Molecular characterisation of behavioural functions in Agrobacterium tumefaciens

Abstract

Tn5 insertion behavioural mutants of A. tumefaciens C58C(^1) were available. Cloning of the kanamycin resistance gene allowed isolation of Tn5 flanking sequences from a number of the mutants. Flanking sequences from five mutants were used to isolate cosmids, overlapping the Tn5 insertion sites of the mutantsfrom a C58C(^1) library. Two cosmids, pDUB1900 and 1905 have been characterised. pDUB1900 contains the insertion sites of eight motility mutants, with another immediately adjacent. The pDUB1905 insert overlaps sequences interrupted in another three mutants. Behavioural genes in Agrobacterium are clustered together on the chromosome, as in other motile bacteria. Restriction maps of the isolated cosmids show that none of motility mutants analysed was the result of mactivation of pscA or chvB which would lead to an altered behavioural phenotype. Flanking sequences from three of the mutants hybridised to R. meliloti chromosomal DNA, but not to DNA from other motile genera. DNA adjacent to the insertion site of fla-11 hybridised to the insert of pRZ-2, a cosmid containing behavioural genes from R. meliloti. Experiments were undertaken to investigate the occurrence of proteins homologous to the MCP's of enteric bacteria in C58C(^1) DNA hybridisation to oligonucleotide probes showed DNA that could potentially code for MCP-like proteins exists m both Agrobacterium and Rhizobium spp. In addition, an antibody raised against the E. coli MCP Tar cross-reacted with a protein of approximately 60kDa. in C58C(^1). In vivo protein methylation experiments using C58C(^1) resulted in the labelling of a 45kDa protein, whose methylation pattern did not change upon addition of chemostimuli. Possible reasons for the difference in size between the labelled protein and that revealed by the antibodv are discussed.