Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71.
In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.
Chatterjee
A
A
Department of Plant Pathology, University of Missouri, Columbia 65211.
McEvoy
J L
JL
Chambost
J P
JP
Blasco
F
F
Chatterjee
A K
AK
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002872:Chromosome Deletion; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics; D004790:Enzyme Induction; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D011133:Polysaccharide-Lyases; C023911:pectate lyase