A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcu
http://www.100md.com
《细菌学杂志》
Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, London, United Kingdom,Cell and Molecular Microbiology Division, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
ABSTRACT
We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.
TEXT
Streptococcus mutans is the principal etiological agent of human dental caries (7). Virulence properties include its ability to grow in the mixed-species biofilm known as dental plaque on tooth surfaces (14, 33) and its ability to tolerate low pH (29). Genetic competence is also thought to be important for survival in the oral cavity, as it promotes genome plasticity and therefore adaptation to changing environments.
Several genes with regulatory roles in biofilm formation, acid tolerance, and genetic competence are known, including the HK11/RR11 and VicRK two-component regulatory systems (17, 30), the histidine kinase CiaH (27), the response regulator TarC (11), trigger factor RopA (32), and the ComCDE quorum-sensing system (5, 15, 16, 18).
Protein phosphorylation is an important mechanism used to translate extracellular signals into cellular responses and is carried out by coupled protein kinases and phosphatases. Although serine/threonine protein kinases (STPK) and their associated phosphatases (STPP) have been known for some time to play major regulatory roles in eukaryotes, their discovery in prokaryotes is relatively recent (13, 22, 31). Eukaryotic-type STPK and STPP have been identified in several bacteria, and some possess multiple kinases and/or phosphatases (3, 12, 25). In gram-positive bacteria, STPK and/or STPP has been found in Bacillus subtilis (20), Listeria monocytogenes (2), Streptococcus pneumoniae (6, 24), Streptococcus agalactiae (28), and Streptococcus pyogenes (4). The S. pneumoniae and S. agalactiae STPK are required for virulence in animal models, and the S. pneumoniae enzyme is required for genetic competence (6, 28).
Identification of STPK- and STPP-encoding genes. Analysis of the genome sequence (1) identified pknB (NC_004350; GeneID 1029535), encoding a homologue of the eukaryotic-like STPK family. The highest similarity is with STPK of S. pyogenes (NCBI accession no. AAM79976; 63% identity), S. agalactiae (AAM99225; 59% identity), and S. pneumoniae (AAM47530; 51% identity) and is most pronounced in the N-terminal region containing the consensus Hanks' subdomains that comprise the catalytic domain (8, 9). Downstream of the kinase domain is a hydrophobic region that may be a transmembrane domain. Topology analysis (http://www.ch.embnet.org/software/TMPRED_form.html) predicts that PknB is an N-in, C-out membrane protein with the catalytic domain in the cytoplasm and the C-terminal, presumably sensory, domain located extracellularly. The SMART software (http://smart.embl-heidelberg.de/) predicts three tandem PASTA (penicillin-binding protein- and serine/threonine kinase-associated) domains (34) in the C terminus. The PASTA domain is thought to bind -lactam antibiotics and their peptidoglycan analogues, suggesting that PknB may sense unlinked peptidoglycan (34).
pknB is located downstream of, and overlaps with, pppL (GeneID 1027981), a gene predicted to encode an STPP of the 2C subfamily (Prosite entry PDOC00792) that is most closely related to the S. pyogenes enzyme (AAM79977; 73% identity). The presence of a consensus promoter sequence (Fig. 1) within the gene preceding pppL and a transcriptional terminator following pknB suggested that pknB and pppL are cotranscribed, and this was confirmed by reverse transcription-PCR (data not shown). The proximity of the hk11/rr11 gene pair encoding a two-component regulatory system involved in biofilm formation and acid resistance (17) is interesting, as STPK genes are sometimes adjacent to genes encoding their substrates (21, 23).
