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 biosynthe sis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulato r RcsA enhanced transcription of the large mRNA from the ams operon, T he nucleotide sequence of this area revealed 12 open reading frames (O RFs), which are all transcribed in the same direction. Five ORFs corre sponded 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 r evealed an amylovoran-deficient phenotype, and the newly identified ge nes were named amsG, amsH, amsI and amsF, respectively. The predicted amino acid sequence of AmsG is highly homologous to galactosyl-1-phosp hate undecaprenyl-phosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD, A signi ficant 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 b e involved in periplasmic processing of the repeating units. Complemen tation experiments located a promoter region required for gene express ion as far as 500 bp upstream of amsG, It is preceded by a typical tra nscriptional termination sequence. A mutation upstream of the terminat or did not affect amylovoran synthesis. Partial nucleotide sequences f urther upstream of the ams region showed homology to genes mapped at 4 5 min on the Escherichia coli chromosome. A termination sequence was a lso found downstream of the ams operon at a distance of 18 kb from the promoter. Between amsF and this terminator, three additional ORFs wer e detected.