Expression of a Cryptic Secondary Sigma Factor Gene Unveils Natural Competence for DNA Transformation in Staphylococcus aureus
A) and B) Southern blot analysis of the sigH locus for the parental strain (RN4220), SigH active mutants (A2, B1, C1) and revertants (A2-r, B1-r, C1-r). Genomic DNA was digested with HindIII or PvuII. A) Blots were probed with a H1p-H2p PCR fragment (upstream region of sigH). B) Probed with H5p-SA0492Rp PCR fragment (sigH coding sequence). Positions of the DNA molecular mass marker (λ HindIII) are indicated in base pairs on the right. C) Schematic representations of the sigH loci of RN4220 and SigH active derivatives B1 and C1. Arrows represent coding sequences, and are colored by gene, e.g. red represents sigH, yellow represents nusG etc. The direct repeat sequences encompassing duplication units are indicated above the gene maps. These direct repeats originally exist in the RN4220 sequence, and the duplication event increases their number from two (in RN4220) to three (in B1/C1). Gene maps are illustrated to scale and the bar represents 1 kbp. D) Western blot analysis showing that SigH is expressed as a fusion protein encoded by the new chimeric genes. A2, B1, and C1 are SigH active derivatives. A2-r, B1-r, and C1-r are the revertants that have lost SigH activity. R: RN4220, RH: RN4220 + pRITsigH. The position of full-length native SigH (calculated molecular weight 23.0 kDa) is indicated by an arrow-head. Signals in B1 and C1 were observed at higher molecular weight positions, in agreement with the calculated molecular weights of the SigH chimeric proteins in strains B1 and C1 (28.3 kDa and 25.5 kDa, respectively). Sizes and positions of the Bench Mark pre-stained protein ladder (Promega) are indicated on the left. The lower panel is a loading control SDS-PAGE gel stained with Coomassie Brilliant Blue staining.