Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase

Abstract

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl1) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by TnS mutagenesis using the R plasmid pJB4JI. Ogl1 Exu+ (Exu+, D-galacturonate utilization) and KduD-Exu-strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Oglmutants and a KduDrecombinant contained a single copy of TnS and that TnS (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl1 strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD-strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate Iyase than the KduD+ parents did. In the KduD-strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Purification and functional characterization of PecS, a regulator of virulence‐factor synthesis in Erwinia chrysanthemi

“…No other factor is known to influence the expression of cellulases (Aymeric et a/., 1988). In contrast, pectate lyase synthesis is subjected to a wide range of environmental conditions such as the presence of pectin degradative products or plant extracts (Chatterjee et a/., 1985;Condemine and Robert-Baudouy, 1986;Bourson ef a/., 1993), anaerobiosis, temperature, osmolarity, nitrogen starvation, catabolic repression (Hugouvieux-Cotte-Pattat efal., 1992) and iron status (Expert and Gill, 1992). Gene-fusion studies have shown that genes involved in the intracellular (e.g.…”

Integration of environmental and host-derived signals with quorum sensing during plant-microbe interactions

“…In E. carotovora , a further level of complexity results from the fact that the expression of many of the plant cell wall‐degrading enzymes is positively regulated by plant cell wall breakdown products that are generated by the action of the bacterial pectinases on the plant tissues. These products include 5‐keto‐4‐deoxouronate, 2,5‐diketo‐3‐deoxygluconate and 2‐keto‐3‐deoxygluconate (KDG) (Chatterjee et al ., 1985; Nasser et al ., 1994). Regulation arises via dissociation of the transcriptional repressor KdgR in the presence of these metabolites (Liu et al ., 1999).…”

Cloning of a galacturonic acid uptake gene from Erwinia chrysanthemi EC16

“…E. chrysanthemi ERH215 cells bearing pOIV3 and cells containing TnphoA insertions into the plasmid were grown at 28"C overnight in 100 ml M-9 salts medium with glycerol and GA as carbon sources. Cells were harvested by centrifugation and lysed on ice by sonication in 15 ml M-9 salts containing 10 mM MgCI 2, 0.3 mM dithiothreitol and 0.2 mM phenylmethyl sulfonylfluoride [4]. Unbroken cells were removed from the cell lysate by centrifugation at 10000 ×g for 10 rain.…”

“…Erwinia chrysanthemi EC16 produces pectolytic enzymes which depolymerize the pectic compounds in plant cell walls into monomers, dimers and multimers, both saturated and unsaturated, of galacturonic acid (GA) [1]. The degraded cell wall compounds are transported into the bacterium and are utilized as carbon and energy sources [2][3][4][5]. While the production of pectolytic enzymes is thought to be involved in virulence, the uptake of degraded plant cell wall products and the role of these uptake systems in virulence are not fully understood [6,7].…”