Rhamnogalacturonate Lyase RhiE Is Secreted by the Out System in Erwinia chrysanthemi

Abstract

Supernatants of rhamnose-induced Erwinia chrysanthemi strain 3937 cultures contain a principal secreted protein named RhiE. A rhiE mutant has been found among a set of rhamnose-induced MudI1681 lacZ fusions. RhiE is a 62-kDa protein that has rhamnogalacturonate lyase activity on rhamnogalacturonan I (RG-I). It does not require a divalent cation for its activity and has an optimal pH of 6.0. rhiE expression is strongly induced in the presence of rhamnose but is also regulated by PecT and Crp, two regulators of the transcription of pectinolytic enzyme genes. RhiE is secreted through the type II Out secretion pathway. RhiE has no disulfide bond. The absence of RhiE secretion in a dsb mutant indicated that disulfide bond formation is required for the biogenesis of the secretion apparatus. RhiE was searched for in several E. chrysanthemi strains by using antibodies, and it was found to be present in one-third of the strains tested. However, the reduced virulence of the rhiE mutant indicates that degradation of the RG-I region of pectin is important for full virulence of E. chrysanthemi.Pectin is one of the main components present in plant cell walls. The pectin molecule can have three types of backbone: homogalacturonan, rhamnogalacturonan I (RG-I), and RG-II. Homogalacturonan (polygalacturonate [PGA]) is a polymer of galacturonic acid residues linked by ␣ 1-4 bonds which are partially methylated and acetylated. Homogalacturonan forms the smooth regions of pectin which are interspersed with hairy regions of RG-I and RG-II. RG-I has a backbone composed of as many as 100 repeats of the disaccharide ␣-L-rhamnose-␣-1,4-galacturonic acid. Side chains of arabinan or galactan are attached to O-4 of the rhamnosyl residues. RG-II is a complex polysaccharide with a short homogalacturonan backbone to which are linked side chains containing more than 10 different types of sugars (1).Plant-pathogenic microorganisms often secrete enzymes that are able to degrade the polysaccharides of cell walls in order to disorganize the plant tissue and obtain carbon sources for their growth. The enzymes that degrade the smooth regions of pectin have been extensively studied (26). The action of pectin methylesterases and of pectin acetylesterases liberates PGA. PGA is then degraded by pectate lyases or polygalacturonases that cleave either in an endo or in an exo mode. These enzymes have been characterized in bacteria as well in fungi. Much less is known about the enzymes degrading RG-I and RG-II. It seems that the presence of side chains prevents the degradation of the homogalacturonan backbone of RG-II by polygalacturonase and pectate lyases, and the debranching enzymes have not yet been characterized. Only a few enzymes that are able to cleave the RG-I backbone have so far been characterized, mostly in Aspergillus aculeatus. A rhamnogalacturonate lyase (18) cleaves inside the chain between a rhamnose and a galacturonate, creating an unsaturation on the galacturonate at the nonreducing end. A rhamnogalacturonate rhamnohydrolase (17) and a rha...

Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4*1

“…In their active form, pectate lyases contain at least one bound calcium ion, which plays an integral part in substrate binding and activation [33]. Though present evidence suggests that calcium ions are not strictly required for the catalytic activity of polysaccharide lyase family 4 [34,35], the calcium ion may have a unique structural role explaining its positive effects on catalysis.…”

“…Low domain I homology (4-9% identity) between fungal and plant sequences may indicate an evolutionary divergent sub-family in which the catalytic machinery has been maintained, while the substrate recognition has undergone major modifications. RhiE, a bacterial RG-lyase from Erwinia chrysanthemi, is more related (18-22% identity) to the A. thaliana MYST sequences than to A. aculeatus RG-lyase (7% identity) and has been shown to cleave to RG-I backbone by a b-elimination mechanism [35]. Such sequence diversity in polysaccharide lyase family 4 may reflect variation in the pattern of arabinose and galactose branching side chains of RG-I from different species.…”

“…This could imply that arginine is also the catalytic base in pectin lyases, though this would require an unprecedented pK a shift of arginine to 6.0-6.5, which could be possible given the appropriate environment. Although pectin lyases contain the equivalent conserved catalytic arginine as the pectate lyases, they have a lower pH optimum 6.0-6.5, which is similar to the family 4 RG-lyases [10,35]. A lysine (Lys150) is the only completely conserved residue found in the sequences in polysaccharide lyase family 4, and the possibility that RG-lyase and pectate/pectin lyases operate by a related mechanism cannot be ruled out.…”

PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937

“…This phase of colonization is followed by the symptomatic phase of the disease only when environmental conditions are favorable for massive bacterial multiplication and production of plant cell wall-degrading enzymes (41). E. chrysanthemi produces different types of pectinases: pectin methyl esterases, pectin acetyl esterases, exo-and endopectate lyases, exopolygalacturonases, and a rhamnogalacturonate lyase (25,51). All of these enzymes have the potential to degrade different parts of pectin, including the linear and branched regions, but are not equally involved in symptom initiation and spreading (3,5,27).…”

Characterization of the Erwinia chrysanthemi gan Locus, Involved in Galactan Catabolism

“…Previous studies showed that E. chrysanthemi is able to degrade and catabolize a range of plant structural polysaccharides, including linear regions of pectin and the backbone of the ramified regions RGI (19,21,28). Galactans are constituents of the side chains attached to RGI.…”