Cloning and Characterization of the Pectate Lyase III Gene of<i>Evwinia carotovora</i>Er

Yoshida, Aruto; Izuta, Miho; Ito, Kazutoshi; Kamio, Yoshiyuki; Izaki, Kazuo

doi:10.1080/00021369.1991.10870733

Cited by 6 publications

(3 citation statements)

References 12 publications

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“…No p-independent-like transcriptional terminators are obser-,.. ved downstream from the pe/9.5 or pell0.5 termination codons. Although such palindromic sequences were described for Ech pel genes (26,53), none have been reported for Ec pel genes (24,25,33,34,63). Partial genes and gene fusions.…”

Section: Resultsmentioning

confidence: 99%

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Site-Specific Inactivation of Bacterial Genes Involved in Plant Rotting

Antonov¹,

Dudov²,

Atanassov³

1995

Biotechnology & Biotechnological Equipment

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The soft rot bacterium Erwinia carotovora subspecies carotovora strain ECJ4 contains multiple pectate lyase genes. Genes pe/9.5 and pell0.5, coding for pectate lyases (pi 9.5 and 10.5), were cloned on a plasmid, transformed into Escherichia coli, and expressed cata~yti ca!ly active products. The isoenzymes were subcloned separate~y and sequenced. Along most of their lengths, the genes are homologous to each other and to the pelBC family of Erwinia extracellular pectate lyase genes. However, the amino acid sequence of the Pell0.5 carboxyterminus diverges markedly from the PelBC consensus. We constructed hybrid genes and their products were found to be pectolytic. The pe/9. 5 and pell 0. 5 genes were mutagenized by marker exchange mutagenesis. A double mutant, pel9.5-pell0.5-, was obtained by mobilization of pLAJJ5 into the pell0.5::kan mutant and exchange recombination by the above method. Hybridization of EcoRI digests of the mutant chromosomal DNAs with pe/9.5-, pell0.5-, kan-, and let-specific probes gave profiles consistent with single-site insertions of the kan and tet genes in the corresponding pel genes. In planta accumulation of Pe/9.5 and Pell 0. 5 mRNAs was induced within 3 to 4 hr after inoculation, reaching maxima between 6 and 12 hr with Pell 0. 5 peaking at 6 hr and Pe/9. 5 at 9 hr. The induction of the two genes was regulated differential~y and the level of their mRNAs was higher during compatible than during incompatible conditions. The three mutants, obtained here, especially the double mutant, have diminished virulence compared to the wild type.

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Section: Resultsmentioning

confidence: 99%

“…In E. carotovora (Ec), three to four pectate lyases with similar chromatographic and hybridization properties have been described (32,52,62) and the genes coding for them have been sequenced (24,25,33,34,63). The first 22 amino acids of the excreted proteins are removed by proteolytic cleavage.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Inactivation of Bacterial Genes Involved in Plant Rotting

Antonov¹,

Dudov²,

Atanassov³

1995

Biotechnology & Biotechnological Equipment

View full text Add to dashboard Cite

show abstract

“…The molecular weight of PL III was estimated to be approximately 39,500 by SDS-PAGE, while that of the processed PL III deduced from the nucleotide sequence of pel III was calculated to be 38,500 2 ) These unexpected slower mobilities of PLs in SDS-PAGE analysis were also observed by other researchers 5 -8) The optimal pH of PL III was 10.0, using 50 mM potassium phosphate buffer (pH 7.0 .. 7.5), 50 mM Tris-HCI buffer (pH 7.4-9.0), and 50mM Caps-NaOH buffer (pH 8.9-10.8). The optimal temperature for activity of PL III Approximately I nmol of the purified PL III excreted by E. caratavora Er harboring pNN3 was used for the identification of N-terminal amino acid sequence.…”

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confidence: 99%