Proteolysis of the McpA chemoreceptor does not require the Caulobacter major chemotaxis operon

Abstract

The degradation of the McpA chemoreceptor in Caulobacter crescentus accompanies the swarmer cell to the stalked-cell differentiation event. To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the mcpA gene and in the mcpA chemotaxis operon. Internal deletions of the mcpA gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis. The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.

FIG. 1

Cell cycle immunoblots of mcpA internal deletions. (A) mcpA internal deletions. The pale grey boxes denote the extent of the McpA protein present in the various chromosomal deletions of the mcpA gene in the named strains (Table 1). The transmembrane domains are denoted by TM1 and TM2 and are colored in grey. The methylation domains (checkered) are named after the K1- and R1-methylated peptides shown in the E. coli Tsr chemoreceptor (11). The HCD (striped box) is the most highly conserved domain in all chemoreceptors (13); it is involved in signalling. The gaps denote the extent of the deletions in the mcpA gene in the named C. crescentus strains. The black box at the C terminus of McpA is the CheR-CheB binding site (R/B) (4, 20). (B) Cell cycle immunoblots. Strains MRKA208, which is the wild-type strain in these experiments, MRKA21, MRKA25, MRKA26, and MRKA24 were synchronized by Percoll (Pharmacia) density centrifugation. Samples were taken at the times (minutes) indicated during the 90-min cell cycle. The same amount of cells was loaded in each lane. The cell extracts were subjected to electrophoresis on a sodium dodecyl sulfate–8% polyacrylamide gel and transferred to nitrocellulose (19); the primary antiserum was to McpA, and the secondary antiserum was anti-rabbit conjugated to horseradish peroxidase.

FIG. 2

The Δche17 deletion strain MRKA580 is defective in chemotaxis. (A) The grey boxes represent the open reading frames for the named genes (AJ006687). The extent of the deletion in Δche17 strains is shown by the black box. (B) A yeast extract swarm agar (0.005% yeast extract, 0.5 mM MgSO₄, 0.5 mM CaCl₂, 0.15% Bacto agar) plate after 48 h inoculated with equal amounts of MRKA208 (wild type [WT]) and MRKA580 (indicated by the no. 17) strains bearing the plasmid pRMCP4.

FIG. 3

McpA methylation and cell cycle degradation in a mcpA operon deletion. (A) Immunoblot of strains MRKA208 (wild type [WT]) and MRKA580 (indicated by the no. 17) using McpA antisera. The arrow indicates the McpA protein band. (B) Immunoprecipitation of McpA in cell extracts from chloramphenicol pretreated cells labelled with [³H]methylmethionine (17). Equal numbers of counts per minute were immunoprecipitated. The cell extracts were from strains MRKA208 (WT) and MRKA580 (no. 17). (C) McpA cell cycle immunoblots of strains MRKA208 (WT) and MRKA580 (no. 17) bearing the plasmid pRMCP4. The numbers above the panel are the time points (minutes) at which the samples were taken.