CHARACTERIZATION OF THE NUCM GENE CODING FOR A NUCLEASE OF THE PHYTOPATHOGENIC BACTERIA ERWINIA-CHRYSANTHEMI

The gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi. The nucM gene was subcloned, and mutagenized by insertion of a uidA-Kan(R) cartridge. This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome. The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity. The nucleotide sequence of nucM was determined. It presents a 798 bp open reading frame, coding for a 266-amino-acid protein, with a predicted molecular mass of 29 91 0 Da. The deduced NucM protein shows 59% sequence identity with the DNaseI precursor from Vibrio cholerae. It contains a typical leader sequence. Experiments of cell fractionation showed that NucM is periplasmic in E. chrysanthemi. The transcription start has been determined by Sl mapping. The -10 and -35 regions do not show homology with consensus sequence of the promoters recognized by sigma70. In fact, the promoter seems to be dependent on the sigma70 , but the first transcription nucleotide is unusually far from the -10 region. nucM seems to be expressed constitutively.