Department of Microbiology and Immunology and Center for Oral Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA1
Author for correspondence: Robert A. Burne. Tel: +1 716 275 0381. Fax: +1 716 473 2679. e-mail: robert_burne{at}urmc.rochester.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: sugar transport, urease, oral streptococci, gene regulation
Abbreviations: EI, EII, enzyme I, II; PEP, phosphoenolpyruvate; PTS, phosphotransferase system
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The PTS is the major high-affinity carbohydrate-transport system in eubacteria (Postma et al., 1993 ), but components of the PTS are also involved in regulation of gene expression and allosteric modulation of the activity of enzymes and transcription factors. Well-established examples of involvement of enzyme I (EI), HPr and sugar-specific enzyme II (EII) components of the PTS include modification of the activity of DNA- and RNA-binding proteins, control of catabolite repression and modulation of the activity of sugar permeases (Deutscher et al., 1995
; Lindner et al., 1999
; Martin-Verstraete et al., 1998
; Reizer et al., 1993
). In oral streptococci, the PTS is regulated by the same environmental factors that control urease expression (Chen & Burne, 1996
), but growth at low pH and in high concentrations of carbohydrate are repressive for PTS-dependent sugar transport (Hamilton, 1987
). Consequently, it was hypothesized that the PTS might play a role in regulation of urease expression through phosphorylation of a putative urease regulatory protein (Chen & Burne, 1996
). To test this hypothesis, spontaneously arising 2-deoxyglucose-resistant mutants that were defective in the production of
were isolated and shown to have about eightfold less urease activity than the wild-type strain when cells were cultured in a chemostat under sugar-limiting conditions (Chen et al., 1998a
). Notably, urease expression in the
-deficient mutant could be restored to wild-type levels when cells were grown under carbohydrate-excess conditions. At that time, a working model was proposed in which a repressor protein that senses cytoplasmic pH binds near the urease promoter in cells growing at neutral pH values and blocks urease gene transcription. Further, it was proposed that the DNA-binding activity of the regulatory protein was enhanced by phosphorylation by a component of the PTS. Specifically, under carbohydrate-limiting conditions, the PTS would preferentially phosphorylate the repressor, whereas under carbohydrate-excess conditions, incoming sugars would receive the phosphate group from the PTS and the repressor would exist in a dephosphorylated state.
EI is involved in the transfer of a phosphate group to a number of different regulatory proteins, including antiterminators (Lindner et al., 1999 ) and transcriptional activators (Martin-Verstraete et al., 1998
). One possible interpretation for the urease expression patterns in
mutants of S. salivarius is that under carbohydrate-limiting conditions, the steady-state rate of sugar transport by the PTS is lower as a result of the EII deficiency. This could result in an accumulation of the phosphorylated form of EI, which is capable of phosphorylation of the urease repressor. When carbohydrate is present in excess, sufficient sugar could flow through the system such that EI preferentially transfers the phosphate to the incoming sugars. Therefore, we hypothesized that EI could play a significant role in regulation of urease gene expression. Although spontaneous EI mutants of S. salivarius ATCC 29575 have been selected by growth on 2-deoxyglucose (Gauthier et al., 1994
), no well-defined, otherwise isogenic EI mutants have been described in S. salivarius, which has traditionally been refractory to genetic manipulation. In this work, a mutant of S. salivarius 57.I lacking all detectable EI was constructed by insertional inactivation of the ptsI gene, the growth characteristics of the mutant on PTS and non-PTS sugars were assessed, and the effects of EI ablation on the expression of urease in steady-state continuous culture were evaluated.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA manipulations.
