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. 2009 Dec 11;192(4):1075–1087. doi: 10.1128/JB.01197-09

FIG. 6.

FIG. 6.

Expression of Crl wild-type (WT) and mutant proteins. (A) Detection of Crl produced from pACcrl-1 and its mutant derivatives in Salmonella ATCCcrl. ATCCcrl strains, harboring the vector pACYC184 and the pACcrl-1 derivatives as indicated below, were grown to stationary phase (OD600 of 4) in LB medium at 37°C and analyzed by Western blotting with anti-Crl antibodies. One microgram of total protein was loaded in slots 1 to 17. Lane 1, pACcrl-1; lane 2, pACYC184; lane 3, pACcrlY22A; lane 4, pACcrlF35A; lane 5, pACcrlC37A; lane 6, pACcrlC41A; lane 7, pACcrlF53A; lane 8, pACcrlW56A; lane 9, pACcrlG74A; lane 10, pACcrlG80A; lane 11, pACcrlW82A; lane 12, pACcrlF103A; lane 13, pACcrlC28A; lane 14, pACcrlC28-37; lane 15, pACcrlC28-41; lane 16, pACcrlC37-41; lane 17, pACcrlC28-37-41. Five micrograms of total protein was loaded in slots 18 to 20. Lane 19, pACcrlC37-41; lane 20, pACcrlC28-37-41; lane 18, a stationary phase culture of ATCC 14028 in LB medium at 37°C was used as control. (B) Expression of wild-type and altered Crl-T18 hybrid proteins. The E. coli BTH101 cya strain harboring pUT18 and its derivatives were grown to stationary phase (OD600 of 3) in LB medium in the presence of 2 mM cAMP and 0.5 mM IPTG to fully induce the lac promoter on pUT18. Five micrograms of total protein was loaded in each slot and analyzed by Western blotting with anti-Crl antibodies. Lane 1, no plasmid; lane 2, pUT18; lane 3, pUTCrl; lane 4, pUTCrlY22A; lane 5, pUTCrlF53A; lane 6, pUTCrlW56A; lane 7, pUTCrlG80A; lane 8, pUTCrlW82A; lane 9, pUTCrlR51A; lane 10, pUTCrlE52A; lane 11, pUTCrlG55A; lane 12, pUTCrlW57A. (C) Detection of Crl in Salmonella ATCC 14028 (lanes 1, 4, 7, 10, and 15) and its mutant derivatives ATCCfur (lanes 2, 5, 8, and 13), ATCCcrl (lanes 3, 6, 9, and 11), ATCCrpoS (lane 12), and ATCCrpoSfur (lane 14). Cultures were grown in LB medium (LB) overnight at 30°C or 37°C or on LB agar at 30°C, as described previously (28), and analyzed by Western blotting with anti-Crl antibodies. One microgram of total protein was loaded in each slot.