Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Allen, Caitilyn; Stromberg, Verlyn K.; Smith, F. D.; Lacy, George H.; Mount, Mark S.

doi:10.1007/bf00331650

Cited by 17 publications

(8 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…E. carotovora subsp. carotovora EC14 and cosmid pCA7 have been described previously (2,43). Escherichia coli DH5a (Bethesda Research Laboratories [BRL], Inc., Gaithersburg, Md.)…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

1991

View full text Add to dashboard Cite

The prtl gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSKI or pSK23, a subclone of pSK1, produces a protease (Prtl) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prtl activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prtl promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prtl transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin.Erwinia carotovora subsp. carotovora EC14 is a gramnegative bacterium which causes soft rot on many plant species (39). Soft rot of potato (Solanum tuberosum) tubers is associated with the production of several degradative enzymes secreted by E. carotovora subsp. carotovora, including pectolytic enzymes, cellulases, proteases, and phospholipases (10, 57). Pectolytic enzymes probably play the most important role in maceration by degrading pectic components of the plant cell wall and middle lamella, resulting in separation of the cells (5). Possible roles for other degradative enzymes have not been established.Several erwinias and pseudomonads causing soft rot secrete proteases. Among soft-rot pseudomonads, extracellular protease production correlates more strongly with the ability to macerate plant tissue than does pectolytic enzyme production (46). In E. carotovora subsp. carotovora, large amounts of extracellular protease are produced when the bacterium is grown in rich broth, on bean (Phaseolus vulgaris) hypocotyls, or on sliced cucumber (Cucumis sativus) fruit (57), but the physiological role of the protease is unknown. Protease may aid in the degradation of plant cell wall components, cytoplasmic membranes, or cytosolic proteins. Purified E. carotovora subsp. carotovora protease causes limited cell death on cucumber disks and lysis of cucumber protoplasts (57). The release of amino acids and small peptides by E. carotovora subsp. carotovora protease may increase the rate of bacterial growth, thereby increasing the ability of the pathogen to colonize its host. The nutritional benefit derived from the proteolytic digestion of host macromolecules may contribute to greater virulen...

show abstract

“…E. carotovora subsp. carotovora EC14 and cosmid pCA7 have been described previously (2,43). Escherichia coli DH5a (Bethesda Research Laboratories [BRL], Inc., Gaithersburg, Md.)…”

Section: Methodsmentioning

confidence: 99%

“…carotovora genomic library and complemented a TnS-induced, protease-deficient mutant, L-763, of E. carotovora subsp. carotovora (2). Here we report subcloning and sequencing of the protease gene (prtl) from pCA7.…”

mentioning

confidence: 99%

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

1991

View full text Add to dashboard Cite

show abstract

“…Chem. 16,1997 S. Palmer et al Table 2. Comparison of clay and loam characteristics used in this study with the Hayter loam used by Orvos and colleagues [14].…”

Section: Media and Enumerationmentioning

confidence: 99%

“…Chem. 16,1997 S. Palmer et al Neither the wildtype nor the engineered strain were detected by plating 24 d after inoculation (detection limit was 10 cells/ml water and 100 cells/ml sediment).…”

Section: Survival In Aquatic Microcosmsmentioning

confidence: 99%

“…The wildtype strains have two endo-pectate lyases focusing isoelectrically at pI 9.5 and 10.4. Genetically engineered E. carotovora L-833 was used in the aquatic microcosms and was constructed from the wildtype by partially removing in vitro an extracellular pectate lyase gene on a plasmid (pDR40) by deletion mutation, inserting a 1.4-kb DNA fragment from Tn903 conferring kanamycin resistance, and chromosomally inserting, by gene replacement, part of the plasmid (pSV41) into the chromosome [9,16]. This strain, L-833, has a 30% reduction in its ability to rot potato tuber tissue compared to the wildtype [9].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Survival and Ecological Effects of Genetically Engineered Erwinia Carotovora in Soil and Aquatic Microcosms

Palmer¹,

Scanferlato²,

Orvos³

et al. 1997

Environ Toxicol Chem

Self Cite

View full text Add to dashboard Cite

Abstract-The release of genetically engineered organisms into the environment has raised concerns about their potential ecological impact. In this study, genetically engineered Erwinia carotovora strains expressing varying levels of reduced phytopathogenicity and wildtype E. carotovora strains were used in aquatic and soil microcosms to assess the survival, intraspecific competition, and effects upon specific groups of indigenous bacteria. In aquatic microcosms, the densities of Erwinia genetically engineered organisms (GEMs) and wildtype strains declined and fell below the detectable limit of plate counts 15 d after the microcosms were inoculated. In aquatic microcosms, engineered E. carotovora did not exhibit a competitive advantage over the wildtype. The effect of engineered and wildtype E. carotovora on densities of total and selected bacteria was not significantly different. Treatment with engineered E. carotovora did not change biomass values of the receiving community but did cause a transitory increase in metabolic activity. In aquatic microcosms, the inability of genetically engineered E. carotovora to persist, displace resident species, or affect the metabolic activity of aquatic communities indicates the low risk of adverse ecological consequences in aquatic ecosystems. Unlike previous investigations involving soil microcosms, densities of both the genetically engineered and wildtype E. carotovora remained at detectable levels over 60 d in both agricultural clay and forest loam soils. The type of soil significantly affected the survival of the GEM and the wildtype. The sorptive properties of clay particles, as well as low concentrations of soil nutrients and organic matter, may have contributed to the unexpected patterns of GEM and wildtype survival.

show abstract

Survival and ecological effects of genetically engineered Erwinia carotovora in soil and aquatic microcosms

Palmer

Scanferlato

Orvos

et al. 1997

Enviro Toxic and Chemistry

Self Cite

View full text Add to dashboard Cite

The release of genetically engineered organisms into the environment has raised concerns about their potential ecological impact. In this study, genetically engineered Erwinia carotovora strains expressing varying levels of reduced phytopathogenicity and wildtype E. carotovora strains were used in aquatic and soil microcosms to assess the survival, intraspecific competition, and effects upon specific groups of indigenous bacteria. In aquatic microcosms, the densities of Erwinia genetically engineered organisms (GEMs) and wildtype strains declined and fell below the detectable limit of plate counts 15 d after the microcosms were inoculated. In aquatic microcosms, engineered E. carotovora did not exhibit a competitive advantage over the wildtype. The effect of engineered and wildtype E. carotovora on densities of total and selected bacteria was not significantly different. Treatment with engineered E. carotovora did not change biomass values of the receiving community but did cause a transitory increase in metabolic activity. In aquatic microcosms, the inability of genetically engineered E. carotovora to persist, displace resident species, or affect the metabolic activity of aquatic communities indicates the low risk of adverse ecological consequences in aquatic ecosystems. Unlike previous investigations involving soil microcosms, densities of both the genetically engineered and wildtype E. carotovora remained at detectable levels over 60 d in both agricultural clay and forest loam soils. The type of soil significantly affected the survival of the GEM and the wildtype. The sorptive properties of clay particles, as well as low concentrations of soil nutrients and organic matter, may have contributed to the unexpected patterns of GEM and wildtype survival.

show abstract

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Cited by 17 publications

References 19 publications

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

Survival and Ecological Effects of Genetically Engineered Erwinia Carotovora in Soil and Aquatic Microcosms

Survival and ecological effects of genetically engineered Erwinia carotovora in soil and aquatic microcosms

Contact Info

Product

Resources

About