Primers for amplification of the ptsI gene from S. salivarius 57.I were designed based on the previously published sequence of the S. salivarius 25975 ptsI (Gagnon et al., 1992 ; GenBank accession no. M81756). SphI and BamHI sites (underlined) were incorporated into the sense (5'-ATGGCATGCATGCTTAAAGGAATCGCAGC-3') and antisense (5'-TTAGGATCCGTTAACGTATTCTTTTGAAAG-3') primers, respectively. PCR reactions were carried out using Taq DNA polymerase (Life Technologies) and Perfect Match Enhancer (Stratagene) with S. salivarius 57.I chromosomal DNA as template. Reactions were performed for five cycles at low stringency (40 °C annealing) and for 30 cycles at high stringency (55 °C annealing). Amplification was completed with a final extension step of 72 °C for 10 min. The resulting 1·7 kbp product was cloned in the pCRII vector (Invitrogen) and the mixture was used to transform E. coli INV
F' cells (Invitrogen). Positive clones were identified by plasmid isolation and restriction mapping. The nucleotide sequence of the cloned DNA was determined to confirm that the insert was the ptsI gene of S. salivarius. The amplified ptsI gene was then cloned into the multiple cloning site of pGEM-7Zf(+) (Promega) at the BamHI and SphI sites. Subsequently, a Kmr determinant (Trieu-Cuot & Courvalin, 1983
) was inserted at the EcoRI site 898 bp from the 5' end of the gene to generate pCW58. The plasmid pCW58 was introduced into S. salivarius 57.I by electroporation using the method of Caparon & Scott (1991)
as modified by Chen et al. (1998b)
, with the exceptions that TY broth was used instead of ToddHewitt broth and the electroporation buffer was composed of 140 mM lactose and 1 mM MgCl2, at pH 6·5. Mutants were selected on TY medium supplemented with 12·5 mM lactose and 750 µg Km ml-1.
Chromosomal DNA was isolated from S. salivarius as previously described (Chen et al., 1996 ), digested with HpaI, electrophoresed through a 0·8% agarose gel, and the fragments transferred to Optitran membrane (Schleicher and Schuell). Purified DNA fragments of the Kmr and ptsI genes were isolated from agarose gels using the Elu-Quik kit (Schleicher and Schuell) and radiolabelled using the Random Primers DNA Labelling System (LTI) and [
-32P]dATP (New England Nuclear). Integration of the Kmr determinant at the ptsI locus by allelic exchange was confirmed by Southern hybridizations (Sambrook et al., 1989
) under stringent conditions.
Western blotting.
Cell extracts for Western blot analysis (Towbin et al., 1979 ) were prepared by mechanical disruption in the presence of an equal volume of glass beads (0·1 mm mean diameter) in a Bead Beater (Biospec Products) for a total of 2 min at 4 °C. The concentration of protein in each lysate was measured by using the Bio-Rad Protein Assay with BSA as standard. Proteins were separated for Western immunoblotting by 12% SDS-PAGE and transferred to Immobilon P membranes (Millipore) as described by Sambrook et al. (1989)
. An anti-S. salivarius EI antibody (generously provided by C. Vadeboncoeur, Université Laval, Québec, Canada) was used at a dilution of 1:500 and immunoreactive proteins were detected by incubation with goat-anti-rabbit IgG (Kirkegaard and Perry Laboratories) followed by disclosure of bound antibody with diaminobenzidine.
PTS assay.
Phosphoenolpyruvate (PEP)-dependent phosphotransferase activities were assayed by the method of Kornberg & Reeves (1972) as modified by LeBlanc et al. (1979)
. Briefly, cells were grown to an OD600 of approximately 0·6 in TY broth supplemented with 12·5 mM lactose or 25 mM glucose for strain 57.I, or 12·5 mM lactose with 750 µg ml-1 Km for strain ptsI18-3. Cells were washed, permeabilized with a mixture of toluene and acetone (1:9), and assays were carried out with glucose (10 mM) as the PTS substrate. The rate of PEP-dependent oxidation of NADH was followed at 340 nm in a Beckman DU640 spectrophotometer at a temperature of 37 °C. Controls for spontaneous oxidation of NADH consisted of assay mixtures without added PEP. PTS activities were expressed as nmol NADH oxidized in a PEP-dependent manner min-1 (mg dry weight of cells)-1.
Measurement of growth.
Growth in FMC medium containing glucose or fructose (25 mM) as PTS sugars, or lactose (12·5 mM) or galactose (25 mM) as non-PTS sugars, was monitored as OD600 using a Spectronic 20 spectrophotometer. Overnight cultures were grown in FMC-lactose, harvested, washed in sterile deionized water, resuspended in sterile deionized water, and used to inoculate FMC containing various carbohydrates. Growth at 37 °C in a 5% CO2, aerobic atmosphere was followed for 48 h.
