MOLECULAR ANALYSIS OF THE AMS OPERON REQUIRED FOR EXOPOLYSACCHARIDE SYNTHESIS OF ERWINIA-AMYLOVORA

A 16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the large mRNA from the ams operon, The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE. Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site-directed mutagenesis of the ORFs 477, 377, 144, and 743 revealed an amylovoran-deficient phenotype, and the newly identified genes were named amsG, amsH, amsI and amsF, respectively. The predicted amino acid sequence of AmsG is highly homologous to galactosyl-1-phosphate undecaprenyl-phosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD, A significant homology to mammalian phosphatases was observed for Amsl, AmsA shows characteristic motifs for membrane association and ATP binding, AmsF carries a secretory signal sequence in the N-terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG, It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45 min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 18 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.