Organization of a pectate lyase gene family in Erwinia chrysanthemi

Abstract

The pelA, pelD and pelE genes encode three of the five major pectate lyase (PL) isoenzymes (PLa, PLd and PLe) in Erwinia chrysanthemi strains B374 and 3937. These genes were previously isolated from genomic libraries or by in vivo cloning as R' factors promoted by the pULB113 plasmid. They are clustered near purE on the chromosomal map of E. chrysanthemi B374 [Van Gijsegem et al., EMBO J. 4 (1985) 787-792]. Genes pelA, pelD and pelE were subcloned separately into pBR322 derivatives, to test their individuality. It then became possible to select specific mutations in each separated gene. Such mutations were obtained using the transposable bacteriophage MudI1734 that allows the construction of lacZ gene fusions. Subcloning experiments and analysis of MudI1734 insertions permitted us to determine the length of each gene, the transcriptional orientation and the location of the promoter. We concluded that the three genes constitute three independent transcriptional units. They are clustered on a 5-kb DNA fragment, in the order: pelD-pelE-pelA. Genes pelD and pelE are transcribed in the same direction, while the transcription of pelA seems to be divergent. Organization of the pel region was very similar in the two strains B374 and 3937. Moreover, lacZ gene fusions were introduced by marker exchange into the chromosome of E. chrysanthemi B374, giving rise to three strains lacking PLa, PLd or PLe. These fusions allowed us to study the regulation of the mutagenized genes.