FIGURE 3.

Abilities of the parental and helix II chimeras to support growth of the arabinose-dependent ΔacpP strain in the absence of arabinose and to be modified by 4′-phosphopantetheine attachment. A, the strains were grown in the presence of IPTG to induce expression of the ACP genes in the presence or absence of Sfp as shown above the plates. The plates were incubated for 20 h at 37 °C. B, the plasmids tested in A plus all of the other constructed plasmids were transformed into the ΔpanD derivative of the arabinose-dependent ΔacpP Sfp-producing strain CY2211. These strains were then starved for β-alanine and labeled with β-[2,3-³H]alanine synthesized from l-[2,3-³H]aspartic acid as described under “Experimental Procedures.” The gels were of 20% polyacrylamide that lacked a denaturant. The mobility of ACP species in these gels is due to their acidic charge plus their Stokes radii (which can be altered by acylation) (26). In the lower gel, the samples of lanes 11–15 were treated with 10 mm dithiothreitol at pH 9.5 for 1 h prior to loading. This treatment cleaves ACP acyl-thioester and disulfide bonds (27). The same treatment of the samples of lanes 1–10 gave essentially identical results. Lanes 12 and 14 are duplicates from separate colonies, whereas lane 13 was another colony from the same plate that grew poorly. Restriction enzyme mapping of the plasmid in this strain showed that part of the lacI gene had been deleted, which seems likely to account for the high expression of the modified protein. The increased labeling observed indicates that Sfp activity is present in excess.