Chemostat culture and urease assays.
S. salivarius strains were grown in a Bio-FloIII chemostat (New Brunswick Scientific) with a working volume of 650 ml in TY base medium supplemented with lactose at 12·5 mM for carbohydrate-limiting conditions, or 100 mM lactose for carbohydrate-excess conditions. Cells were grown for a minimum of ten generations for each set of growth parameters before the culture was considered to be at steady state. Urease activity was determined by measuring the amount of ammonia released from urea by intact cells using Nesslers Reagent (Aldrich) with ammonium sulfate as the standard. Urease activity was expressed as nmol urea hydrolysed min-1 (mg cell dry weight)-1.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The generation times for ptsI18-3 and 57.I did not differ significantly for growth on lactose (5055 min), which can be transported by a non-PTS route. In contrast, growth of ptsI18-3 on galactose, also a sugar which can be taken up by a non-PTS permease (Gauthier et al., 1994 ), was consistently better than that of the parent strain, and strain 57.I reached a lower final OD600 in galactose than did ptsI18-3 in an equivalent time period. One possible explanation for the differences in the characteristics of the mutant and the parent growing on galactose is that EI, or a factor modified by EI, participates in repression or allosteric regulation of galactose transport or the catabolic pathway(s), which are inducible (Vadeboncoeur & Pelletier, 1997
).
Urease activity in S. salivarius 57.I and ptsI18-3
Urease activity was measured in cells growing at steady state in continuous chemostat culture at pH values of 7·0, 6·0 and 5·5, under carbohydrate-limiting or carbohydrate-excess conditions, with lactose as the carbohydrate source (Table 1). Regardless of the growth conditions, urease expression was almost completely repressed at pH 7·0 and urease activity increased in both the mutant and the parent as the pH became more acidic. This result is consistent with previous observations that pH is the dominant influence governing urease expression in S. salivarius 57.I (Chen & Burne, 1996
; Chen et al., 1998a
). When lactose was supplied to strain 57.I in carbohydrate-limiting concentrations, urease activities were markedly lower than in cells growing on glucose. In contrast, urease levels in ptsI18-3 were about the same at pH 7·0, but were 22-fold higher at pH 6·0, and 11-fold higher at pH 5·5 than in the wild-type growing under the same conditions. Furthermore, under carbohydrate-excess conditions (100 mM lactose), urease activities were approximately twofold higher at pH 7·0 and pH 6·0, and threefold higher at pH 5·5 in the mutant as compared with the parent. Of note, urease activity in 57.I growing on lactose was always less than that in 57.I growing on either glucose or fructose at equivalent concentrations when carbohydrate was the limiting growth substrate. Urease activity in the mutant, growing on 12·5 mM lactose at pH values of 6·0 or 5·5, was significantly higher than the urease activity of 57.I in either glucose or fructose at equivalent concentrations of total carbohydrate. Urease activity in ptsI18-3 growing on 100 mM lactose was also significantly higher than that found in 57.I growing on glucose or fructose at equivalent concentrations of total carbohydrate at pH 5·5.
|
It is possible that EI, perhaps in conjunction with HPr, plays a direct role in governing the activity of the urease repressor. First, it is generally agreed that lactose is a non-PTS sugar for S. salivarius (Vadeboncoeur & Pelletier, 1997 ). Supporting this notion is the observation that insertional inactivation of the endogenous ß-galactosidase in S. salivarius 57.I eliminates the ability of the organism to grow with lactose as the sole carbohydrate (Y. M. Chen & R. A. Burne, unpublished). Consequently, as proposed in the model of urease regulation and consistent with data presented in Table 1
, growth on a non-PTS sugar resulted in lower levels of urease expression, presumably because no sugar is coming in through the PTS. In turn, the accumulation of phosphorylated EI and subsequent transfer of a phosphate group to the urease regulatory element, perhaps via HPr, causes diminished urease expression. Secondly, mutants of S. salivarius lacking EI have much higher levels of urease expression at low pH when carbohydrate is limiting, suggesting that EI, particularly at low carbohydrate concentrations, may be an important modulator of urease gene transcription.
