Identification and molecular characterisation of chemotaxis genes in agrobacterium tumefaciens

Abstract

Using heterologous probing, with fragments from the S. meliloti che operon, putative chemotaxis genes were identified in A. tumefaciens. The cosmid pDUB1911, from a representative genomic library of C58C1, was identified and found to contain a cluster of chemotaxis-related genes. A 9.6kb region of pDUB1911 was completely sequenced (GenBank Accession No. AF044495), in both directions, and found to contain an 8kb chemotaxis cluster. The cluster begins with orf1, followed by orf2, cheYl, cheA, cheR, cheB, cheY2, orf9 and orflO. All of the identified homologues showed a high degree of sequence conservation with their counterparts in the chemosensory regions of the related bacteria S. meliloti and R. sphaeroides, and were arranged in a similar order. A homologue of the flagellar gene fliF was identified directly downstream of the che cluster. This arrangement is similar to that seen in S. meliloti, where the che operon is followed by a large region containing flagellar/motility-related genes. It was therefore postulated that the region identified in this work could be linked to the cluster of flagellar/motihty-related genes previously identified in A. tumefaciens. Mutant strains were created by in-frame deletion of cheA and orflO, and insertion of a neomycin resistance cassette in orfl, cheA and fliF. The oifl and cheA mutants showed wild type motility, but impaired chemotactic capabilities. Deletion of orflO appeared to have no effect on either motility or chemotaxis, under the conditions studied. Mutation of fliF resulted in a non-motile, non-flagellate phenotype. A "gutted" strain was created by deletion of the entire che cluster. As with the orfl and cheA mutant strains, the gutted strain showed severely impaired chemotaxis, but wild type patterns of motility. Preliminary work was conducted on the construction of a selectively-infective phage (SIP) system for studying bimolecular interactions within, and between, the che and vir systems of A. tumefaciens. A phage vector was constructed, which following further testing, should allow such work to begin. Probing with a fragment coding for the conserved region of an MCP recently identified m R. leguminosarum, suggested that A. tumefaciens could contain a number of proteins resembling the classical MCPs of E. coli. A putative MCP homologue was also identified in pDUB1911, downstream of the main che cluster. Although the che cluster was found not to contain a homologue of cheW, heterologous probing and PGR using consensus primers indicated that c/ieWmaps elsewhere in the A. tumefaciens genome.