Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development

Abstract

ComP is a sensor histidine kinase of Bacillus subtilis required for the signal transduction pathway that initiates the development of competence for genetic transformation. It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the srfA promoter. In the present study, fusions to the Escherichia coli proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP. These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain. Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction. The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX. The implications of these data are discussed.

FIG. 1

Model for ComP topology. The eight putative membrane-spanning segments are (counting from the amino terminus) are as follows: 1, I10 to I33; 2, Y114 to F134; 3, S145 to G167; 4, L236 to L257; 5, K272 to F295; 6, I300 to A323; 7, Y338 to F357; and 8, I362 to F383. Sites of fusion with alkaline phosphatase (H, F, I, L, M, N, Z, P, G, and Y) and with β-galactosidase (R, S, W, T, U, V, and X) are indicated. The conserved histidine kinase domain is shown as a rectangle. The predicted kallikrein cleavage sites are indicated by dots. The transmembrane segments are numbered, and the cytoplasmic and extracellular loops referred to in the text are labeled for convenience.

FIG. 2

srfA-lacZ expression in comP mutants. In each panel the diagram at the top presents the extent and position of the relevant deletion. The dots on these diagrams indicate the positions of the S and R insertions introduced during construction of the deletions. In each panel, β-galactosidase activities are presented for the strains grown in the absence of IPTG (▵, □); the solid symbols are for strains grown in the presence of IPTG. Strains are designated as follows: The comP wild-type strain (BD1890) in a comX⁺ background (●) and the comP mutant strains in comX⁺ (□, ■) and comX backgrounds (▵, ▴) are as indicated. (A) ΔLabc comP BD2757 (comX⁺) and BD2744 (comX). (B) ΔLbc comP BD2758 (comX⁺) and BD2745 (comX). (C) ΔLc comP BD2759 (comX⁺) and BD2746 (comX). (D) XbaI comP mutant strains BD2760 (comX⁺) and BD2747 (comX). Time is given in hours before or after T₀, the point of transition from exponential to stationary phase.

FIG. 3

Response of srfA-lacZ expression to conditioned medium in strains carrying wild-type and mutant comP. The open symbols indicate β-galactosidase activities for strains grown with conditioned medium obtained from a comX strain (BD2356). The solid symbols indicate β-galactosidase activities for strains grown with conditioned medium from a comX⁺ strain (IS75). The strains grown in these conditioned media carried the XbaI comP (●, ○; BD2747) or the ΔLc comP (■, □; BD2746) mutations. Time is given in hours before or after T₀.

FIG. 4

Detection of ComP-PhoA hybrid proteins in B. subtilis membranes. Membrane vesicles were prepared from B. subtilis strains carrying comP-phoA hybrid genes in a single copy on the chromosome. Equal amounts (10 μg of protein/lane) of membrane vesicles were separated by SDS-PAGE on a 10% gel, and monoclonal anti-alkaline phosphatase antibody was used for the immunoblotting. Fusion proteins are indicated by letters, and their positions are denoted by asterisks. The numbers in parentheses represent alkaline phosphatase values normalized to that associated with fusion H (Table 2). Strain BD630, which lacks a comP-phoA fusion, was used as a negative control (−). The positions of molecular size standards (in kilodaltons) are shown, as is the position of mature alkaline phosphatase (AP).

FIG. 5

Detection of ComP-PhoA hybrid proteins in E. coli by Western blotting. Total cell extracts containing equal amounts of protein were separated by SDS-PAGE on a 10% gel and visualized by immunoblotting with monoclonal anti-alkaline phosphatase antibody. Fusion proteins are indicated by letters, and their positions are denoted by arrows. The numbers in parentheses represent alkaline phosphatase values normalized to that associated with fusion H (Table 2). A strain carrying the phoA vector with no comP insert was used as a negative control (−). The positions of molecular size standards (in kilodaltons) are shown, as is the position of mature alkaline phosphatase (AP).

FIG. 6

Proteolysis of ComP with kallikrein. Protoplasts of B. subtilis were prepared from strain BD2358 (lanes 3 to 8), which carries comP on a multicopy plasmid, and from strain BD2362 (lanes 1 and 2), which carries a complete deletion of comP. Samples were incubated at 37°C with the protease kallikrein (K) at a final concentration of 100 μg/ml (+) or 200 μg/ml (++). Triton X-100 (T) was added to some of the samples to permeabilize the protoplasts. Proteins were transferred to nitrocellulose and analyzed by immunoblotting with anti-ComP antisera. Arrows indicate the positions of ComP and of the main proteolytic product derived from ComP. The positions of molecular size standards (in kilodaltons) are shown.