<i>Erwinia Carotovora</i>: Molecular Cloning of a 3.4 Kilobase DNA Fragment Mediating Production of Pectate Lyases

Roberts, Daniel P.; Berman, Phyllis; Allen, Caitilyn; Stromberg, Verlyn K.; Lacy, George H.; Mount, Mark S.

doi:10.1080/07060668609501836

Cited by 17 publications

(6 citation statements)

References 23 publications

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“…Potato tuber tissue inoculated with the wild-type pathogen (E. carotovora L-543) and the genetically engineered strain (E. carotovora L-833). Tuber slices were inoculated by the eye-ofthe-needle method (21). from the same site.…”

Section: Methodsmentioning

confidence: 99%

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

Scanferlato

Orvos

Cairns

et al. 1989

Appl Environ Microbiol

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The survival of genetically engineered Erwinia carotovora L-864, with a kanamycin resistance gene inserted in its chromosome, was monitored in the water and sediment of aquatic microcosms. The density of genetically engineered and wild-type E. carotovora strains declined at the same rate, falling in 32 days below the level of detection by viable counts. We examined the impact of the addition of genetically engineered and wild-type strains on indigenous bacteria belonging to specific functional groups important in nutrient cycling. For up to 16 days, the densities of total and proteolytic bacteria were significantly higher (P < 0.05) in microcosms inoculated with genetically engineered or wild-type E. carotovora, but by 32 days after inoculation, they had decreased to densities similar to those in control microcosms. Inoculation of genetically engineered or wild-type E. carotovora had no apparent effect on the density of amylolytic and pectolytic bacteria in water and sediment. Genetically engineered and wild-type E. carotovora did not have significantly different effects on the densities of specific functional groups of indigenous bacteria (P > 0.05).

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Section: Methodsmentioning

confidence: 99%

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

Scanferlato

Orvos

Cairns

et al. 1989

Appl Environ Microbiol

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“…In addition, the preparations were also tested for hydrolytic pectic enzyme activity by using 100 mM phosphate buffer (pH 6.5) with 10 mM EDTA (7), but no activity was detected. However, polygalacturonase has been detected in E. carotovora previously (8,9), and we detected low levels of this enzyme in 48-h-old culture supernatants.…”

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confidence: 60%

“…Several research groups have cloned the pectate lyase genes from E. carotovora subsp. carotovora into Escherichia coli (5,9,11).…”

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confidence: 99%

A Rapid Procedure for Purifying Pectate Lyase from Erwinia carotovora Based on Substrate Affinity

Ward

Boer

1987

Appl Environ Microbiol

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To purify pectate lyase produced by Erwinia carotovora subsp. carotovora, we used the supernatant from 48-hold cultures grown in broth containing sodium polypectate and yeast extract. The supernatant was combined with the enzyme substrates sodium polypectate and polygalacturonic acid, which were then precipitated with CaC12. After the precipitate was washed, pectate lyase was eluted with 1.0 M NaCl.

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“…Bacterial strains, plasmids, media, and chemicals. Eca strain EC 14 and plasmids pDR40 and pDR1 have been described previously (44,45). Escherichia coli strain DH5a (BRL) was used as a plasmid host.…”

Section: Methodsmentioning

confidence: 99%

“…We report here studies on pectate lyases associated with an 8.4-kb DNA fragment cloned from Ecc strain EC14 (45). E. coli cells transformed with pDR40 produce two catalytically active pectate lyases with pis measured at 9.5 and 10.5 (44,45,60). We report nucleotide sequences for pel genes (pe/9.5 and pel10.5), compare them to the PelBC family of extracellular pectate lyases, and find that Pel9.5/l0.5 and Pell0.5/9.5 fusion proteins have pectate lyase activity.…”

Section: Introductionmentioning

confidence: 99%

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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Erwinia Carotovora: Molecular Cloning of a 3.4 Kilobase DNA Fragment Mediating Production of Pectate Lyases

Cited by 17 publications

References 23 publications

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

A Rapid Procedure for Purifying Pectate Lyase from Erwinia carotovora Based on Substrate Affinity

Site-Specific Inactivation of Bacterial Genes Involved in Plant Rotting

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