deletion_cmr insertion mutant), the efficiency of sporulation was approximately 25% of that observed for the parental strain JH642 or for strains which carried plasmids that do not inactivate the kina gene (e.g., pjm8102). The lower frequency of the strain carrying the interrupted kina gene was similar to that of the strain with the transposon kina mutation, JH AbrB and protease regulation in a kina mutant strain. The regulation of the transition state in a kina-deficient strain was tested by analyzing the transcription of the transition state regulator abrb gene (26) and of the apre gene, which is known to be activated at this stage of cell growth (5). Strains JH12604 and JH12315 (Table 1) carried a transcriptional fusion between the abrb promoter region and the apre promoter region, respectively, with the lacz gene of E. coli inserted in single copy in the amye region. Chromosomal BS PhoR MNK YRVRLFSVFVVWCILVFC VLGLFLOOLFETSDNRKAEEHIEKEAKYLASLLDAGNLNNQANEKIIKDAGGALDVSASVIDTDGKVLYGSNGR BS KinA NEQOTQHVKPLQTKTDIHAVLASNGRIIYISANSKLHLGYLOGEMIGSFLKTFLHEEDQFLVESYF-----YNEHHLMPCTFRFIKKDHTIVWVEAAVEIVTTRAERTEREIILKMKV *. ** * * * * BS PhoR SADSQKV ALVSGHEHILSTT-DNKLYYGLSLRSEGEKTGYVL-LSASEKSDGLKGELW1LTASLCTAFIVIVYFYSSMTSRYKRSIES-ATNVATELSKGNYDA---RTYGGYIRR BS KinA LEEETGHOSLNCEKHEIEPASPESTTYITDDYERLVENLPSPLCISVKGKIVYVNSAMLSMLGAK-SKDAIIGKSSYEFIEEEYHDIVKNRIIRMQKGMEVGMIEQTWKRLDGTPVHL *. *... * * * * * * ** * * * * * * * BS PhoR SDKLGHAMNS LAIDLMENTRTQEM---ORDRLLTVIENIGSGLIMIDGRGFINLVNRSYAKQFHINPNHML--RRLYHD--AFEHEEVIQLVEDIF--MTETKKCKLLRLP BS KinA EVKASPTVYKNOOELLLLIDISSRKKFQTILQKSRERYQLLIONSIDTIAVIHNGKWV-FMNESGISLFEAATYEDLIGKNIYDoLHPCDHEDVKERIoNIAEQKTESEIvKQSWFT * * ** * * * * * *.* * * * *** * ** * BS PhoR IKIERRYFEVDGVPIMGPDDEWKGIVLVFHDMTETKKLEOMR KDFVANVSHELKTPITSIKGFTETLLDGAMEDKEALSEFLSIILKESERLQSLVQDLLDLSKIEOQN BS KinA FQNRVIYTEI4VCIPTTFFGEA--AVQVILRDISERKOTEELMLKSEKLSIAGQLAAGIAHEIRNPLTAIKGFLO-L4KPTMEGNE ---HYFDIVFSELSRIELILSELLMLAKPOQNA * * * * * * * * ** * ***** * ** * * * * ** ** * ~ I BS PhoR FTLSIETFEPAKE4LGEIETLLKHKADEKGISLHLNVPKDPOYVSGDPYRLKOVFLNLVNALTYTPEGGSVAINVKPREKDIQIEVADSGIGIQKEEIPRIFERFYRVDKDRSRNSGG BS KinA VK---EYLNLKKLIGEVSALLETOANLNGIFIRTSYEKDSIYINGDQONQLKQVFINLIKNAVESMPDGGTVDIIITEDEHSVHVTVKDEGEGIPEKVLNRIGEPFL-TTKEK-----G BS PhoR TGLGLAIVKHLIEAHEGKIDVTSELGRGTVFTVTLKRAAEKSA BS KinA TGLGLMVTFNIIENHQGVIHVDSHPEKGTAFKISFPKK----- ***** ** ** * ** ** * I I FIG. 4. Homology of KinA to PhoR protein. The homology of KinA to the B. subtilis (BS) PhoR protein (28) is shown as determined by the CLUSTAL4 program of Higgins and Sharp (11). Asterisks indicate identical residues, and dots show related residues. The residues generally common to transmitter kinases (24) are indicated by bars.

