Azospirillum irakense produces a novel type of pectate lyase

The peL4 gene from the N-2-fixing plant-associated bacterium Azospirillum i rakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing peL4 indicated an open reading frame of 1,296 bp, coding for a preprotein o f 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the pro tein in E. coli at the signal peptidase cleavage site predicted by nucleoti de sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a ne w pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pit optimum, and requires Ca2+ for its activity. Of several divalent catio ns tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as s ubstrates. Characterization of the degradation products formed upon incubat ion with polygalacturonate Indicated that PelA is an endopectate lyase gene rating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion, T ranscription of this fusion is low under all growth conditions tested and i s dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pecta te lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.