Azospirillum irakense produces a novel type of pectate lyase.
The pelA gene from the N2-fixing plant-associated bacterium Azospirillum irakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing pelA indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in E. coli at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca2+ for its activity. Of several divalent cations tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.
Bekri
M A
MA
F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, 3001 Heverlee, Belgium.
Desair
J
J
Keijers
V
V
Proost
P
P
Searle-van Leeuwen
M
M
Vanderleyden
J
J
Vande Broek
A
A
D016949:Azospirillum / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D015806:Azospirillum brasilense / Q000201:enzymology / Q000235:genetics; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D010935:Plant Diseases / Q000382:microbiology; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D011994:Recombinant Proteins; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase