Genetic analysis of the Staphylococcus epidermidis Macromolecular Synthesis Operon: Serp1129 is an ATP binding protein and sigA transcription is regulated by both σA- and σB-dependent promoters

摘要

Background The highly conserved macromolecular synthesis operon (MMSO) contains both dnaG (primase) and sigA (primary sigma factor). However, in previously evaluated gram-positive species, the MMSO is divergent upstream of dnaG. The MMSO of Bacillus subtilis contains three open reading frames (ORFs) that are differentially regulated by multiple promoters. In conjunction with studies to determine the expression profile of dnaG, the MMSO of Staphylococus epidermidis was characterized. Results The ORFs of S. epidermidis were compared to the previously described MMSO of B. subtilis and two additional ORFs in S. epidermidis, serp1129 and serp1130, were identified. The largest transcript, 4.8 kb in length, was expressed only in exponential growth and encompassed all four ORFs (serp1130, serp1129, dnaG, and sigA). A separate transcript (1.5 kb) comprising serp1130 and serp1129 was expressed in early exponential growth. Two smaller transcripts 1.3 and 1.2 kb in size were detected with a sigA probe in both exponential and post-exponential phases of growth. Western blot analysis correlated with the transcriptional profile and demonstrated that Serp1129 was detected only in the exponential phase of growth. Computational analysis identified that Serp1130 contained a CBS motif whereas Serp1129 contained an ATP/GTP binding motif. Functional studies of Serp1129 demonstrated that it was capable of binding both ATP and GTP. Comparisons with a sigB:dhfr mutant revealed that the 1.3 kb sigA transcript was regulated by a σB-dependent promoter. Conclusions These studies demonstrated that the S. epidermidis 1457 MMSO contains two ORFs (serp1129 and serp1130) not described within the B. subtilis MMSO and at least three promoters, one of which is σβ-dependent. The transcriptional regulation of sigA by σB provides evidence that the staphylococcal σB-dependent response is controlled at both the transcriptional and post-transcriptional level. The conservation of serp1129 across multiple gram-positive organisms and its capability to bind ATP and GTP support the need for further investigation of its role in bacterial growth.