Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco.

Abstract

Ralstonia solanacearum K60 was mutagenized with the transposon Tn5, and two mutants, M2 and M88, were isolated. Both mutants were selected based on their increased sensitivity to thionins, and they had the Tn5 insertion in the same gene, 34 bp apart. Sequence analysis of the interrupted gene showed clear homology with the rfaF gene from Escherichia coli and Salmonella typhimurium (66% similarity), which encodes a heptosyltransferase involved in the synthesis of the lipopolysaccharide (LPS) core. Mutants M2 and M88 had an altered LPS electrophoretic pattern, consistent with synthesis of incomplete LPS cores. For these reasons, the R. solanacearum gene was designated rfaF. The mutants were also sensitive to purified lipid transfer proteins (LTPs) and to an LTP-enriched, cell wall extract from tobacco leaves. Mutants M2 and M88 died rapidly in planta and failed to produce necrosis when infiltrated in tobacco leaves or to cause wilting when injected in tobacco stems. Complemented strains M2* and M88* were respectively obtained from mutants M2 and M88 by transformation with a DNA fragment harboring gene rfaF. They had a different degree of wild-type reconstituted phenotype. Both strains retained the rough phenotype of the mutants, and their LPS electrophoretic patterns were intermediate between those of the wild type and those of the mutants.