Evidence that SpoIVFB is a novel type of membrane metalloprotease governing intercompartmental communication during Bacillus subtilis sporulation

Abstract

Processing of pro-sigma(K) in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.

FIG. 1

Model showing proteins involved in the signal transduction pathway that governs pro-ς^K processing. The top part shows a sporangium in which engulfment of the forespore (FS) has been completed. Dots represent pro-ς^K associated with the mother cell (MC) membrane and the outermost membrane surrounding the FS (39). Triangles represent SpoIVFA and SpoIVFB associated with the outermost membrane surrounding the FS (26). The bottom part shows an expanded view of the two membranes surrounding the FS. The topologies depicted for BofA, SpoIVFA, and SpoIVFB are based on analysis of lacZ and phoA fusions in E. coli (8, 35). The HELGH and DG sequences of SpoIVFB subjected to mutational analysis appear to be in transmembrane segments. The model depicts the hydrophobic prosequence of pro-ς^K (13, 33) inserted into the outermost membrane surrounding the FS, but it is not known how pro-ς^K associates with membranes (39). Cleavage of pro-ς^K releases ς^K into the MC, where it binds core RNAP and directs transcription.

FIG. 2

Rescue of pro-ς^K processing by pSL16 and mutant derivatives. Extracts were prepared from cells (10) collected 5 h after the onset of sporulation in DSM medium (21). Proteins (10 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis with polyclonal anti-pro-ς^K antibodies diluted 1:10,000 (39). Lane 1, PY79; lane 2, BSL51/pDG148; lane 3, BSL51/pSL16; lane 4, OR745/pDG148; lane 5, OR745/pSL16; lane 6, OR745/pSL16 I42P; lane 7, OR745/pSL16 E44D.

FIG. 3

Detection of SpoIVFB produced from pSL16 and mutant derivatives. Extracts were prepared from cells (10) collected 3 h after the onset of sporulation. Proteins (10 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis (39) with affinity-purified anti-SpoIVFB antibodies diluted 1:500 (26). The generation of anti-SpoIVFB antiserum and affinity purification of antibodies is described at our web site (http://www.bch.msu.edu/faculty/kroos.htm). (A) Wild-type PY79 and spoIVFB mutant OR745 containing pDG148, pSL16, or pSL16 encoding the indicated amino acid substitution. Sporulation was done in DSM medium (21). (B) Wild-type PY79 and spoIVFAB mutant BSL51 containing pDG148, pSL16, or pSL16 encoding the indicated amino acid substitution. Sporulation was done in SM medium (21).