Resolution of four pectate lyase structural genes of Erwinia chrysanthemi (EC16) and characterization of the enzymes produced in Escherichia coli

Barras, Frédéric; Thurn, Kerry K.; Chatterjee, Arun

doi:10.1007/bf00329660

Cited by 75 publications

(47 citation statements)

References 23 publications

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“…PelA exhibits a very poor macerating ability and seems always to be produced at very low levels (22,224). Nevertheless, a mutation in the pelA gene results in a delay of symptoms (29).…”

Section: Effect Of Ph On the Expression Of Pectinolytic Enzymesmentioning

confidence: 99%

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

Brencic

Winans

2005

Microbiol Mol Biol Rev

339

208

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Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described

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“…PelA exhibits a very poor macerating ability and seems always to be produced at very low levels (22,224). Nevertheless, a mutation in the pelA gene results in a delay of symptoms (29).…”

Section: Effect Of Ph On the Expression Of Pectinolytic Enzymesmentioning

confidence: 99%

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

Brencic

Winans

2005

Microbiol Mol Biol Rev

339

208

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“…Unlike most saccharidases, the Pels have an unusually high pH optimum, ranging from 8 to 10 (Kotoujansky, 1987). The four Pel proteins of Erwinia chrysanthemi have a pH optimum between 8.9 and 9.5 (Barras et al, 1987). AI1 Pels are Ca2+ dependent and require Ca2' for in vitro activity.…”

mentioning

confidence: 99%

The Refined Three-Dimensional Structure of Pectate Lyase C from Erwinia chrysanthemi at 2.2 Angstrom Resolution (Implications for an Enzymatic Mechanism)

Yoder

Jurnak

1995

Plant Physiol.

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“…Four structural genes coding for the PLs secreted by E. chrysanthemi EC16 have been cloned and sequenced (13). These have been described as catalyzing the endolytic depolymerization of polygalaturonate (PGA), with PLa, PLb, and PLc catalyzing the formation of unsaturated dimer, trimer, and tetramer, respectively, and PLe catalyzing the formation of dimer, tetramer, and larger oligomers (1). By using ion-pair reverse-phase high-performance liquid chromatography (HPLC) and a chromatographic system employing an automatic sample injector, the kinetics of the formation of individual unsaturated oligogalacturonates, ranging in degree of polymerization (DP) from DP 2 to 13, has been determined (10).…”

mentioning

confidence: 99%

“…If this is the case, the evolutionary changes that have occurred have provided very specific changes in the depolymerization mechanism, along with changes in the pI of the protein products and upstream regulatory sequences. The very basic property of the PLe (pI 10), along with its depolymerization mechanism, may account for its potent macerating activity, especially in relation to PLa (1,13).…”

mentioning

confidence: 99%

Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16

et al. 1992

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The four pectate lyases (EC 4 The phytopathogenic activities of many Erwinia species are related to their abilities to macerate nonlignified tissues during infection. The initiation of the maceration process involves the depolymerization of pectin and pectate components of the rhamnogalacturonan fraction of the cell walls. Strains of Erwinia chrysanthemi effect this depolymerization through the secretion of a combination of pectin esterases and pectate lyases (PLs), along with a regulatory contribution by an exolytic pectate hydrolase activity (3,5,6,8). In addition to these macerating activities, E. chrysanthemi strains secrete cellulolytic and proteolytic enzymes that may contribute further to the pathogenic process (2,4,14).The application of molecular genetics has provided detailed information on the organization of the genes coding for pectinolytic enzymes in different strains of E. chrysanthemi and Erwinia carotovora as well as the regulation of their expression (3,5,8). Four structural genes coding for the PLs secreted by E. chrysanthemi EC16 have been cloned and sequenced (13). These have been described as catalyzing the endolytic depolymerization of polygalaturonate (PGA), with PLa, PLb, and PLc catalyzing the formation of unsaturated dimer, trimer, and tetramer, respectively, and PLe catalyzing the formation of dimer, tetramer, and larger oligomers (1). By using ion-pair reverse-phase high-performance liquid chromatography (HPLC) and a chromatographic system employing an automatic sample injector, the kinetics of the formation of individual unsaturated oligogalacturonates, ranging in degree of polymerization (DP) from DP 2 to 13, has been determined (10). By using this approach, the recombinant trimer-generating PLb derived from E. chrysanthemi EC16 has been shown to have a depolymerization mechanism similar to those of PLs secreted by other bacteria (11,12) for the different PLs were derived from E. chrysanthemi EC16 and expressed from plasmids in E. coli HB101 as previously described (7, 13). PLa, PLb, PLc, and PLe were expressed from plasmids pPEL812, pPEL344, pPEL410, and pPEL748, respectively. Batch cultures (1 liter in 2.8-liter Fernbach flasks) were grown with shaking (140 rpm; New Brunswick G10 gyrotary shaker) at room temperature in Luria broth containing ampicillin (50 ,ug/ml). Isopropyl-oi-Dthiogalactopyranoside was added to a concentration of 1 mM when the culture was initiated (for the overexpression of PLa and PLc) or when the culture turbidity (optical density at 600 nm) was 0.5 to 0.7 (for the overexpression of PLb and PLe). Cells were harvested at early stationary phase (optical density at 600 nm = 2.1 to 2.4) by centrifugation at 5,500 rpm (GSA rotor; DuPont-Sorvall), and the pellets were washed by resuspension with 0.2 M Tris-HCl, pH 8.3, followed by recentrifugation. Periplasmic enzymes were released from cells following spheroplast formation (7,15).PLa and PLb were purified from periplasmic fractions by chromatofocusing, as previously described for the purification of analogous act...

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Resolution of four pectate lyase structural genes of Erwinia chrysanthemi (EC16) and characterization of the enzymes produced in Escherichia coli

Cited by 75 publications

References 23 publications

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

The Refined Three-Dimensional Structure of Pectate Lyase C from Erwinia chrysanthemi at 2.2 Angstrom Resolution (Implications for an Enzymatic Mechanism)

Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16

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