MOLECULAR CHARACTERIZATION OF THE LEVANSUCRASE GENE FROM THE ENDOPHYTIC SUGARCANE BACTERIUM ACETOBACTER-DIAZOTROPHICUS SRT4

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4 .1.10) (IsdA) was isolated from a genomic library. The nucleotide sequ ence of a 2.3 kb DNA fragment sufficient for complementation of a leva nsucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid seque nce of the extracellular levansucrase indicated the presence of a prec ursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps, Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production o f a protein with levansucrase activity. The deduced amino acid sequenc e of the IsdA gene was 48% and 46% identical with the levansucrases fr om the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram- positive bacteria. Multiple alignments of published levansucrase seque nces from Gram-negative and Gram-positive bacteria revealed eight cons erved motifs. A comparison of the catalytic properties and the sequenc e of the A. diazotrophicus levansucrase with those of the Bacillus sub tilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthesized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking b oth levansucrase activity and the ability to utilize sucrose as a carb on source, suggesting that levansucrase is the key enzyme in sucrose m etabolism of A. diazotrophicus.