CHARACTERIZATION OF FLAGELLA GENES OF AGROBACTERIUM-TUMEFACIENS, AND THE EFFECT OF A BALD STRAIN ON VIRULENCE

Agrobacterium tumefaciens produces flagella that are arranged circumth ecally near one end of the bacilliform cell, The flagella are required for motility to facilitate reaching the root surface, and possibly ai d in orientating the bacterial cells at various sites for infection, W e have identified three flagella genes designated flaA, flaB, and flaC , Mutations in flaA, flaB and flaC result in abberant swimming behavio ur, Electron microscopic examination of these mutants revealed the def ective flagella, A non-motile, bald mutant strain was generated by del eting all three fla genes, Nucleotide sequencing of flaA, flaB, and fl aC showed that they have a potential coding capacity for polypeptides of 307, 321, and 314 amino acid residues, respectively, The predicted amino acid sequences of the A, tumefaciens FlaA and FlaB proteins are similar (66% average identity) to the FlaA and FlaB proteins encoded b y flaA and flaB genes, respectively, in Rhizobium meliloti. There was no counterpart FlaC protein reported in R. meliloti, but the A. tumefa ciens FlaC is similar in amino acid sequence to the R. meliloti FlaA ( 59.8% identity) and FlaB (66.7% identity), Distinct from FlaA and FlaB of R. meliloti is the absence of histidine and cysteine residues and their shorter length (by 88 amino acid residues fewer than FlaA and Fl aB of R. meliloti), The transcriptional start sites of each fla gene d etermined by primer extension revealed consensus-sequence boxes repres enting potential binding sites for sigma(28) RNA polymerase (RNAP) ups tream of the transcriptional start of each fla gene, Besides the poten tial sigma(28)-binding site upstream of flaC, also present are additio nal putative conserved sequences, GC at -11 and GG at -21 from the tra nscriptional start, that resemble potential binding motifs for sigma(5 4). Because the sigma(54) promoter is associated with genes regulated by physiological changes in various bacteria, the flaC gene might be s imilarly regulated in response to A. tumefaciens responding to host pl ant stimuli, Virulence studies showed that the bald strain was consist ently reduced in virulence below that of the parental wild-type strain by at least 38%. The difference is statistically significant and sugg ests that the flagella may play a role in facilitating virulence.