The impact of the expression of wild type or mutated Gcp in trans on the growth of the Pspac-regulated gcp expression strain.

<p>(A) Construction of Gcp complementation system using the <i>Pspac</i>-regulated <i>gcp</i> expression strain and Western blot analysis of <i>gcp</i> expression <i>in trans</i>. Lane 1, purified recombinant Gcp; lane 2, negative control (JRN0210); lane 3, Gcp complementary strain (JNR0110). Without an inducer, ATc, Gcp is constitutively expressed due to the leakage of <i>Pxyl/tetO</i> promoter. (B–H) Growth curves of Gcp complementary strains. The <i>Pspac</i>-regulated <i>gcp</i> expression <i>S. aureus</i> strain carrying the staphylococcal wild-type <i>gcp</i> complementary plasmid, pXL108 was used as positive control (B); carrying parental plasmid, pMY1107, as a negative control (C); carrying the <i>gcpseg1</i> complementary plasmid, pLT109 (D); carrying the <i>gcpseg2</i> complementary plasmid, pLT209 (E); carrying the <i>gcp1-1</i> complementary plasmid, pLT309 (F); carrying the <i>gcp1-2</i> complementary plasmid, pLT409 (G); carrying the <i>gcp1-3</i> complementary plasmid, pLT509 (H). The above strains were incubated overnight in TSB in the presence of appropriate antibiotics and 0.2 mM IPTG. Bacteria were diluted and incubated in fresh TSB containing appropriate antibiotics and different concentration of IPTG at 37°C with shaking. The cell growth was monitored by measuring OD_{600 nm} every 15 min for 18 h with 1 min mixing before each reading.