Enzymatic And Molecular Mechanism Of Tissue Maceration By Softrot Bacteria

5.4.1 Biochemical Characterization of Pectate Lyase (PL) Analysis of PL Isozymes

Soft-rot erwinia including Ech, Ecc, and Eca are characterized by their ability to produce an array of pectolytic enzymes including pectin methylesterase (PME), polygalacturonase (PG), pectin lyase (PNL), and pectate lyase (PL). These enzymes can be readily detected in filtrates of bacterial cultures and assayed by the standard biochemical procedures [58]. The PLs produced by Ech, Ecc, and Eca are usually present in multiple (three to five) isozymic forms in culture filtrates, which can be readily identified by isoelectric focusing (IEF) gel electrophoresis and overlay enzyme staining techniques [59]. In Ech, a second set of PL isozymes, inducible only in the presence of plant constituents, have been identified using molecular genetic and enzyme analyses [60]. The biological and pathological function of each pectic enzyme produced by soft-rot erwinia has not been fully determined. It is also unclear if production of certain pectic enzymes is restricted to specific tissues or organs or limited to specific stages of plant development. With the aid of molecular genetic technologies, experimental results [61-63] have shown that no single pectic enzyme produced by soft-rot erwinia is absolutely required for the pathogen to initiate disease development. However, the PL isozymes, especially alkaline PLe, usually display the highest degree of tissue macerating ability in vitro [64] and are assumed to be the principal enzymes required for development of soft rot by erwinia in vivo [62].

Because of their complex pectic enzyme system, it is difficult to purify a single PL isozyme from culture filtrates of Erwinia spp. However, due to the simplicity of the pectic enzyme system in other spoilage bacteria, including P.fluorescens, P. viridiflava, and Xanthomonas campestris, it is relatively easy to purify the PLs from their culture filtrates [65]. Normally, following two simple steps (ammonium sulfate precipitation and anion exchange chromatography), the PL can be purified from culture filtrates of these two PF pseudomonads to near homogeneity [65,66]. Enzymological properties of PLs purified from culture filtrates of P.fluorescens and P. viridiflava have been characterized, and a minute amount of purified enzyme was capable of causing total maceration of potato tuber tissue even in the absence of live bacteria [67]. Production of Other Pectic Enzymes

In addition to PLs, soft-rot erwinia produces an array of other pectic enzymes including pectin methyesterase (PME), polygalacturonase (PG) and pectin lyase (PNL). Production of PME, PG, and PNL by soft-rot pathogens does not seem to play a significant role in initiating the maceration of plant tissues. However, they may be required for interactions with host plants or coping with adverse environments [68]. It has been reported that purified PG, but not purified PME or PNL by itself, is sufficient to induce soft rot of potato tuber slices. Production of PNL by soft-rot Erwinia spp. [69] and Pseudomonas spp. [70] is inducible only after exposing the bacteria to DNA-damaging agents such as ultraviolet radiation and mitomycin C. The ecological and pathological significance of producing PNL by erwinia and pseudomonas remains obscure. The role of PME in soft-rot pathogenesis is minimal and probably not required. However, it has been suggested that a coordinated action between PME and PL may be necessary for complete degradation of native pectins in plant cell walls.More information about the enzymatic mechanism of soft-rot pathogenesis by Erwinia spp. can be found in earlier reviews [61-63,68].

5.4.2 PL as the Principal Tissue-Macerating Factor Transposon Mutagenesis

The notion that PL is the principal or sole pathogenicity factor of soft-rotting pseudomonads can be supported by a series of molecular genetic studies. By using transposon (Tn5)-mediated mutagenesis, Liao et al. [66] isolated several types of P. viridiflava mutants that became defective in production or secretion of PL. When assayed on plants, nonpectolytic P. viridiflava mutants were unable to induce soft rot on potato tuber slices. The loss in the ability to produce or secrete PL is accompanied by the loss in the ability to induce soft rot. This result provides the first unequivocal evidence that PL is the sole enzyme required for the induction of soft rot by P. viridiflava [66]. Cloning and Analysis of PL Genes

The gene encoding PL has been cloned from the genomes of P. fluorescens [71], P. viridiflava [72], and Xanthomonas campestris [73]. When cloned PL gene was mobilized into nonpectolytic mutants of P. viridiflava or P. fluorescens, the PL-producing and soft-rotting ability of nonpectolytic mutants was restored [74-76]. These results provide direct genetic evidence that the gene coding for PL is the principal or sole pathogenicity or virulence determinant of soft-rotting P. viridiflava or P. fluorescens.

