Identification of two new cell division genes that affect a high-molecular-weight penicillin-binding protein in Caulobacter crescentus

Abstract

Penicillin-binding proteins (PBPs) are membrane proteins associated with the synthesis of the bacterial cell wall. We report the characterization of 14 PBPs in Caulobacter crescentus, using in vivo and in vitro penicillin-binding assays and experiments to determine their possible role in cell division. New conditional cell cycle mutants were isolated by selecting cephalosporin-C-resistant mutants of the beta-lactamase strain SC1107 at 30 degrees C that are also defective in cell division at 37 degrees C. They fall into two classes, represented by strains PC8002 and PC8003. Strain PC8002 produced short cells arrested at all stages of cell division at 37 degrees C and was found to contain a high-molecular-weight PBP 1B which was temperature sensitive when assayed in vivo and in vitro. Strain PC8003 was blocked at an early stage of cell division and formed tightly coiled, unpinched filaments. This cephalosporin-C-resistant strain was also defective in PBP 1B, but only when assayed in vivo. PBP 1B behaved like a high-affinity PBP, and in competition assays, beta-lactams that induced filamentation bound preferentially to PBP 1B. These results and the phenotype of mutant PC8002 suggest that PBP 1B is required for cell division, as well as for cell growth, in C. crescentus. The behavior of strain PC8003 suggests that it contains a conditionally defective gene product that interacts in some way with PBP 1B at an early stage of cell division. None of the mutants showed an allele-specific PBP pattern when assayed in vitro at the nonpermissive temperature, but all of them displayed temperature-sensitive PBP 1C (102 kilodaltons) activity. Thus, it appears that PBP 1C is inhibited at 37 degree C as a consequence of filamentous growth.