It has also been demonstrated that lack of phosphorylation of HPr at histidine residue 15 in bacteria with mutations in ptsI could lead to accumulation of the form of HPr which is phosphorylated at serine 46 (Deutscher & Engelmann, 1984 ; Reizer et al., 1984
). Seryl-phosphorylated HPr has multiple regulatory roles, including allosteric modulation of the global regulator of carbon catabolite repression CcpA (Deutscher et al., 1995
; Reizer et al., 1993
). A CcpA-like protein has been detected by immunological means in S. salivarius using antibodies raised to Bacillus megaterium CcpA (Küster et al., 1996
) and specifically in S. salivarius 57.I using an antibody raised to histidine-tagged, purified S. mutans CcpA (Y. M. Chen & R. A. Burne, unpublished). Although there are no identifiable consensus catabolite-response elements near the ure gene promoter, it cannot be excluded that the effects of EI ablation may be exerted, directly or more likely indirectly, through HPr- or CcpA-dependent regulatory circuits.
There is also reason to believe that factors other than EI may exert control over carbohydrate-concentration-dependent expression of S. salivarius urease. Specifically, if EI were entirely responsible for the observed effects, the concentration of lactose on which the wild-type cells were grown should not have had a dramatic influence on urease levels (Table 1), and in particular, the amount of urease expressed in the EI mutant should not have varied at all as carbohydrate concentrations were altered. As suggested above, enhancements in urease expression could occur indirectly through the HPr/CcpA pathway at high carbohydrate concentrations, such as CcpA-dependent repression of the urease regulatory gene. Given the rapidity of induction of urease at low pH (Y.H. Li & R. A. Burne, unpublished), it is more likely that the level of glycolytic intermediates, perhaps fructose 1,6-bisphosphate or glucose 6-phosphate, may also affect the DNA-binding activity of the urease repressor. It also cannot yet be excluded that enhanced urease expression in cells growing in excess carbohydrate may occur through a regulatory pathway involving transcriptional activation once the repressor is dissociated from the promoter region. Preliminary data from deletion analyses indicate the potential for a region upstream of the urease promoter which may be required for optimal expression (Chen et al., 1999
), although there is no evidence yet to suggest that the regulation by pH and carbohydrate occur through separate entities.
Concluding remarks
In conclusion, the PTS plays an intimate role in the regulation of expression of the urease gene cluster of S. salivarius. Although pH is the dominant influence and repression occurs at neutrality regardless of the sugar concentration in which the cells are growing, transcription of the urease operon becomes very sensitive to carbohydrate availability once the operon is derepressed. Whether this occurs through the urease repressor as proposed in our working model (Chen et al., 1998b ) or through separate pathways can only be determined once the regulatory protein is isolated. Efforts to do so are currently under way.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caparon, M. G. & Scott, J. R. (1991). Genetic manipulation of pathogenic streptococci.Methods Enzymol 204, 556-586.[Medline]
Chen, Y. M. & Burne, R. A. (1996). Analysis of Streptococcus salivarius urease expression using continuous chemostat culture.FEMS Microbiol Lett 135, 223-229.[Medline]
Chen, Y. M., Clancy, K. A. & Burne, R. A. (1996). Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus.Infect Immun 64, 585-592.[Abstract]
Chen, Y. M., Hall, T. H. & Burne, R. A. (1998a). Streptococcus salivarius urease expression: involvement of the phosphoenolpyruvate:sugar phosphotransferase system.FEMS Microbiol Lett 165, 117-122.[Medline]
Chen, Y. M., Weaver, C. A., Mendelsohn, D. R. & Burne, R. A. (1998b). Transcriptional regulation of the Streptococcus salivarius 57.I urease operon.J Bacteriol 180, 5769-5775.
Chen, Y. M., Weaver, C. A. & Burne, R. A. (1999). A cis-element required for the negative regulation of urease expression in Streptococcus salivarius. 99th General Meeting of the American Society for Microbiology, Chicago, IL, Abstract H153, p. 358.
