H-074. Transcriptioanal Analysis of the S. epidermis Marcomolecular Synthesis Operon

K. A. Bryant, S. H. Hinrichs, P. D. Fey;
Univ. of Nebraska Med. Ctr., Omaha, NE.

Background: A bacterium’s ability to adapt by rapidly changing its transcriptional profile is a key component to survival. Little is known about the transcriptional regulation of key replicative enzymes. In order to better understand the transcriptional control of replication enzymes within bacteria, the macromolecular synthesis operon (MMSO) of Staphylococcus epidermidis 1457, which contains the essential replication enzyme primase, was studied. The MMSO of all gram positive bacteria studied thus far contain open reading frames (ORFs) coding for the replicative DNA primase (dnaG) and the major sigma factor of RNA polymerase (sigA). We hypothesize that the MMSO of S. epidermidis will be regulated differently from B. subtilis, the most well characterized gram positive MMSO, because the staphylococci are non-sporulating and contains only 3 sigma factors (σA , σB ,and σH ) Methods: Northern, RT-PCR, and primer extension analysis were used to characterize the S. epidermidis strain 1457 MMSO Results: A large 4.9 kb transcript was observed and suggests, unlike B. subtilis , that the first gene in the S. epidermidis MMSO is a protein of unknown function containing a CBS motif (cbs) followed by an 816 bp gene encoding for a separate protein of unknown function (puf). A smaller transcript of 1.8 kb is also detected, which contains the cbs and puf ORFs. Small transcripts containing sigA are also observed. Transcriptional comparisons between wild type 1457 and a 1457 σB knockout mutant demonstrate sigA is transcribed in late exponential phase by a σB dependent promoter. Conclusions: These data suggest that the S. epidermidis MMSO is regulated differently than that of B. subtilis; and in addition, sigA is regulated at the transcriptional level by the alternative sigma factor σB. This additional layer of regulation may function to ensure an adequate supply of σA is present during times of stress and could be critical for the return to a normal transcriptional state after stress. Finally in contrast to the B. subtilis MMSO, the S. epidermidis MMSO encodes for a CBS domain protein and an additional protein of unknown function.