Construction and complementation of a pknB mutant. pknB and flanking DNA were PCR amplified from strain UA159 and cloned into pGEM-T-Easy (Table 1). A 1.7-kb fragment of pknB was deleted, and a unique SmaI site was introduced by inverse PCR (Table 1) and used to clone a kanamycin resistance gene (aphA3), producing pEA72. The insert was released from pEA72 and transformed into UA159 as a linear fragment. Allelic replacement was confirmed by PCR using primers flanking pknB (data not shown).
pVA838 (19) was used to make two constructs for complementation: pEA78, containing pknB regulated by the erythromycin resistance gene promoter (Perm) from pVA838, and pEA74, identical to pEA78 except that it lacked a ribosome binding site for pknB. Transformation (10) of the pknB mutant (PKNB) with pEA74 and pEA78 yielded a single Ermr colony in each case.
The pknB mutant has a reduced early growth rate. Comparison of the growth of strains PKNB and UA159 in brain heart infusion (BHI) broth showed that PKNB had a reduced growth rate early in the growth cycle, but by exponential phase the growth rate of PKNB was not significantly different from that of UA159 (data not shown). As the mutant had a tendency to clump, cultures were vortexed before removal of samples for optical density (OD) measurement. After overnight culture in tryptone soy broth containing 0.5% (wt/vol) yeast extract, UA159 grew as a uniformly turbid suspension, whereas PKNB accumulated at the bottom of the culture vessel (data not shown). Interestingly, aggregation of PKNB was less apparent in BHI broth (data not shown). Both defects were partially restored by complementation in strain PKNBC1, whereas PKNBC2 exhibited similar growth kinetics and a propensity to aggregate in the same way as PKNB.
The pknB mutant has a transformation defect. pVA838 (19) or genomic DNA from a spontaneous streptomycin-resistant (Strr) strain of UA159, each at 1 μg/ml, was used to measure transformation (10) (Table 2). To eliminate the possibility that the transformation defect was a result of the reduced growth rate of PKNB, assays were carried out that ensured that PKNB and UA159 had reached the same density (OD at 600 nm [OD600], 0.2) before DNA addition. These assays gave results similar to those in Table 2 (not shown). Addition of synthetic competence-stimulating peptide (500 ng/ml; Sigma-Genosys Ltd.) (18) did not improve the transformation efficiency of PKNB, whereas UA159 showed a 10-fold increase (data not shown). Reintroduction of pknB on a plasmid in PKNBC1 resulted in a transformation frequency 100-fold greater than that of the parent (Table 2), presumably as a result of the increased gene dosage. PKNBC2 gave similar levels of transformation as PKNB.
Mutation of pknB results in defects in biofilm formation. Biofilms were grown on hydroxyapatite disks in 24-well culture clusters (Corning Inc., from Fisher Scientific, Loughborough, United Kingdom). Two milliliters of undiluted and diluted (1:50) exponential-phase culture (OD600, 0.5) in BHI broth was added to the wells containing the hydroxyapatite disks. After incubation for 48 h the disks were rinsed, treated with BacLight LIVE/DEAD stain (Molecular Probes), and visualized by confocal laser scanning microscopy (35). Marked differences in biofilm depth and coverage were apparent between the mutant and parent biofilms (Fig. 2). The parent strain produced a thicker biofilm (typically around 28 μm) than the mutant (typically around 13 μm) and produced more biomass. The structure of the parent biofilm is typical of a mature oral biofilm (26), comprising areas covered by stacks and areas with little or no coverage that form channels between the regions of biomass. The mutant produced smaller stacks and clusters with less coverage and more intercellular gaps, more typical of a biofilm during early development. The reduced biofilm density in PKNB is not due to its reduced early growth rate, as prolonged incubation did not result in an increase in biofilm density. In PKNBC1, biofilm density was restored to approximately 70% that of the parent biofilm (data not shown). The fact that PKNBC1 showed only a partial restoration of the biofilm defect whereas it gave levels of transformation greater than the parent is presumably due to perturbation of the STPK-controlled signal transduction pathways that results from overexpression of pknB on a multicopy plasmid.