7 VOL. 171, 1989 PHOSPHORYLATION OF B. SUBTILIS SpoOA AND SpoOF 6193 A :: * B C FIG. 5. PAGE of purified KinA, SpoOA, and SpoOF proteins. The KinA (lane A), SpoOA (lane B), and SpoOF (lane C) proteins used in the phosphorylation reactions were subjected to SDS-PAGE along with prestained high-molecular-mass markers (in kilodaltons) from Bethesda Research Laboratories. The proteins were stained with Coomassie blue. DNA from these strains was used to transform strain JH12638 to Cmr in order to transfer the fusion constructs from the wild-type strains into the transposon insertion kina mutant. One Cmr transformant obtained for each promoterlacz fusion was purified and analyzed, and the P-galactosidase activity was tested together with the respective donor strains as a control. Figure 7 shows the results of the assay. Transcription from the abrb promoter and the apre promoter in the kina mutant showed normal temporal regulation in comparison with the wild-type strains. The rate of transcription from the subtilisin promoter in the sporulationdeficient strain was two- to threefold higher than in the Spo+ strain. AbrB regulation was normal; transcription decreased in parallel with a wild-type strain. Effect of the multicopy kina gene. In order to investigate the effect of a multicopy kinase gene on sporulation, chromosomal DNA fragments carrying the kina gene were cloned in the replicative vector pbs19 and transformed into B. subtilis JH642 and isogenic spoo mutant strains. Plasmid pjm8116 contained the kina gene, and plasmid pjm8122 contained the promoter and upstream region for the kina A B C 1-41' FIG. 6. Phosphorylation of SpoOA and SpoOF proteins in the presence of KinA protein. Reactions were performed as described in Materials and Methods and subjected to electrophoresis in a 15% polyacrylamide gel (17) without prior boiling. The undried gel was exposed to Kodak XRP-5 film for 2 h at -70 C with one intensifying screen. Purified KinA protein was autophosphorylated in the presence of [_y-32p]atp (lane A-1). Addition of purified SpoOA or SpoOF protein resulted in the transfer of the phosphate from the KinA to SpoOA (lane B-2) or SpoOF (lane C-3). TABLE 2. Efficiency of sporulation in kina-defective strains Strain Total no. of No. of cells/ml spores/ml % Spores' JH642(pJM8104) 4 x X JH642(pJM8115) 4 x x JH x x JH642(pJM8102) 3 x x JH642 3 x x a The percentage is expressed as 100 times the ratio between spores per milliliter and total cells per milliliter from the same culture. gene (Fig. 1). These plasmids were transformed into the Spo- strains, and one colony from each transformation was subjected to growth in sporulation conditions for 24 h in the presence of chloramphenicol. A comparison between the number of spores obtained and the total viable cells for each strain is shown in Table 3. The presence of the kina gene on a multicopy vector did not suppress the sporulation-deficient phenotype in an spooa strain (JH646), but it restored the ability to sporulate in the spooe (JH647), spoob (JH648), and spoof (JH649) strains. In the spooh mutant strain JH651, the presence of the replicative vectors with the kina gene resulted in inhibition of growth, and a very low level of suppression was observed. In all cases, the entire kina gene was required to restore sporulation, as the plasmid containing only the kina promoter was ineffective. DISCUSSION The kina gene codes for a protein of 69,170 Da which shows strong homology in its carboxyl sequence to the transmitter kinases of two component regulatory systems (15, 24). The carboxyl terminus of these proteins is thought to interact with the conserved amino terminus of receiver proteins, and it is these portions of the molecules that may contain the kinase and phosphoacceptor enzymatic functions (23). The amino-terminal regions of transmitter kinases are of variable length and unique sequence. Presumably this portion of the molecule interacts with a component of the signal transduction mechanisms unique to each kinase. Among the transmitter proteins so far sequenced, the kina gene product is most closely related in overall sequence to the B. subtilis PhoR protein. We believe this represents a close evolutionary relationship rather than any similarity in signal transduction function. The kina gene product does not contain stretches of hydrophobic residues characteristic of membrane proteins and is found as a soluble protein when produced in E. coli. In this feature, KinA resembles NtrB and CheA, which are not membrane associated and receive their sensory input from other signaling proteins rather than directly from the environment (24). Kofoid and Parkinson (24) have shown that both of these soluble transmitters have receiver modules in the same protein which may serve to control the transmitter activity of the molecule. The KinA protein, however, does not contain a recognizable receiver module and therefore differs in this respect from NtrB and CheA (J. S. Parkinson, personal communication). Evidence from plasmid integration studies suggests that the kina gene is the sole cistron in its transcription unit. Thus, the second component of this potential two-component regulatory system was not obvious as a gene cotranscribed with kina. Genes for both components of twocomponent systems are usually found in the same operon in a variety of bacteria, including the B. subtilis two-component pairs phor-phob (28) and degs-degu (9, 16). The

