The secretome of the plant pathogenic bacterium Erwinia chrysanthemi

Abstract

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

“…In Erwinia chrysanthemi, genes involved in the type II secretion pathway, outF and outG, are induced during spinach infection (299). Pectinases, which are major virulence factors in the soft rot-causing Erwinia chrysanthemi, are secreted through the Out system (123,273). It was also demonstrated with IVET that genes involved in pectin degradation, plyD and pme, are upregulated during plant infection by the plant pathogens Pseudomonas syringae (16) and Ralstonia solanacearum (26), respectively.…”

Structural Biology of Pectin Degradation byEnterobacteriaceae

“…However, there are examples of downstream periplasmic CEs in Erwinia spp. and Vibrio parahaemolyticus (PaeX/ CE12X and PemB/CE8B) (40,56,60), which suggests some plasticity in the different stages of pectin degradation compared between species. In this regard, it is important to consider that organisms with periplasmic CEs also contain pectate depolymerases in the same cellular compartment, which may alter the required stringency of extracellular de-esterification events.…”

“…Although the molecular determinants of selective extracellular secretion verses periplasmic localization are still unclear, two polypeptide components of the OUT system, OutC and OutD, have been identified to specifically interact with exoproteins and likely participate in this process (12,46). Proteomic analysis of the E. chrysanthemi secretome detected 25 unique polypeptides involved in pectin degradation (40). These included eight endo-acting extracellular Pels (PelA to -E/ EchPL1A to -E, PelZ/EchPL1Z, PelI/EchPL3I, and PelL/ EchPL9A), an intracellular exo-acting Pel (PelX/EchPL9X), one extracellular polygalacturonase (PehN/EchGH28N), three periplasmic exopolygalacturonases (PehV to -X/EchGH28V to -X), one extracellular (PaeY/EchCE12Y) and one periplasmic (PaeX/EchCE12X) Pae, and one extracellular Pem (PemA/ EchCE8A) (40) ( Table 1).…”

“…Closer analysis, however, does expose species-specific localization and isozyme differences. For example, E. chrysanthemi contains one extracellular ␣-endo-poly-D-galacturonosidase and three periplasmic ␣-exo-poly-D-galacturonosidases that releases disaccharides (40,64), and Y. enterocolitica contains only one periplasmic ␣-exo-poly-D-galacturonosidase with homologous activity (Table 1) (56). This distinction is in agreement with potential substrate accumulation within each environment, as endo-acting enzymes would degrade the extracellular highly polymerized forms of polygalacturonate present within the plant cell wall, and exo-acting enzymes would be most efficient at producing small oligogalacturonides for intracellular transport from pectic fragments that accumulate in the periplasm (see below).…”

“…In Erwinia spp., there is both an extracellular Pae (PaeY/CE12Y) and a methylesterase (PemA/CE8A), the latter of which is also present in Yersinia spp. (40,56). The acetylesterase removes acetylations at C-2 and C-3 (62), and the methylesterase demethylates the methoxylated uronate groups, releasing methanol and restoring the inherent negative charge on the galacturonide product (9,60) (Fig.…”