The pknB mutant is sensitive to low pH. The abilities of UA159 and its derivatives to grow at low pH were examined by growing them on agar adjusted to pH 5.0 (17). Compared to the parent, PKNB showed reduced growth on pH 5.0 agar, whereas it grew as well as the parent on agar at pH 7.0. PKNBC1 grew as well as the parent at both pH 5.0 and pH 7.0 (Fig. 3).
Summary. Disruption of pknB, encoding a eukaryotic-type STPK in S. mutans, causes defects in biofilm formation, acid resistance, and genetic competence. Other genes with regulatory roles in these phenotypes are known, raising the possibility that the defects in the pknB mutant are the result of altered expression or activity of one or more of these proteins.
REFERENCES
Ajdi, D., W. M. McShan, R. E. McLaughlin, G. Savi, J. Chang, M. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H. Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 99:14434-14439.
Archambaud, C., E. Gouin, J. Pizzaro-Cerda, P. Cossart, and O. Dussurget. 2005. Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes. Mol. Microbiol. 56:383-396.
Av-Gay, Y., and M. Everett. 2000. The eukaryotic-like serine/threonine protein kinases of Mycobacterium tuberculosis. Trends Microbiol. 8:238-244.
Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M.-Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, J. K. McCormick, D. Y. M. Leung, P. M. Schlievert, and J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99:10078-10083.
Cvitkovitch, D. G. 2001. Genetic competence and transformation in oral streptococci. Crit. Rev. Oral Biol. Med. 12:217-243.
Echenique, J., A. Kadioglu, S. Romao, P. W. Andrew, and M.-C. Trombe. 2004. Protein serine/threonine kinase StkP positively controls virulence and competence in Streptococcus pneumoniae. Infect. Immun. 72:2434-2437.
Hamada, S., and H. D. Slade. 1980. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44:331-384.
Hanks, S. K., A. M. Quinn, and T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42-52.
Hanks, S. K., and T. Hunter. 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9:576-596.
Hudson, M. C., and R. Curtiss III. 1990. Regulation of expression of Streptococcus mutans genes important to virulence. Infect. Immun. 58:464-470.
Idone, V., S. Brendtro, R. Gillespie, S. Kocaj, E. Peterson, M. Rendi, W. Warren, S. Michalek, K. Krastel, D. Cvitkovitch, and G. Spatafora. 2003. Effect of an orphan response regulator on Streptococcus mutans sucrose-dependent adherence and cariogenesis. Infect. Immun. 71:4351-4360.
Inouye, S., R. Jain, T. Ueki, H. Nariya, C. Y. Xu, M. Y. Hsu, B. A. Fernandez-Luque, J. Munoz-Dorado, E. Farez-Vidal, and M. Inouye. 2000. A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium. Microb. Comp. Genomics 5:103-120.
Kennelly, P. J., and M. Potts. 1996. Fancy meeting you here. A fresh look at "prokaryotic" protein phosphorylation. J. Bacteriol. 178:4759-4764.
Kuramitsu, H. K. 1993. Virulence factors of mutans streptococci: role of molecular genetics. Crit. Rev. Oral Biol. Med. 4:159-176.
Li, Y.-H., M. N. Hanna, G. Svenster, R. P. Ellen, and D. G. Cvitkovitch. 2001. Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms. J. Bacteriol. 183:6875-6884.
Li, Y.-H., P. C. Lau, J. H. Lee, R. P. Ellen, and D. G. Cvitkovitch. 2001. Natural genetic transformation of Streptococcus mutans growing in biofilms. J. Bacteriol. 183:897-908.
Li, Y.-H., P. C. Lau, N. Tang, G. Svenster, R. P. Ellen, and D. G. Cvitkovitch. 2002. Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans. J. Bacteriol. 184:6333-6342.
Li, Y.-H., N. Tang, M. B. Aspiras, P. C. Y. Lau, J. H. Lee, R. P. Ellen, and D. G. Cvitkovitch. 2002. A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J. Bacteriol. 184:2699-2708.
