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Research Paper |
Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA1
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA2
Author for correspondence: Robert A. Burne. Tel: +1 352 392 4370. Fax: +1 352 392 7357. e-mail: rburne{at}dental.ufl.edu
Differential expression of the Streptococcus salivarius 57.I urease operon in response to pH is effected by repression of transcription from a proximal promoter, PureI. To localize the cis-acting elements involved in the regulation of the urease operon, the intact promoter region and its derivatives were generated and fused to a promoterless chloramphenicol acetyltransferase (cat) gene. The promoter–cat fusions were established in the lacZ gene of S. salivarius by using a newly constructed integration vector. CAT-specific activities were examined in batch-grown cells at pH 7·5 and 5·5. The results indicated that a 21 bp region immediately 5' to the -35 element was required for efficient repression of PureI at neutral pH and that the 39 bp (-57 to -95) 5' to this region contained sequences required for optimal expression of PureI. A potential secondary repressor-binding site was tentatively identified further upstream of the -35 element (-96 to -115). To further analyse the cis-acting elements, base changes were introduced into two AT-rich repeats within the primary repressor-binding site. One such derivative, S. salivarius M1, with five base substitutions immediately 5' to the -35 element, expressed 20-fold more CAT-specific activity at neutral pH than the strain carrying wild-type PureI–cat. Also, the pH sensitivity of strain M1 was greatly reduced, suggesting that this AT-rich region is crucial for repression of the urease operon. Deletion of three consecutive 15- or 16-base segments from -52 to -96 in the S. salivarius M1 background resulted in lower activities compared to strain M1, confirming the presence of sequences required for optimal expression of the operon. All of the PureI–cat fusions were also integrated into the gtfG gene of Streptococcus gordonii DL1, a non-ureolytic oral Streptococcus sp. Repression of PureI was observed at neutral pH in S. gordonii and the effects of the various mutations of the repressor-binding site largely paralleled those seen in S. salivarius, suggesting that the cis-elements may be a target for a global regulatory circuit that controls gene expression in streptococci in response to pH.
Keywords: cis-elements, pHregulation, urease gene expression, oral streptococci
Abbreviations: cat and CAT, chloramphenicol acetyltransferase; PureI, promoter of the ure operon
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