The F-ATPase Operon Promoter of Streptococcus mutans Is Transcriptionally Regulated in Response to External pH

Abstract

Streptococcus mutans F-ATPase, the major component of the acid-adaptive response of the organism, is transcriptionally upregulated at low pH. Fusions of the F-ATPase promoter to chloramphenicol acetyltransferase indicated that pH-dependent expression is still observed with a short promoter that contains a domain conserved between streptococcal ATPase operons.During growth at acidic pH values, Streptococcus mutans and S. sanguis both increase production of their F-ATPases (4,5,12). By pumping protons out of cells, the enzymes function to assist in maintaining internal pH values (6,8,13,14). The ATPases from S. mutans and S. sanguis differ in their innate abilities to function in acidic conditions, as evidenced by the pH optima of the enzymes: approximately 6.0 for S. mutans and 7.0 for S. sanguis, with the maximal induction of the S. mutans enzyme occurring at pH 5. Thus, the ATPase is likely a significant reason why the mutans streptococci are able to outcompete S. sanguis during growth in low-pH environments (21) and to cause dental caries (18,24).The present study was undertaken to determine whether control of ATPase production in oral streptococci occurs at the transcriptional level. RNA slot blots, reporter gene fusions, and computer modeling were used to examine the pH-dependent expression of the S. mutans F-ATPase promoter in S. mutans and S. sanguis.(The material presented here was used in partial fulfillment of the Ph.D. requirements of the University of Rochester by W. L. Kuhnert.).RNA samples from cells grown at pH 5 have greater abundance of ATPase ␤-subunit transcript. We first determined whether ATPase-specific mRNA levels differed in RNA samples prepared from cells grown at pH 5 or 7. S. mutans UA159 was grown in continuous culture to steady state at a dilution rate of 0.24 h Ϫ1 , as described previously (10, 15). RNA slot blots hybridized to a ␤-subunit-specific DNA probe (amplified with primers ␤ Fwd and ␤ Rev; Table 1) showed an approximately twofold increase in the abundance of ATPase message in the cells grown at pH 5 (Fig. 1).ATPase operon transcriptional rate is greater at pH 5 than at pH 7. Three reporter gene fusions were constructed using PCR (25), both in multicopy (plasmid-borne) and single-copy (chromosomal) fusions, to determine the transcriptional activity of the operon. These included (i) an intact promoter fragment of the S. mutans GS-5 ATPase operon fused to a cat gene (contained on pDL556/CAT and in S. mutans UR123); (ii) a fragment containing the Ϫ10 and Ϫ35 sequences and promoter sequence that extended only into the predicted inverted DNA repeat proximal to the structural gene (carried on pDL680/CAT and in S. mutans UR124); and (iii) a promoterless cat gene fusion (contained on pDLCAT and in S. mutans UR122). The appropriate clones of promoter fragments were verified by nucleotide sequencing (15, 26) and were subsequently subcloned into pDL278 (16,17), resulting in the constructs listed above (detailed in Fig. 2).Single-copy fusions were created by integrating the promoter-chlo...

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

“…F-type ATPases that use an electrochemical gradient of H + or Na + to synthesize ATP, or hydrolyse ATP to reverse an electrochemical gradient, are known to play important roles in maintaining internal pH (pH i ) homeostasis (Cotter & Hill, 2003;Quivey et al, 2001). For example, the protontranslocation F 1 F 0 -H/ATPase operon in S. mutans consists of eight genes, atpEBF and atpHAGDC, which respectively encode the c, a and b subunits of the membrane-embedded F 0 complex and the d, a, c, b, and e subunits of the peripherally bound F 1 protein (Kuhnert et al, 2004). The F 0 complex has proton-translocating activity, while the F 1 complex has ATPase activity.…”

“…Previous studies suggested that the F 1 F 0 -ATPase in S. mutans might be constitutively expressed, since the promoter analysis of the F 1 F 0 -ATPase operon showed a putative Pribnow box (TAAACT) and a clear 235 sequence of TTGACA, homologous with the canonical s 70 promoter in E. coli, suggesting a housekeeping function for F 1 F 0 -ATPase synthesis (Quivey et al, 2001). However, using transcriptional reporter gene fusions to the promoter of the atp operon, Quivey and colleagues found that the increase in F 1 F 0 -H/ATPase activity was correlated with increased transcription of the atp operon of S. mutans grown at low pH (Kuhnert et al, 2004 (Kakinuma, 1998;Magalhaes et al, 2005). For instance, the copYAZ operon in S. mutans encodes a Ptype ATPase responsible for transport of copper, allowing this organism to tolerate the high concentrations of copper potentially released from dental amalgam (Vats & Lee, 2001).…”

Smx Nuclease Is the Major, Low-pH-Inducible Apurinic/Apyrimidinic Endonuclease in Streptococcus mutans

“…The organism is able to persist in the oral cavity by adapting to environmental stresses, particularly the acidification of its milieu (18,30). It has been established that the F-ATPase is transcriptionally up-regulated during growth at pH values below 7 (16,17). Recently, we have shown that the membrane fatty acid composition of S. mutans changes dramatically during growth at low pH values (7,8,28).…”

“…RNA was extracted from overnight batch cultures of S. mutans UA159 with a QIAGEN RNeasy mini kit with modifications, as previously described (17). The Promega (Madison, Wis.) avian myeloblastosis virus reverse transcriptase primer extension system was used to label an oligonucleotide primer, SmnPE3 (Table 2), with [␥-32 P]ATP (6,000 Ci/mmol) (Perkin Elmer, Boston, Mass.).…”

Identification of genes associated with mutacin I production in Streptococcus mutans using random insertional mutagenesis

“…The internal acidification also reduces the activity of acidlabile enzymes and damages proteins and DNA (van de Guchte et al, 2002). To stabilize the transmembrane pH during an acidic challenge, the multimeric F 0 F 1 -ATPase is utilized to expel protons out of the cell (Kuhnert et al, 2004;Suzuki et al, 2000). Mutations in the b (AtpD) and c (UncG) subunits of the F 0 F 1 -ATPase resulted in mutacin I defective phenotypes.…”