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Rajagopal, L., A. Clancy, and C. E. Rubens. 2003. A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence. J. Biol. Chem. 278:14429-14441.
Sansone, C., J. van Houte, K. Joshipura, R. Kent, and H. C. Margolis. 1993. The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J. Dent. Res. 72:508-516.
Senadheera, M. D., B. Guggenheim, G. A. Spatafora, Y.-C. Huang, J. Choi, D. C. I. Hung, J. S. Treglown, S. D. Goodman, R. P. Ellen, and D. G. Cvitkovitch. 2005. A VicRK signal transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development. J. Bacteriol. 187:4064-4076.
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ABSTRACT
We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.
TEXT
Streptococcus mutans is the principal etiological agent of human dental caries (7). Virulence properties include its ability to grow in the mixed-species biofilm known as dental plaque on tooth surfaces (14, 33) and its ability to tolerate low pH (29). Genetic competence is also thought to be important for survival in the oral cavity, as it promotes genome plasticity and therefore adaptation to changing environments.
Several genes with regulatory roles in biofilm formation, acid tolerance, and genetic competence are known, including the HK11/RR11 and VicRK two-component regulatory systems (17, 30), the histidine kinase CiaH (27), the response regulator TarC (11), trigger factor RopA (32), and the ComCDE quorum-sensing system (5, 15, 16, 18).
Protein phosphorylation is an important mechanism used to translate extracellular signals into cellular responses and is carried out by coupled protein kinases and phosphatases. Although serine/threonine protein kinases (STPK) and their associated phosphatases (STPP) have been known for some time to play major regulatory roles in eukaryotes, their discovery in prokaryotes is relatively recent (13, 22, 31). Eukaryotic-type STPK and STPP have been identified in several bacteria, and some possess multiple kinases and/or phosphatases (3, 12, 25). In gram-positive bacteria, STPK and/or STPP has been found in Bacillus subtilis (20), Listeria monocytogenes (2), Streptococcus pneumoniae (6, 24), Streptococcus agalactiae (28), and Streptococcus pyogenes (4). The S. pneumoniae and S. agalactiae STPK are required for virulence in animal models, and the S. pneumoniae enzyme is required for genetic competence (6, 28).
Identification of STPK- and STPP-encoding genes. Analysis of the genome sequence (1) identified pknB (NC_004350; GeneID 1029535), encoding a homologue of the eukaryotic-like STPK family. The highest similarity is with STPK of S. pyogenes (NCBI accession no. AAM79976; 63% identity), S. agalactiae (AAM99225; 59% identity), and S. pneumoniae (AAM47530; 51% identity) and is most pronounced in the N-terminal region containing the consensus Hanks' subdomains that comprise the catalytic domain (8, 9). Downstream of the kinase domain is a hydrophobic region that may be a transmembrane domain. Topology analysis (http://www.ch.embnet.org/software/TMPRED_form.html) predicts that PknB is an N-in, C-out membrane protein with the catalytic domain in the cytoplasm and the C-terminal, presumably sensory, domain located extracellularly. The SMART software (http://smart.embl-heidelberg.de/) predicts three tandem PASTA (penicillin-binding protein- and serine/threonine kinase-associated) domains (34) in the C terminus. The PASTA domain is thought to bind -lactam antibiotics and their peptidoglycan analogues, suggesting that PknB may sense unlinked peptidoglycan (34).
pknB is located downstream of, and overlaps with, pppL (GeneID 1027981), a gene predicted to encode an STPP of the 2C subfamily (Prosite entry PDOC00792) that is most closely related to the S. pyogenes enzyme (AAM79977; 73% identity). The presence of a consensus promoter sequence (Fig. 1) within the gene preceding pppL and a transcriptional terminator following pknB suggested that pknB and pppL are cotranscribed, and this was confirmed by reverse transcription-PCR (data not shown). The proximity of the hk11/rr11 gene pair encoding a two-component regulatory system involved in biofilm formation and acid resistance (17) is interesting, as STPK genes are sometimes adjacent to genes encoding their substrates (21, 23).
