FIGURE 1.

H-NS is required for repression of PhoP-dependent transcription in high Mg²⁺ conditions. A, RT-PCR analysis of mRNA level of PhoP-activated genes: type I, phoP, pcgL, pagK, and phoN; type II, pagC and ugtL; synthesized from bacteria was determined in wild-type (wt, 14028s), hns mutant (YS11708), hns mutant (YS11708) harboring pUHE21–2lac^q with a wild-type copy of the hns gene (phns, pYS1118), phoP mutant (YS11590), and hns phoP mutant (YS11945). Wild-type chromosomal DNA (chr) was used as control. The constitutively transcribed rpoD gene indicated that similar amounts of total RNA were used. The protein levels of H-NS were determined in the same growth conditions from strains harboring hns-HA fusion in the chromosome or in plasmid pYS1119, whose sequence is identical to pYS1118 except a HA tag sequence at the C terminus of the hns gene (Table 1). B, P_phoP1-lacZ transcriptional fusion expressed by bacteria harboring pYS1100 was determined in wild-type (14028s), phoP mutant (YS11590), hns mutant (YS11708), hns mutant (YS11708) harboring pYS1118, and hns phoP mutant (YS11945) strains. C, pcgL-lacZ transcriptional fusion expressed by bacteria was determined in wild-type (YS10382), phoP mutant (YS11743), hns mutant (YS11744), hns mutant (YS11744) harboring pYS1118, and hns phoP mutant (YS11745) strains. D, pagC-lacZ transcriptional fusion expressed by bacteria was determined in wild-type (YS11644), slyA mutant (YS11664), phoP mutant (YS11782), hns mutant (YS11780), hns mutant (YS11780) harboring pYS1118, hns slyA mutant (YS11935), hns phoP mutant (YS11932), hns phoP slyA mutant (YS11938), and hns phoP slyA mutant (YS11938) harboring pYS1118 strains. Bacteria were grown in N minimal medium, pH 7.4, with 0.01 mm (low or L) and 10 mm (high or H) Mg²⁺. Complementation required the addition of 0.2 mm IPTG. All graphed values (Miller unit) are mean ± S.D., and data correspond to three independent assays conducted in duplicate.