Chapter 15 : The Pho Regulon

(A) PhoP core binding region found between bp −20 and −60 in promoters activated by PhoP∼P. The core contains four repeats of conserved 6-bp sequence that is believed to bind two dimers based largely on mutagenesis of the PhoD promoter. (B) Determination of the conserved sequence for B. subtilis PhoP binding site repeats. The TTAACA-like sequences from 11 Pho regulon promoters were aligned. The tabulated numbers represent the number of times a specific nucleotide appears at that position. The boldface number identifies the most frequent nucleotide at that position. The promoters and the number of consensus repeats used from each included tu&A (8 consensus repeats) ( 31 ), phoA (6 repeats) ( 32 ), pstS (4 repeats) ( 32 ), pfioB (4 repeats) ( 30 ), tagA (4 repeats) ( 29 ), tagD (2 repeats) ( 29 ), phoD (6 repeats) ( 9 ), resA (2 repeats) ( 5 ), resD (2 repeats) ( 5 ), ykoL (4 repeats) ( 48 ), and glpQ (4 repeats) ( 1 ). (C) Tally of the frequency at which the assigned consensus nucleotide appears at each position in the 46 repeats.

Insertion and/or deletion mutagenesis of phoD promoter. (A) The wild-type promoter. Solid rectangles represent 1 1 1 ACA-like sequences at approximately −25, −35, −45, −55, −185, and −195 relative to the transcription start site. A straight arrow (→ or ←) shows the 5′ to 3′ direction of two consensus repeats (two solid rectangles separated by nonconserved sequence) believed to bind one PhoP dimer. Two dimer-binding sites separated by approximately five nonconserved bp make up the PhoP core binding region. Diagonal lines (//) indicates that the distance between upstream binding site and core binding region is not to scale. The bent arrow indicates the start of the PhoD coding sequence. Percent of transcription activity is designated 100% for the wild-type promoter. (B) Insertion mutation between consensus repeats in a putative dimer-binding site. Triangle (Δ) indicates the point of insertion of 5 or 10 bp. (C) Insertion between two dimer binding sites (Δ). (D) Insertion between core binding region and upstream secondary binding site (Δ). (E) Deletion of upstream secondary PhoP binding site. (F) Inverted orientation of secondary 5′ PhoP dimer binding site (↔). The effect of each promoter mutation (B, C., D, E, and F) is given as percent transcription activity compared with the wild-type promoter. Where two numbers are given, the first number reports results for a 5-bp insertion that would change the face of the helix, and the second number reports results for a 10-bp insertion that should restore the face of the helix, albeit with a 10-bp distance change.

Model depicting the signal transduction network leading to Pho induction in B. subtilis a phosphate deficiency response. Proteins are indicated by ovals, and genes/operons are symbolized by rectangles. Solid lines indicate that direct interaction has been demonstrated. Dashed lines are utilized for interactions that could either be direct or indirect. Positive regulation is labeled with an arrow and a plus sign (+), while repression is noted by an arrow and a minus sign (−). Two-component system members are labeled as either the histidine kinase (HK) or response regulator (RR).

The PhoR protein. The domains and the predicted topology of wild-type PhoR protein. Numbers indicate the approximate boundaries of each domain. Solid boxes represent the transmembrane sequence. The hatched box represents the conserved catalytic domain. TM1, the first transmembrane sequence; P, periplasmic or extracytoplasmic domain; TM2, the second transmembrane sequence; C2, the cytoplasmic linking domain containing a putative PAS domain adjacent to the catalytic domain; diagonal-striped box, the conserved catalytic domain; H360, the conserved histidine residue; O, outside of membrane; I, inside cytoplasmic membrane.