Construction and complementation of a pknB mutant. pknB and flanking DNA were PCR amplified from strain UA159 and cloned into pGEM-T-Easy (Table 1). A 1.7-kb fragment of pknB was deleted, and a unique SmaI site was introduced by inverse PCR (Table 1) and used to clone a kanamycin resistance gene (aphA3), producing pEA72. The insert was released from pEA72 and transformed into UA159 as a linear fragment. Allelic replacement was confirmed by PCR using primers flanking pknB (data not shown).
pVA838 (19) was used to make two constructs for complementation: pEA78, containing pknB regulated by the erythromycin resistance gene promoter (Perm) from pVA838, and pEA74, identical to pEA78 except that it lacked a ribosome binding site for pknB. Transformation (10) of the pknB mutant (PKNB) with pEA74 and pEA78 yielded a single Ermr colony in each case.
The pknB mutant has a reduced early growth rate. Comparison of the growth of strains PKNB and UA159 in brain heart infusion (BHI) broth showed that PKNB had a reduced growth rate early in the growth cycle, but by exponential phase the growth rate of PKNB was not significantly different from that of UA159 (data not shown). As the mutant had a tendency to clump, cultures were vortexed before removal of samples for optical density (OD) measurement. After overnight culture in tryptone soy broth containing 0.5% (wt/vol) yeast extract, UA159 grew as a uniformly turbid suspension, whereas PKNB accumulated at the bottom of the culture vessel (data not shown). Interestingly, aggregation of PKNB was less apparent in BHI broth (data not shown). Both defects were partially restored by complementation in strain PKNBC1, whereas PKNBC2 exhibited similar growth kinetics and a propensity to aggregate in the same way as PKNB.
The pknB mutant has a transformation defect. pVA838 (19) or genomic DNA from a spontaneous streptomycin-resistant (Strr) strain of UA159, each at 1 μg/ml, was used to measure transformation (10) (Table 2). To eliminate the possibility that the transformation defect was a result of the reduced growth rate of PKNB, assays were carried out that ensured that PKNB and UA159 had reached the same density (OD at 600 nm [OD600], 0.2) before DNA addition. These assays gave results similar to those in Table 2 (not shown). Addition of synthetic competence-stimulating peptide (500 ng/ml; Sigma-Genosys Ltd.) (18) did not improve the transformation efficiency of PKNB, whereas UA159 showed a 10-fold increase (data not shown). Reintroduction of pknB on a plasmid in PKNBC1 resulted in a transformation frequency 100-fold greater than that of the parent (Table 2), presumably as a result of the increased gene dosage. PKNBC2 gave similar levels of transformation as PKNB.
Mutation of pknB results in defects in biofilm formation. Biofilms were grown on hydroxyapatite disks in 24-well culture clusters (Corning Inc., from Fisher Scientific, Loughborough, United Kingdom). Two milliliters of undiluted and diluted (1:50) exponential-phase culture (OD600, 0.5) in BHI broth was added to the wells containing the hydroxyapatite disks. After incubation for 48 h the disks were rinsed, treated with BacLight LIVE/DEAD stain (Molecular Probes), and visualized by confocal laser scanning microscopy (35). Marked differences in biofilm depth and coverage were apparent between the mutant and parent biofilms (Fig. 2). The parent strain produced a thicker biofilm (typically around 28 μm) than the mutant (typically around 13 μm) and produced more biomass. The structure of the parent biofilm is typical of a mature oral biofilm (26), comprising areas covered by stacks and areas with little or no coverage that form channels between the regions of biomass. The mutant produced smaller stacks and clusters with less coverage and more intercellular gaps, more typical of a biofilm during early development. The reduced biofilm density in PKNB is not due to its reduced early growth rate, as prolonged incubation did not result in an increase in biofilm density. In PKNBC1, biofilm density was restored to approximately 70% that of the parent biofilm (data not shown). The fact that PKNBC1 showed only a partial restoration of the biofilm defect whereas it gave levels of transformation greater than the parent is presumably due to perturbation of the STPK-controlled signal transduction pathways that results from overexpression of pknB on a multicopy plasmid.
