cis-Acting elements that regulate the low-pH-inducible urease operon of Streptococcus salivarius.
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.
Chen
Yi-Ywan M
YM
Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA1.
Betzenhauser
Matthew J
MJ
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA2.
Burne
Robert A
RA
Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA1.
D024506:5' Flanking Region / Q000235:genetics; D020856:AT Rich Sequence / Q000502:physiology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics; D014510:Urease / Q000096:biosynthesis / Q000235:genetics*
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012097:Repressor Proteins; D014510:Urease