aureus database sequences and 97–98% identity amongst other staphylococci, including S. haemolyticus, S. epidermidis and S. saprophyticus, indicating that SA1665 is highly conserved. Conversely, there were no orfs highly similar to SA1665 found in other bacterial species, with the most similar sequences found in Bacillus licheniformis DSM13 and Desulfitobacterium hafniense Y51, which shared only 64% and 59% similarity, Milciclib cell line respectively. Figure 1 DNA-binding protein purification assay using mec operator DNA region as a bait. A, Silver stained SDS-polyacrylamide protein gel containing the elutions from DNA-binding protein capture assays performed with either DNA-coated
(+) or uncoated (-) Pifithrin-�� in vitro streptavidin magnetic beads. One protein band, indicated by the arrow, was only captured by the DNA-coated beads, indicating that it bound specifically to the mec operator
Selleckchem Oligomycin A DNA. The protein size marker (M) is shown on the left. B, Organisation of the genomic region surrounding SA1665. The regions used to construct the deletion mutants are indicated by lines framed by inverted arrow, which represent the positions of primers used for their amplification. The chromosomal organisation, after deletion of SA1665 is shown beneath. The position of the SA1665 transcriptional terminator, which remained intact after SA1665 markerless deletion is indicated (⫯). Electro mobility shift assays (EMSA) EMSA was used to confirm binding of SA1665 to the mec operator region. Crude protein extracts of E. coli strain BL21, carrying for the empty plasmid (pET28nHis6) or pME20 (pET28nHis6-SA1665) which expressed nHis6-SA1665 upon induction with IPTG, were incubated with
the 161-bp biotinylated-DNA fragment previously used as bait in the DNA-binding protein assay. A band shift was observed with extracts from the strain expressing recombinant nHis6-SA1665 but not from the control strain carrying the empty plasmid. Several bands resulted from the shift, which is most likely due to protein oligomerisation (Figure 2A). The specifiCity of the gel shift was also demonstrated by the addition of increasing concentrations of purified nHis6-SA1665 protein to the biotinylated-DNA fragment (Figure 2B). Band-shift of the biotinylated DNA was inhibited in the presence of specific competitor DNA but not by the presence of the non-specific competitor DNA, confirming that nHis6-SA1665 had a specific binding affinity for the 161-bp DNA fragment. Figure 2 Electromobility shift of mec operator DNA by SA1665. A, Gel shift using biotinylated DNA (6 ng) and crude protein extracts. Lane 1, DNA only control; lanes 2 and 3, DNA incubated with 200 ng and 500 ng of crude protein extract from E. coli BL21 pET28nHis6, respectively; lanes 4 and 5, DNA incubated with 200 ng and 500 ng of crude protein extract from E. coli BL21 pME20, expressing SA1665, respectively. B, Gel shift of biotinylated DNA (6 ng) with purified SA1665 protein.