Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major or-factor r 7~ To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of ~r 7~ Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.

Induction of vancomycin resistance in the Gram-positive Enterococci requires a two-componet regulatory system, VanS and VanR, for transcriptional activation of three genes (vanH, A, X) that encode enzymes for a cell wall biosynthetic pathway that produces an altered peptidoglycan intermediate with lower affinity for the antibiotic. The catalytic efficiency (kcat/KM) has been determined for phosphotransfer from the phosphohistidyl form of VanS to both its homologous partner VanR and the heterologous (Escherichia coli) response regulator Phob. The rate of formation of the phosphoaapartyl forms of VanR and PhoB were determined as well as the rate of appearance of inorganic phosphate. Using PhoB in excess of P-VanS, a pseudo-first-order rate constant (kxfer) of 0.2 min-1 for phosphotransfer and a KM for PhoB of 100 microM were readily determined. The corresponding kxfer of 96 min-1 for phosphotransfer from P-VanS to VanR required quench kinetics. A KM of 3 microM was estimated for VanR, leading to a 10(4)-fold preference in kxfer/KM for phosphotransfer to VanR compared to PhoB. No phosphotransfer was detachable to three other E. coli response regulators, OmpR, ArcB, or CreB, providing some sense of the selectivity against two-component regulatory system cross-talk. In the phosphotransfer from P-VanS to PhoB and VanR, there was evidence of competition between water, to give Pi, and the specific aspartyl beta-COO- moiety of either PhoB or VanR with about 25% of the initial flux generating inorganic phosphate. The kinetics of phosphotransfer from P-VanS to VanR were complicated by inhibition by free VanS but, the inhibition pattern could be modeled to yield at KD of 30 nM for VanR binding to free VanS, an affinity similar to that of the CheA-CheY pair in E. coli chemotaxis.

By making operon fusions with XplacMu53, we identified, cloned, and analyzed the phoH gene belonging to the phosphate (pho) regulon. We mapped the phoH gene at 23.6 min in the Escherichia coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50: [495][496][497][498][499][500][501][502][503][504][505][506][507][508] 1987 dium supplemented with excess phosphate (TGHP) or limiting phosphate (TGLP) were described previously (2). The media used for the routine preparation of M13 phages were described previously (1). To detect P-galactosidase (P-Gal) activity of the colony, 40 pug of 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-Gal) per ml was added to agar plates (28). The activities of chloramphenicol acetyltrans-1316