Nucleotide sequence of pnl gene from Erwinia carotovora Er

doi:10.1016/0006-291X(91)90927-Y

Biochemical and Biophysical Research Communications

Volume 176, Issue 1, 15 April 1991, Pages 321-327

https://doi.org/10.1016/0006-291X(91)90927-Y Get rights and content

Abstract

The nucleotide sequence of pnl gene encoding pectin lyase (PNL; EC4.2.2.10)from Erwinia carotovora Er was determined. The structural gene of pnl consisted of 942 base pairs. An open reading frame that could encode a 33,700 dalton polypeptide consisting 314 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of PNL determined in E.carotovora Er. The nucleotide sequence of the 5′-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in E.carotovora and Escherichia coli. Between the presumed Pribnow box and the ribosome binding site, two pairs of inverted repeats were found. By comparing the predicted amino acid sequences of pnl, several reported bacterial pectate lyases and Aspergillus niger pectin lyase, short regions of homology were found despite the different substrate specificities of these enzymes.

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-Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv. marginalis MAFF 03-01173 with total 33% recovery of the initial activity. From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase. The M_rs of pectin lyase and pectate lyase were estimated to be 34 000 and 43 000, respectively, by SDS polyacrylamide gel electrophoresis. Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3. © 1997 Elsevier Science Ltd. All rights reserved
Molecular cloning and nucleotide sequence of the gene encoding phosphate-inducible pectin lyase of Bacillus subtilis
1996, FEBS Letters
The gene encoding the pectin lyase (PNL; EC 4.2.2.10) of Bacillus subtilis has been cloned, sequenced, and characterized. A coding sequence for the PNL composed of 345 amino acids including a 24-amino-acid signal peptide was assigned. No sequence resembling a LexA binding site was found upstream of the structural gene. Furthermore, PNL activity of the gene product expressed in Escherichia coli DH5α was detected intracellularly, which might suggest that expression of the gene was not controlled by RecA. Regulation of the gene expression seemed to be quite different from that of other bacterial PNL genes previously reported.
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Erwinia chrysanthemi synthesizes and secretes a large number of pectinases. The major pectinases include a pectin methylesterase PemA and five isoenzymes of endo-pectate lyases PelA, PelB, PelC, PelD and PelE. In addition, secondary pectinases were identified: a pectin methylesterase PemB, two endopectate lyases PelL and PelZ, an exo-pectate lyase PelX and an exopolygalacturonase, PehX. The regulation of pectinase synthesis is very complex and dependent on many environmental conditions. It is induced by pectin catabolic products and affected by growth phase, catabolite repression, osmolarity, iron or oxygen starvation…
Three regulators were identified by genetic analysis. The main repressor, KdgR, controls the transcription of pectinase genes, the intracellular catabolic pathway and the secretion machinery. The PecS repressor controls the production of pectate lyases and cellulases, the secretion machinery and the biosynthesis of a blue pigment. PecT acts as a repressor of the production of some pectate lyases. Other proteins are involved in the regulation of pectinase synthesis but their role is not well characterized.
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A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonasmarginalis N6301 was cloned and expressed in Escherichiacoli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2.1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5α. A plasmid thus obtained was designated as pPNL6301. E. coli DH5α harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pnl gene in the plasmid pPNL6301 encoding PNL from P.marginalis N6301 was determined. The structural gene of pnl consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P.marginalis N6301 determined. The nucleotide sequence of the 5′-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichiacoli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pnl gene between P.marginalis N6301 and E.carotovora Er were 60.8% and 57.2%, respectively.
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The nucleotide sequence of lukS gene encoding S-component of Staphylococcal leukocidin from methicillin resistant Staphylococcus aureus (MRSA) was determined. The structural gene of lukS consisted of 857 base pairs. An open reading frame that could encode a 35,556 dalton polypeptide consisting of 315 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of pre-matured S-component determined in DNA-directed transcription/translation system. Inspection of the amino acid sequence deduced from nucleotide sequence of lukS and that from S-component of leukocidin clarified that pre-matured S-component contains a typical signal sequence at the NH₂terminus. The amino acid sequence of predicted matured S-component correlated exactly with the known N-terminal 50 amino acid sequence of S-component from MRSA and S. aureus V8. The molecular size of the predicted matured protein was also close to the value of S-component determined in both MRSA and S. aureus V8. The nucleotide sequence of the 5′-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in Escherichia coli.

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Nucleotide sequence of pnl gene from Erwinia carotovora Er

Abstract

Biochem. Biophys. Res. Commun

Gene

J. Mol. Biol

Gene

Gene

Agric. Biol. Chem

Agric. Biol. Chem

J. Bacteriol