Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis

Abstract

The phosphate starvation response in Bacillus subtilis was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of sigma(B), the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhF confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a sigma(A) promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed with phoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in both phoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by sigma(B)-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a sigma(B)-dependent manner and in the sigB mutant probably via sigma(H). YxiE was induced by phosphate starvation independently of sigma(B) and PhoPR.

FIG. 1

Synthesis of cytoplasmic phosphate starvation-inducible proteins in the B. subtilis wild type labeled with l-[³⁵S]methionine before (168 control) and 1 h after entry into the stationary phase provoked by phosphate starvation (168 1h phosphate starvation). PhoPR-dependent proteins are indicated by boxes, PhoPR-independent proteins are indicated by broken underlining, and proteins which are controlled by both ς^B and other sigma factors are indicated by solid underlining. The remaining proteins are ς^B-dependent Gsp proteins.

FIG. 2

Synthesis of cytoplasmic phosphate starvation-inducible proteins in the B. subtilis phoR mutant labeled with l-[³⁵S]methionine before (ΔphoR control) and 1 h after entry into the stationary phase provoked by phosphate starvation (ΔphoR 1h phosphate starvation). For details, see the legend to Fig. 1.

FIG. 3

Synthesis of cytoplasmic phosphate starvation-inducible proteins in the B. subtilis sigB mutant labeled with l[³⁵S]methionine before (ML6 control) and 1 h after entry into the stationary phase provoked by phosphate starvation (ML6 1h phosphate starvation). For details, see the legend to Fig. 1.

FIG. 4

Extracellular phosphate starvation-inducible proteins in the B. subtilis wild type. PhoPR-dependent proteins are indicated by boxes, and the other labeled proteins are PhoPR-independent phosphate starvation-inducible proteins. The 2D gels were stained with Coomassie blue R-250.

FIG. 5

Physical organization of glpTQ (A), transcript analyses of glpQ (B and C), and sequence of the glpQ promoter region (D). For Northern blot (B) and primer extension (C) experiments, 10 μg of RNA each was isolated from wild-type B. subtilis 168 and the phoR mutant before (control) and at different times after (30, 60, 90, 120, and 150 min) entry into the transient phase (t₀) provoked by phosphate starvation. The probable 5′ end of the ς^A-dependent glpQ message is marked by +1 (C and D). The −10 and −35 promoter sequences of the ς^A-dependent transcript are indicated by boxes, and the putative PhoP binding sites are underlined (D). The dideoxy sequencing ladder (ACGT) (C) extends from the same primer as that used for the primer extension experiments and is complementary to that determined by DNA sequencing.

FIG. 6

Physical organization of the phoB-ydhF operon (A) and transcript analysis of ydhF (B). For Northern blot experiments (B), 10 μg of RNA was isolated from wild-type B. subtilis 168 and the phoR mutant before (control) and at different times after (30, 60, 90, 120, and 150 min) entry into the transient phase (t₀) provoked by phosphate starvation.

FIG. 7

Physical organization of yjbCD (A), transcript analyses of yjbC (B and C), and sequence of the yjbC promoter region (D). For Northern blot (B) and primer extension (C) experiments, 10 μg of RNA each was isolated from wild-type B. subtilis 168 and the sigB mutant ML6 before (control) and at different times after (30, 60, 90, and 120 min) entry into the transient phase (t₀) provoked by phosphate starvation. The probable 5′ ends of the yjbC-specific transcripts are marked by +1 (C and D). The −10 and −35 promoter sequences are indicated by boxes (D). The dideoxy sequencing ladder (ACGT) (C) extends from the same primer as that used for the primer extension experiments and is complementary to that determined by DNA sequencing.

FIG. 8

Physical organization of the yfhKLM operon (A) and transcript analysis of yfhM (B). For Northern blot experiments (B), 10 μg of RNA was isolated from wild-type B. subtilis 168, the sigB mutant ML6, and the phoR mutant before (control) and at different times after (30, 60, and 90 min) entry into the transient phase (t₀) provoked by phosphate starvation.

FIG. 9

Physical organization of the yxiE gene (A) and transcript analysis of yxiE (B). For Northern blot experiments (B), 10 μg of RNA was isolated from wild-type B. subtilis 168, the sigB mutant ML6, and the phoR mutant before (control) and at different times after (30, 60, and 90 min) entry into the transient phase (t₀) provoked by phosphate starvation.