The two-component system PhoPR of Clostridium acetobutylicum is involved in phosphate-dependent gene regulation

Abstract

The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in P(i)-limited chemostat cultures and in response to P(i) pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external P(i) concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under P(i)-limiting growth conditions compared to growth with an excess of P(i). At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

FIG. 1.

Gene architecture and localization of the phoPR operon of C. acetobutylicum. Numbers above the black line indicate the localization of the gene region on the chromosome. Arrows symbolize the orientation of transcription of the annotated open reading frames on the chromosome of C. acetobutylicum, and known gene names are given below in boldface letters.

FIG. 2.

PhoR proteins (A) and PhoP proteins (B) of E. coli (Ec), B. subtilis (Bs), and C. acetobutylicum (Ca). Schemes with the domain architectures of the polypeptides are presented at the tops of panels A and B. TM, transmembrane domain; HiskA, histidine kinase A dimerization/phosphoacceptor domain. Below the schemes, alignments of selected protein regions (I to IV in the case of PhoP and the N and C termini in the case of PhoR) are shown. Letters represent the amino acid residues, gray boxes highlight homologous or identical residues (accepted as homologous substitutions are A and G; R and K; F and Y; S and T; D and E; N and Q; and I, L, V, and M), and asterisks indicate conserved phosphorylation sites.

FIG. 3.

Transcription of the phoPR operon of C. acetobutylicum in response to phosphate depletion. Individual RT-PCR analyses of genes phoP, phoR, and ptb with total RNA of cells after a phosphate pulse are shown. External phosphate concentrations are given below the lanes (0 mM P_i symbolizes steady-state conditions). The ptb gene encoding phosphotransbutyrylase, which is expected to be constitutively expressed under experimental conditions, was used as the positive control. Bars represent relative signal intensity (rel. intensity), and the maximum signal intensity was set as 100%.

FIG. 4.

Expression of PhoP under different phosphate concentrations. (A) Shown are sections of 2-DE gels with PhoP proteins with a surplus of P_i (6.7 mM) or P_i limitation (0 mM). The lines mark the spots of PhoP. The other spots serve orientation purposes (9309, dihydrodipicolinate reductase [dapB, cac2379]; 9317, acetoacetate decarboxylase [adc, cap0165]; 9324, triosephosphate isomerase [tpi, cac0711]; 9344, adenylate kinase [adk, cac3112]; 9318, not identified [score below 65]). (B) Distribution of normalized spot volumes (arbitrary units [a.u.]; n = 3) for PhoP (see Fig. 4A) are shown as box-and-whisker plots. The boxes represent the upper and lower quartiles. The separation line in the box marks the 50% value (mean; not visible). The whiskers indicate the 5th and 95th percentiles, respectively. Average values are depicted as little triangles. Upper and lower extreme values are indicated with a minus sign. The × symbol shows the 99% and 1% range of the individual values, respectively.

FIG. 5.

Transcription start points of phoP of C. acetobutylicum. The products of primer extension reactions using IRD800-labeled oligonucleotides were run on polyacrylamide gels alongside the corresponding sequencing reactions (T, G, C, A) generated with the same primer (A). (B) Locations of the identified transcription start points, PE1 (primer, phoP-neu-PE2), PE2 (phoP-neu-PE3), and PE3 (phoP-neu-PE3) are highlighted (gray circles) within the DNA sequence of the promoter region upstream of the phoP gene. The boldface ATG symbolizes the start codon of the phoP gene, and the arrow indicates its orientation; its dedicated ribosome binding site is underlined. Putative regulatory transcriptional elements of the transcription start points are boxed with a −10 or −35 (see text for details). Numbers in parentheses indicate their assignment to the corresponding transcription start points. Broken lines appear above possible regulatory repeats.

FIG. 6.

Purification of heterologously expressed PhoP Strep-Tag (A) and MBP-*PhoR (B). Coomassie-stained SDS-PAGE (12.5%). M, protein molecular weight marker; W, washing fractions; E, elution fractions.

FIG. 7.

Autophosphorylation of MBP-*PhoR and phosphate transfer to PhoP Strep-Tag. Ten μCi of [γ-³²P]ATP was incubated with 2 μM MBP-*PhoR (lane 1) or with 2 μM MBP-*PhoR and 17 μM Strep-Tag-PhoP (lane 2) for 15 min at room temperature and was separated by SDS-PAGE (12.5%). Shown is an autoradiogram; relative signal intensities referring to the certain proteins are given below the lanes.

FIG. 8.

Binding of PhoP to the pst promoter. Electromobility shift assays were carried out with 0.5 ng 3′ DIG-labeled pst promoter fragment P3 (A), fragment P1 (B), and fragment P4 (C) in the presence of 5 mM ATP. Fragments were incubated without protein (lane 1), with 5 μM Strep-Tag-PhoP (lane 2), with 5 μM Strep-Tag-PhoP and 1.5 μM MBP-*PhoR (lane 3), or with 5 μM Strep-Tag-PhoP, 1.5 μM MBP-*PhoR, and 100 ng unlabeled fragment. (D) As negative controls, unspecific DIG-labeled DNA fragments were incubated under the same conditions without protein (lane 1) and with 5 μM Strep-Tag-PhoP and 1.5 μM MBP-*PhoR (lane 2). Additionally DIG-labeled fragment P3 was incubated without protein (lane 3) and with 3 μM MBP-*PhoR (lane 4).