Abstract
Serratia entomophila (Se) causes amber disease in the soil-dwelling pest, Costelytra zealandica (Cz). The disease presents two main signs: anti-feeding effect (AFE) and development of amber coloration (AC). To identify the genetic loci involved in pathogenicity, non-pathogenic (Path −) Se mutants were created by transposon (Tn phoA) mutagenesis [Upadhyaya et al., J. Bacteriol. 174 (1992) 1020–1028]. The mutant UC24 lost the ability to produce amber disease signs and it was shown to contain a single Tn phoA insertion. The Tn phoA insertion site was mapped in a 5.3-kb DNA fragment, which was named amb2 locus. Cosmids containing amb2 fully restored AFE and partially restored AC in UC24. Escherichia coli (Ec) HB101 bearing the amb2 locus was able to cause AFE in a multiple-dose bioassay. SDS-PAGE analysis of the amb2 gene products produced in minicells showed the synthesis of two proteins of 16 and 19.5 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis. Pathogenicity tests with insect larvae fed with bacteria carrying the anfA and anfB gene regions separately showed that both regions are essential for AFE. It is proposed that the AnfA and AnfB proteins are virulence factors (toxin-like proteins) causing AFE in Cz.
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