Use of Tn5tac1 to clone a pel gene encoding a highly alkaline, asparagine-rich pectate lyase isozyme from an Erwinia chrysanthemi EC16 mutant with deletions affecting the major pectate lyase isozymes.
Erwinia chrysanthemi mutant CUCPB5047, delta(pelA pelE) delta(pelB pelC)::28bp delta(pelX) delta 4bp pehX::omega Cmr, was constructed, mutated with Tn5tac1, and screened for isopropyl-beta-D-thiogalactopyranoside-dependent pectate lyase (Pel) production. A Kmr SacI fragment from the hyperexpressing Pel+ mutant CUCPB5066 was cloned into Escherichia coli and sequenced. The gene identified, pelL, encodes a novel, asparagine-rich, highly alkaline enzyme that is similar in primary structure to PelX and in enzymological properties to PelE.
Alfano
J R
JR
Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203, USA.
Ham
J H
JH
Collmer
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000201:enzymology / Q000235:genetics*; D017353:Gene Deletion; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000235:genetics*
D007527:Isoenzymes; D011133:Polysaccharide-Lyases; C023911:pectate lyase