The pknB mutant is sensitive to low pH. The abilities of UA159 and its derivatives to grow at low pH were examined by growing them on agar adjusted to pH 5.0 (17). Compared to the parent, PKNB showed reduced growth on pH 5.0 agar, whereas it grew as well as the parent on agar at pH 7.0. PKNBC1 grew as well as the parent at both pH 5.0 and pH 7.0 (Fig. 3).
Summary. Disruption of pknB, encoding a eukaryotic-type STPK in S. mutans, causes defects in biofilm formation, acid resistance, and genetic competence. Other genes with regulatory roles in these phenotypes are known, raising the possibility that the defects in the pknB mutant are the result of altered expression or activity of one or more of these proteins.
REFERENCES
Ajdi, D., W. M. McShan, R. E. McLaughlin, G. Savi, J. Chang, M. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H. Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 99:14434-14439.
Archambaud, C., E. Gouin, J. Pizzaro-Cerda, P. Cossart, and O. Dussurget. 2005. Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes. Mol. Microbiol. 56:383-396.
Av-Gay, Y., and M. Everett. 2000. The eukaryotic-like serine/threonine protein kinases of Mycobacterium tuberculosis. Trends Microbiol. 8:238-244.
Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M.-Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, J. K. McCormick, D. Y. M. Leung, P. M. Schlievert, and J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99:10078-10083.
Cvitkovitch, D. G. 2001. Genetic competence and transformation in oral streptococci. Crit. Rev. Oral Biol. Med. 12:217-243.
Echenique, J., A. Kadioglu, S. Romao, P. W. Andrew, and M.-C. Trombe. 2004. Protein serine/threonine kinase StkP positively controls virulence and competence in Streptococcus pneumoniae. Infect. Immun. 72:2434-2437.
Hamada, S., and H. D. Slade. 1980. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44:331-384.
Hanks, S. K., A. M. Quinn, and T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42-52.
Hanks, S. K., and T. Hunter. 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9:576-596.
Hudson, M. C., and R. Curtiss III. 1990. Regulation of expression of Streptococcus mutans genes important to virulence. Infect. Immun. 58:464-470.
Idone, V., S. Brendtro, R. Gillespie, S. Kocaj, E. Peterson, M. Rendi, W. Warren, S. Michalek, K. Krastel, D. Cvitkovitch, and G. Spatafora. 2003. Effect of an orphan response regulator on Streptococcus mutans sucrose-dependent adherence and cariogenesis. Infect. Immun. 71:4351-4360.
Inouye, S., R. Jain, T. Ueki, H. Nariya, C. Y. Xu, M. Y. Hsu, B. A. Fernandez-Luque, J. Munoz-Dorado, E. Farez-Vidal, and M. Inouye. 2000. A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium. Microb. Comp. Genomics 5:103-120.
Kennelly, P. J., and M. Potts. 1996. Fancy meeting you here. A fresh look at "prokaryotic" protein phosphorylation. J. Bacteriol. 178:4759-4764.
Kuramitsu, H. K. 1993. Virulence factors of mutans streptococci: role of molecular genetics. Crit. Rev. Oral Biol. Med. 4:159-176.
Li, Y.-H., M. N. Hanna, G. Svenster, R. P. Ellen, and D. G. Cvitkovitch. 2001. Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms. J. Bacteriol. 183:6875-6884.
Li, Y.-H., P. C. Lau, J. H. Lee, R. P. Ellen, and D. G. Cvitkovitch. 2001. Natural genetic transformation of Streptococcus mutans growing in biofilms. J. Bacteriol. 183:897-908.
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