Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pglA and its involvement in phytopathogenicity

Abstract

A major endopolygalacturonase excreted by Pseudomonas solanacearum was purified to >95 % homogeneity and shown to have an isoelectric point of 9.0 and a subunit molecular mass of 52 kilodaltons (kDa). The gene encoding this enzyme (pglA) was isolated from a genomic library of P. solanacearum DNA based on its expression in Escherichia coli and shown to be contained on a 1.8-kilobase DNA fragment. The identity of the pglA gene product and the 52-kDa polygalacturonase was demonstrated by immunoadsorption and isoelectric focusing experiments. The cloned pglA gene was apparently expressed from its own promoter in E. coli and its product was partially secreted into the periplasm. The pglA gene was insertionally inactivated in vitro and used to mutate the chromosomal pgL4 gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant strain was deficient in production of the 52-kDa polygalacturonase and took twice as long to wilt and kill tomato plants as the wild-type parent in plant bioassay experiments. Complementation in trans with the wild-type cloned pg4 gene restored virulence to near wild-type levels. The data indicate that the pglA gene is important, but not absolutely necessary, for pathogenesis.

The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility, pectate lyase activity, and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209

“…After incubation at 30°C for 24 h, 1 mL of 0.5% soft agar containing 1% hexadecyltrimethylammonium bromide was overlaid on the culture medium to visualize pectate lyase activity. 28) Pectate lyase activity was assessed by measuring the length which pulled the diameter of the colony from that of clear zone formed by pectate lyase activity. Pectate lyase assay was repeated five times.…”

X-ray analysis of crystals of polygalacturonase A fromPseudomonas solanacearum

“…In this study, PG was purified from P. solanacearum AR containing pDR340, a broad host range plasmid construct overexpressing the pglA gene from a lac promoter (Schell et al, 1988). To simplify purification P. solanacearum strain AR was used as host because it harbors a phcA mutation that simultaneously increases pglA expression and blocks expression of many other extracellular proteins and viscous extracellular polysaccharides (Schell et al, 1994;Huang, Denny, & Schell, 1993).…”

“…Pseudomonas solanacearum is a soil-borne plant pathogen that causes wilting diseases in many crop plants worldwide (Hayward, 1991). Schell, Roberts & Denny (1988) and Denny, Carney & Schell (1990) were able to demonstrate that a major secreted PG encoded by the pglA gene plays a significant role in disease, but is not absolutely necessary for pathogenesis. P. solanacearum produces at least two other PG's besides PglA, both are apparently exopolygalacturonases (Schell, Denny & Huang, 1994).…”

“…All three structures are composed of three parallel /3-sheets in a parallel /3-helix motif. Structural analysis by Goodenough et al (1991) of a fungal PG from Clostridium thermocellum based on far UV-CD spectra suggests that PG will also contain predominately /3 structure, with very little a structure.In this study, PG was purified from P. solanacearum AR containing pDR340, a broad host range plasmid construct overexpressing the pglA gene from a lac promoter (Schell et al, 1988). To simplify purification P. solanacearum strain AR was used as host because it harbors a phcA mutation that simultaneously increases pglA expression and blocks expression of many other extracellular proteins and viscous extracellular polysaccharides (Schell et al, 1994;Huang, Denny, & Schell, 1993).…”

“…To simplify purification P. solanacearum strain AR was used as host because it harbors a phcA mutation that simultaneously increases pglA expression and blocks expression of many other extracellular proteins and viscous extracellular polysaccharides (Schell et al, 1994;Huang, Denny, & Schell, 1993). The protein was purified based on the procedure described by Schell et al (1988). To a culture medium composed of 20 mM sodium-potassium phosphate, pH 7.0, 0.1% ammonium sulfate, 0.2% glucose, 0.1% yeast extract, 0.1% casamino acids, and 20 ~tg ml -I tetracycline, the P.…”