Identification and analysis of a flagellar gene operon of Agrobacterium tumefaciens

Abstract

The cosmid, pDUB1900, from a representative genomic library of the Agrobacterium tumefaciens C58C1 chromosome has previously been partly mapped and found to contain genes concerned with flagellar structure, assembly and function. In this study a region of 4470 nucleotides from the largest HindIII fragment of pDUB1900 was sequenced completely in both directions. This sequenced region unified two previously identified operons which were transcribed in oppposite directions, motA leftward and flgB rightward. This sequence was compared against databases to identify homologous regions. The comparison results and other computer analyses revealed two flagellar gene homologues, flgF and fliI, and two open reading frames, orfX and orfZ with no significant sequence identity to any previously identified genes. Computer analysis also indicated that all four ORFs may be transcribed together as a putative operon, independent of the two flanking operons. Previously identified A. tumefaciens flagellar genes were found to be homologous to flagellar genes in Rhizobium meliloti and have the same gene order, therefore, the four ORFs identified here were also compared to R. meliloti DNA. The ORFs were isolated, radiolabelled and used as probes against Southern blots containing R. meliloti DNA from the pMB7 plasmid and pRZ cosmids, known to contain motility genes. All four of the A. tumefaciens ORFs hybridised to homologous DNA in R. meliloti and were found in the same order in both species. Recently released DNA sequence from R. meliloti, homologous to this region, confirmed these findings. This suggests that there is a high degree of conservation between the two species at the molecular level with regards to motility and chemotaxis. Mutants were created in the last ORF (orfZ) of the putative operon to determine the effects on motility. The orfZ mutants produced were all motile suggesting that the gene product of orfZ is not essential for flagella function. Alternatively, enough of orfZ may have remained before the insertion site to ensure it was still functional and thus, die putative operon transcribed as normal.