Analysis of the Spontaneous Mutation to Avirulence by Pseudomonas Solanacearum

Abstract

A gene from P. solanacearum encoding an endoglucanase (EGase) enzyme was cloned on a 30-kb DNA fragment in the cosmid pLAFR3 by virtue of its expression in E. coli. Activity inhibition analysis with antiserum against a purified EGase of P. solanacearum showed that this gene (egl) encodes the major endoglucanase excreted by virulent P. solanacearum. This 38-kDA EGase enzyme is produced at 25-fold lower levels in spontaneous, non-mucoid, avirulent mutants. After mapping and subcloning, Southern hybridization analysis showed that there are no major structural differences between the egl gene in the wild type virulent parent and in avirulent mutants. However, RNA measurements showed that the egl gene was transcribed nearly 10-fold less in avirulent mutants. Transfer of the wild type egl gene into avirulent mutants resulted in non-mucoid strains which produce nearly normal levels of egl gene product. Attempts to spontaneously mutate the cloned egl gene in merodiploids were unsuccessful. The available evidence suggests that a small cis-acting promoter mutation results in the reduced egl expression in the spontaneous, pleiotrophic mutants.