Molecular characterisation of flagellar genes from agrobacterium tumefaciens

Abstract

Three behavioural mutants of A. tumefaciens C58C1 (mot-l, mot-12 and fla-15) generated by transposon (Tn5) mutagenesis were studied. Analysis was initially at the molecular level, as a cosmid, pDUB1900, from a representative genomic library of C58C1 had been isolated that complemented the mutants. A region of 8624 nucleotides to which the Tn5 insertion sites of the three mutants had been mapped was sequenced completely in both directions. The comparison of this sequence with sequence databases and other computer analyses revealed six flagellar gene homologues (flgI,flgH,fliP,flaA,flaB,flaC), three open reading frames (ORFA, B and C) with no significant sequence identity to any open reading frames in the databases and the partial sequence of the flagellar gene homologue flgG. Computer analysis also showed that theflgH,flgI andfliP homologues, and ORFs A, B and C, could form the downstream region of a larger operon involved in chemotactic and motility functions. However putative transcription signals were also found within the operon. A new mutant (MANl) was created in the last gene (fliP) of the putative operon to investigate the function of possible transcription signals in the open reading frame immediately upstream of it (ORFC). The mot-12 mutant phenotype of fully synthesised but paralysed flagella is brought about by the insertion of Tn5 in ORFC. ORFC contains a possible promoter for fliP. The Tn5 insertion in ORFC should have polar effects upon the expression of fliP, unless the putative promoter can cause expression of fliP. The MANl mutant had a flagella-less phenotype. FliP in other bacteria is required early in the synthesis of flagella and the null phenotype is/7a-. Thus for flagella to be present in mot-12 suggests fliP must have a promoter. The ORFC sequence is highly conserved in R. meliloti and the overall regulation of these flagellar gene homologues may be as an operon with other regulatory signals. Evidence from other operons (including motility operons) with multiple transcription signals is discussed. The flaABC homologues were multiple copies of the gene encoding the flagellin protein of the flagellum. The mot-l phenotype of severely truncated filaments was caused by a Tn5 insertion in flaA. Analysis of the sequence showed flaABC to each have transcription signals that could lead to separate transcription. Transcription analysis by Northern blotting showed flaA to be transcribed monocistronically. Flagella were isolated from A. twnefaciens and the flagellins separated by SDS-PAGE. The migrated distances (relative to those of markers) was not as predicted from the nucleotide sequence. This anomaly could be caused by unequivalent binding of SDS or post-translational modification of FlaA. The A. tumefaciens flagellar genes were most similar to those of R. meliloti. However A. tumefaciens flagella do not exhibit the characteristic cross-hatching of the complex flagella of R. meliloti. This study also showed A. tumefaciens flagella not to be dependent on divalent cations for subunit associations unlike R. meliloti. These properties of A. tumefaciens flagella were similar to those of R. leguminosarum.The open reading frames found were isolated, radiolabelled and used as probesagainst Southern blots containing chromosomal DNA from a variety of soil bacteria, and cosmids known to contain motility genes in R. meliloti. Hybridisation revealed homologous DNA sequences in a number of these bacteria. All the A. tumefaciens open reading frames hybridised to homologous DNA in R. meliloti and are found in the same order in both species. This suggests that there are similarities at the molecular level in motility and chemotaxis functions between R. meliloti and A. tumefaciens as well as in the patterns of chemotaxis and motility observed previously.