8 6194 PEREGO ET AL. J. BACTERIOL..t X 2000 C 1000 r Ca, O M Ca Ca A T-3 T-2 T-1 TO Ti T2 T3 FIG. 7. Time course of,3-galactosidase activity in strains carrying the abrb-lacz (A) and apre-lacz (B) transcriptional fusion plasmids. Symbols: (A) 0, JH12604; 0, JH12679; (B) U, JH12315; O, JH TO, End of logarithmic growth; T-1, T-2, and T-3, hourly intervals before TO; Tl, T2, and T3, hourly intervals after TO. receiver protein genes spooa and spoof are single-cistron transcription units with no hint of a transmitter kinase gene flanking them (7, 30). Thus, linkage relationships have not been informative in uncovering the substrate for kina or for identifying the kinase for SpoOA or SpoOF protein. The purified KinA protein was autophosphorylated in the presence of ATP, an enzymatic function inherent to this class of kinases. The KinA protein mediates the transfer of phosphate from ATP to the SpoOA and SpoOF proteins, both of which contain the amino-terminal homology characteristic of receiver proteins (31). Under the assay conditions used, 25- to 50-fold more phosphate label was found in SpoOF than in SpoOA. It would be easy to conclude from this result that SpoOF was the "real" substrate for KinA and that the low level of SpoOA phosphorylation represented crosstalk (23). However, this conclusion would ignore several other possibilities, including that the in vitro conditions used do not favor the phosphorylation of the SpoOA protein. Perhaps the kinase needs to be "activated" to function effectively on SpoOA. If the kinase is activated in vivo by signal transduction to function on its receiver protein, it seems unlikely that purified kinase would retain this active state unless the activation were a covalent modification and could occur in E. coli, from which the kinase was purified. It is also possible that purified SpoOA protein does not reflect the state of the SpoOA substrate in vivo. Either of these hypothetical states TABLE 3. Suppression of defective-sporulation phenotypes by a multicopy plasmid carrying the kina gene Strain Total no. of celis No. of spores/ml JH642(pJM8116) 2.7 x x 107 JH642(pJM8122) 2.3 x x 107 JH642(pBS19) 1.5 x X 107 JH646(pJM8116) 2.0 x JH646(pJM8122) 6.1 x JH646(pBS19) 3.3 x JH647(pJM8116) 2.0 x x 106 JH647(pJM8122) 1.7 X X 104 JH647(pBS19) 1.6 X X 104 JH648(pJM8116) 3.0 x x 107 JH648(pJM8122) 1.1 x JH648(pBS19) 5.2 x JH649(pJM8116) 1.5 x x 107 JH649(pJM8122) 2.0 x JH649(PBS19) 2.5 x JH651(pJM8116) 3.0 x x 103 JH651(pJM8122) 1.2 x JH651(pBS19) 1.1 x 108 0

9 VOL. 171, 1989 PHOSPHORYLATION OF B. SUBTILIS SpoOA AND SpoOF 6195 for KinA and SpoOA might be mimicked by alterations in the reaction conditions. Experiments of this type are under way. There seems to be little doubt that both SpoOA and SpoOF proteins are capable of being phosphorylated in vitro and therefore in vivo. Genetics has proven to be the only means of assigning a known kinase to a receiver protein. All of the transmitter kinases now known were identified on the basis of homology and phenotypic dependency. In the case of the sporulationregulatory proteins SpoOA and SpoOF, we have not identified a kinase gene by homology or phenotypic dependency among the spoo genes. The question whether KinA functions in vivo to phosphorylate SpoOA and/or SpoOF is difficult to answer. Clearly, kina mutants are not completely dependent on the KinA kinase for sporulation, indicating that if phosphorylation is a prerequisite for SpoOA and/or SpoOF function, another kinase must effectively fulfill this role. Strains carrying deletion mutations in the kina gene are only slightly deficient in sporulation. They produced about one-third as many spores in liquid culture after 24 h as the wild-type strain. On solid medium, we classified the mutants as delayed type, since after 72 h there was no noticeable difference between the level of spores seen in the wild-type and mutant strains. The delayed phenotype is also characteristic of Sco- mutants. The kina mutant also overproduces protease, which is another feature of Sco- mutations (4). Sco- mutations have been mapped in this region of the chromosome (20), and we found that plasmid pjm8118 could transform the scod4 mutation to prototrophy (unpublished results), suggesting that the scod locus is identical to kina. Activation of the SpoOA protein is thought to be the final step in the action of the SpoOB, SpoOE, and SpoOF proteins on sporulation (unpublished results). A multicopy vector containing the kina gene was able to overcome mutations in the spoob, spooe, and spoof genes but not a mutation in the spooa gene. These results suggest that the phosphorylation of SpoOA is the ultimate target of spoob, spooe, and spoof action. A similar plasmid carrying the DegS kinase (9) was ineffective (unpublished results), suggesting some specificity in KinA function. The results are most consistent with the possibility that multiple kinases exist for the SpoOA protein and that KinA may be one of this group. ACKNOWLEDGMENTS We thank Jonathan Day for performing the,b-galactosidase assays. This research was supported in part by Public Health Service grants GM19416 and GM39442 from the National Institute of General Medical Services. LITERATURE CITED 1. Anagnostopoulos, C., and J. 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