Genetic analysis of a temporally transcribed chemotaxis gene cluster in Caulobacter crescentus

Abstract

Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division.