5.4.3 Control of PL Production and Pseudomonas Rot Two-Component Regulatory Gene System

The enzymatic and molecular genetic mechanism of soft-rot pathogenesis caused by erwinia has been extensively investigated and reviewed [61-63,68]. However, very little is known about the mechanism by which PF pseudo-monads regulate the production of PL and induction of tissue maceration in plants. Pleotropic mutants of P. fluorescens and P. viridiflava showing simultaneous loss of production of both pectolytic and proteolytic enzymes have been identified by transposon mutagenesis [74-76]. Results from Southern Blot analysis revealed that mutants were derived from the insertion of Tn5 into one of two distinct genomic fragments. Two genes regulating the production of pectolytic enzyme and induction of soft rot, designated as gacS (=repA or lemA) and gacA (=repB), have been identified in these two fragments and subsequently cloned and confirmed by complementation studies [74-76].

Based on the nucleotide sequence analyses, the gacS and gacA genes were respectively predicted to encode a sensory and a regulator protein in the two-component regulatory protein family [74,76]. The gacS/gacA genes were predicted to act in pairs to mediate the production of an array of extracellular compounds including PL, protease (PRT), exopolysaccharide (EPS), and ion-chelating siderophores [74-76], possibly in response to environmental signals. The gacS/gacA genes in biological control strains of P. fluorescens have also been shown to regulate the production of phospholipase C [77], lipase [78], and antibiotics [79-81]. Proper function of the gacS/gacA gene system is also required for the formation of disease lesions on snap beans by Pseudomonas syringae pv. syringae [82]. This two-component gacS/gacA gene system can also interact with the stationary-phase factor 8s (encoded on rpoS) in a biological control strain of P. fluorescens to control the responses of this strain to environmental stimuli [83]. In P. aeruginosa, the activator GacA will interact with two quorum sensing proteins (LuxR, LuxI) to regulate the production of an autoinducer (butylhomoserine lactone) [84]. It has not yet been investigated, however, if RpoS, LuxR, and LuxI would act in concert to regulate the production of PL and other extracellular compounds in soft-rotting P. fluorescens and P. viridiflava.

A group of P. viridiflava mutants failing to excrete PL and Prt across the outer membrane have also been generated by transposon mutagenesis during the isolation of nonpectolytic mutants [66]. These secretion-defective mutants (designated Out ) were assumed to result from the insertion of Tn5 into a gene belonging to the Type II secretory gene family [85,86]. Out mutants were also unable to induce soft rot on potato tuber slices and bell pepper fruits [66]. This indicates that the synthesis and the secretion of PL are two critical steps required for induction of soft rot. Role of Calcium Ions

Production of PL in certain strains of P. fluorescens is inducible by pectic substrates [87,88] or plant tissue extracts [89-91]. However, in other P. fluorescens strains production of PL is not affected by the type of carbon source included in the medium [91]. Recently, we investigated the mode of PL production in 24 strains of P. fluorescens and found that production of PL in certain P. fluorescens strains (4 out of 24) was not induced by pectic substrates but by Ca2+ [92]. These four strains produce ten times more PL in medium containing 1 mM CaCl2 than in one containing no CaCl2 supplement. Supplement of CaCl2 in the medium not only affects the amount but also the final destination of PL. Over 86% of total PL produced by strain CY091 in CaCl2-supplemented medium was excreted into the culture fluid. By comparison, only 13% of total PL produced by this strain in CaCl2-deficient medium was detected in the extracellular fraction. The effect of Ca2+ on PL production is concentration-dependent and can be replaced by Sr2+, but not by Zn2+, Fe2+, Mn2+, Mg2+, or Ba2+ [92]. Use of Ion-Chelating Agents for Control of Pseudomonas Rot

Because of the indispensable role of Ca2+ in the production, secretion, and catalytic activity of PLs, the potential of using ion-chelating agents such as EDTA for control of pseudomonas rot has been investigated [92]. Application of ion-chelating agents such as EDTA to limit the availability of Ca2+ to P. fluorescens infecting the plants thus offers a potential strategy for control of soft rot caused by pseudomonads. We have demonstrated that application of 0.05 ^M (or 40 ppm) of EDTA, alone [92] or in combination with a bacteriocin (nisin) [93], suppresses the induction of soft rot by P. fluorescens. Zucker and Hankin [94] also reported that EDTA treatments reduced the soft rot potential of potato tubers.

It should be noted, however, that the EDTA treatment would not be effective for control of soft rot caused by erwinia, because Erwinia spp. produce not only Ca2+-dependent PL but also Ca2+-independent PG. However, infiltration of potato tubers or apple fruits with CaCl2 can enhance their resistance to attack by Ecc or Eca [95] or Penicillium expansum [96]. Changes in calcium fertilization in potato fields could also affect the susceptibility of potato tubers to bacterial soft rot [97]. Infiltration of potato tubers and fruits with Ca2+ was thought to strengthen the cell walls and consequently increase their resistance to postharvest rot pathogens [98]. None of the above control strategies have been applied on a large scale for commercial operations.

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