Collins, C. M. & DOrazio, S. E. F. (1993). Bacterial ureases: structure, regulation of expression and role in pathogenesis.Mol Microbiol 9, 907-913.[Medline]
Collins, C. M., Gutman, D. M. & Laman, H. (1993). Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes.Mol Microbiol 8, 187-198.[Medline]
Cvitkovitch, D. G., Boyd, D. A., Thevenot, T. & Hamilton, I. R. (1995). Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system.J Bacteriol 177, 2251-2258.[Abstract]
Deutscher, J. & Engelmann, R. (1984). Purification and characterization of an ATP-dependent protein kinase from Streptococcus faecalis.FEMS Microbiol Lett 23, 157-162.
Deutscher, J., Küster, E., Bergstedt, U., Charrier, V. & Hillen, W. (1995). Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria.Mol Microbiol 15, 1049-1053.[Medline]
DOrazio, S. E. F. & Collins, C. M. (1993). The plasmid-encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR, a member of the AraC family of transcriptional activators.J Bacteriol 175, 3459-3467.[Abstract]
Gagnon, G., Vadeboncoeur, C., Levesque, R. C. & Frenette, M. (1992). Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.Gene 121, 71-78.[Medline]
Gauthier, L., Thomas, S., Gagnon, G., Frenette, M., Trahan, L. & Vadeboncoeur, C. (1994). Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsI missense mutants.Mol Microbiol 13, 1101-1109.[Medline]
Hamilton, I. R. (1987). Effect of changing growth conditions on sugar transport and metabolism by oral bacteria. In Sugar Transport and Metabolism by Gram-positive Bacteria, pp. 94-133. Edited by J. Reizer & A. Peterofsky. Chichester: Ellis Horwood.
Kornberg, H. L. & Reeves, R. E. (1972). Inducible phosphoenolpyruvate-dependent hexose phosphotransferase activities in Escherichia coli.Biochem J 128, 1339-1344.[Medline]
Küster, E., Luesnick, E. J., de Vos, W. M. & Hillen, W. (1996). Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria.FEMS Microbiol Lett 139, 109-115.[Medline]
LeBlanc, D. J., Crow, V. L., Lee, L. N. & Garon, C. F. (1979). Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis. J. Bacteriol 137, 878-884.[Medline]
Lindner, C., Galinier, A., Hecker, M. & Deutscher, J. (1999). Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation.Mol Microbiol 31, 995-1006.[Medline]
Martin-Verstraete, I., Charrier, V., Stülke, J., Galinier, A., Erni, B., Rapoport, G. & Deutscher, J. (1998). Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR.Mol Microbiol 28, 293-303.[Medline]
Mobley, H. L. T. & Hausinger, R. P. (1989). Microbial ureases: significance, regulation, and molecular characterization.Microbiol Rev 53, 85-108.
Mobley, H. L. T., Island, M. D. & Hausinger, R. P. (1995). Molecular biology of ureases.Microbiol Rev 59, 451-480.[Abstract]
Postma, P. W., Lengeler, J. W. & Jacobsen, G. R. (1993). Phosphoenolpyruvate:carbohydrate phosphotransferase of bacteria.Microbiol Rev 57, 543-594.[Abstract]
Reizer, J., Novotny, M. J., Hegstenberg, W. & Saier, M. H.Jr (1984). Properties of ATP-dependent protein kinase from Streptococcus pyogenes that phosphorylates a seryl residue in HPr, a phosphocarrier protein of the phosphotransferase system.J Bacteriol 160, 333-340.[Medline]
Reizer, J., Romano, A. H. & Deutscher, J. (1993). The role of phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, in the regulation of carbon metabolism in Gram-positive bacteria.J Cell Biochem 51, 19-24.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Terleckyj, B., Willett, N. P. & Shockman, G. D. (1975). Growth of several cariogenic strains of oral streptococci in a chemically defined medium.Infect Immun 11, 649-655.[Medline]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA 76, 4350-4354.[Abstract]
Trieu-Cuot, P. & Courvalin, P. (1983). Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5'-aminoglycoside phosphotransferase type III.Gene 23, 331-341.[Medline]
Vadeboncoeur, C. & Pelletier, M. (1997). The phosphoenolpyruvate:sugar phosphotransferase of oral streptococci and its role in the control of sugar metabolism.FEMS Microbiol Rev 19, 187-207.[Medline]
Received 12 November 1999;
revised 14 January 2000;
accepted 18 January 2000.