Regulation of transcription by SMU.1349, a TetR family regulator, in Streptococcus mutans

Abstract

The TetR family of transcriptional regulators is ubiquitous in bacteria, where it plays an important role in bacterial gene expression. Streptococcus mutans, a gram-positive pathogen considered to be the primary etiological agent in the formation of dental caries, encodes at least 18 TetR regulators. Here we characterized one such TetR regulator, SMU.1349, encoded by the TnSmu2 operon, which appeared to be acquired by the organism via horizontal gene transfer. SMU.1349 is transcribed divergently from the rest of the genes encoded by the operon. By the use of a transcriptional reporter system and semiquantitative reverse transcription-PCR (RT-PCR), we demonstrated that SMU.1349 activates the transcription of several genes that are encoded within the TnSmu2 operon. Gel mobility shift and DNase I footprinting assays with purified SMU.1349 protein demonstrated binding to the intergenic region between SMU.1349 and the TnSmu2 operon; therefore, SMU.1349 is directly involved in gene transcription. Using purified S. mutans RpoD and Escherichia coli RNA polymerase, we also demonstrated in an in vitro transcription assay that SMU.1349 could activate transcription from the TnSmu2 operon promoter. Furthermore, we showed that SMU.1349 could also repress transcription from its own promoter by binding to the intergenic region, suggesting that SMU.1349 acts as both an activator and a repressor. Thus, unlike most of the TetR family proteins, which generally function as transcriptional repressors, SMU.1349 is unique in that it can function as both.

Fig. 1.

Protein profile of the S. mutans wild-type strain (UA159/pIB184), the SMU.1349 mutant strain (IBSC07/pIB184), and the complemented strain (IBSC07/pIBC55). Whole-cell lysates were prepared from strains grown overnight in THY medium. Equal amounts of protein were resolved in a 4 to 20% gradient SDS-PAGE gel and were stained with Coomassie brilliant blue stain. The band, indicated by the arrow, was excised and identified by mass spectrometry as described previously (6). M, prestained molecular weight marker (Fermentas).

Fig. 2.

Regulation of transcription by SMU.1349 at the TnSmu2 locus. (A) Schematic representation of the TnSmu2 locus. Open reading frames are represented by shaded arrows, and the orientation indicates the direction of transcription. Bent arrows indicate the putative transcription start site, and asterisks indicate the genes used for qRT-PCR analysis. (B) Glucuronidase assay results demonstrating the effect of SMU.1349 mutation on the P_SMU.1348 promoter. (C) Quantitative RT-PCR assay indicating the effects of SMU.1349 mutation on the expression of the SMU.1348, SMU.1342, and SMU.1339 genes in the TnSmu2 locus. The results are representative of two to three independent experiments each. The expression of genes was normalized to 1 in the wild-type strain with respect to gyrA expression. See the text for details.

Fig. 3.

Interaction of the SMU.1349 protein at the SMU.1348 promoter region. (A) A gel shift assay was performed using 1 pmol of a DNA fragment containing the putative SMU.1348 promoter region (∼275 bp), incubated with increasing amounts of His-tagged SMU.1349. Lane 1, no protein; lane 2, 28 pmol; lane 3, 56 pmol; lane 4, 84 pmol; lane 5, 112 pmol; lane 6, 140 pmol; lane 7, specific competition using 112 pmol protein and 20 pmol of cold P_SMU.1348; lane 8, nonspecific competition using 112 pmol protein and 20 pmol of cold P_nlmA (212 bp). (B) DNase I protection assay demonstrating the interaction of the SMU.1349 protein at the SMU.1348 promoter region. The position of the protected region is indicated with reference to the translation start site of SMU.1348. Asterisks indicate DNase I-hypersensitive sites. The results are representative of two independent experiments. See the text for details.

Fig. 4.

In vitro transcription runoff assay done using the putative promoter region of SMU.1348 (200 ng) in the presence of increasing amounts of SMU.1349 protein (45 to 675 pmol) with E. coli core RNA polymerase (Epicentre) and purified S. mutans sigma factor (RpoD). Transcription products were resolved on an 8% denaturing urea gel. This gel is representative of two independent experiments.

Fig. 5.

Interaction of SMU.1349 at its own promoter. (A) A GUS assay indicates that SMU.1349 acts as a repressor of its own expression. (B) SMU.1349 interacts directly at its own promoter, as indicated by a gel shift assay. EMSA was performed using 1 pmol of a DNA fragment containing the putative promoter region of SMU.1349 (189 bp), incubated with increasing amounts of His-tagged SMU.1349 protein. Lane 1, no protein; lane 2, 28 pmol; lane 3, 56 pmol; lane 4, 84 pmol; lane 5, 112 pmol; lane 6, 140 pmol; lane 7, specific competition using 112 pmol protein and 20 pmol of cold P_SMU.1349; lane 8: nonspecific competition using 112 pmol protein and 20 pmol of cold P_nlmA (212 bp). (C) DNase I protection assay demonstrating the interaction of SMU.1349 protein at the SMU.1349 promoter region. The positions of the protected regions are indicated with reference to the translation start site of SMU.1349. Asterisks indicate DNase I-hypersensitive sites. The results are representative of two independent experiments. See the text for details.

Fig. 6.

Schematic representation of the possible model of transcription regulation by the SMU.1349 protein at the SMU.1349–SMU.1348 intergenic region. Binding of the monomeric or oligomeric forms of the regulator causes localized bending of the SMU.1349 promoter, leading to transcription repression, whereas the regulator recruits the RNA polymerase (RNAP) to the SMU.1348 promoter region, initiating transcription at this locus. The bent arrows indicate the putative transcription start site.