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PF-MglA(T47A)-V, and pFMglA(Y63A)-V, respectively, except that they confer resistance to nourseothricin. The plasmid pF-MglA has been previously described (10). The pF3 plasmids listed above were generated by replacing the kanamycin resistance gene in pF-MglA-V, pF-MglA, pF-MglA(T47A)-V, and pF-MglA(Y63A)-V with the nourseothricin resistance gene from the previously described pF3 plasmid (21). Genetic screens for mutants of MglA or SspA specifically defective for interaction with PigR. The mglA portion of the fusion in the vector pBR-MglA- was mutagenized using error-prone PCR with Taq polymerase and primers flanking the mglA gene. The PCR product was then digested with the restriction enzymes NdeI and NotI and inserted in the pBR-MglA- vector to generate a library of plasmids that direct the synthesis of MglA- fusion proteins with random mutations in the mglA moiety of the mglA- fusion gene. This library was transformed into KDZif1 Z cells along with plasmids pCL-SspA and pACTR-PigR. Cells were plated on LB containing carbenicillin, spectinomycin, tetracycline, X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside; 50 g/ml), IPTG (isopropyl- -D-thiogalactopyranoside; 50 g/ml), and the X-Gal inhibitor tPEG (2-phenylethyl -D-thiogalactoside; 125 g/ml). Approximately 120 colonies were selected in which cells had low levels of lacZ expression compared to cells expressing wild-type MglA- along with PigR-Zif and LVS SspA. These colonies were struck out on LB plates containing carbenicillin to select for only those cells containing the pBRMglA- vector. The pBR-MglA- plasmids expressing various mutant MglA- fusion proteins were then isolated, pooled, and subsequently transformed into KDZif1 Z cells along with pACTR-SspA-Zif. These cells were then plated on LB containing carbenicillin, tetracycline, X-Gal (50 g/ml), and IPTG (50 g/ml). Approximately 40 colonies were selected in which cells expressed levels of lacZ similar to those in cells expressing the wild-type MglA- fusion protein and SspA-Zif. pBRMglA- plasmids were isolated from these colonies and transformed back into KDZif1 Z cells with either pCL-SspA and pACTR-PigR-Zif or pACTR-SspA-Zif and assayed for -galactosidase activity. Plasmids directing the synthesis of MglA- mutant proteins that were specifically defective for interaction with PigR were then sequenced to determine the corresponding mutation. The genetic screen for SspA mutants that were specifically defective for interaction with PigR was essentially the same as that described above forOctober 2014 Volume 196 Numberjb.asm.orgRohlfing and DoveMglA mutants, except that a library of SspA- mutants was generated using error-prone PCR with Taq polymerase and vector pBR-SspA- . This library was analyzed first in KDZif1 Z cells along with the vectors pCL-MglA and pACTR-PigR-Zif. Then, approximately 100 candidate mutants were screened in KDZif1 Z cells containing plasmid pACTRMglA-Zif. Finally, 40 colonies were selected in which cells expressed lacZ levels similar to those in cells expressing the wild-type SspA- fusion protein in the presence of MglA-Zif. Plasmids were isolated from cells from these colonies and tested in the bridge-hybrid assay and two-hybrid assays to confirm that the isolated mutants were specifically defective for interaction with PigR. Plasmids directing the synthesis of SspA- mutant fusion proteins that were specifically defective for interaction with PigR were sequenced to identify the corresponding mutation.