Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.     

Pectate lyase gene regulatory mutants of Erwinia chrysanthemi.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.     

Processing of the pectate lyase PelI by extracellular proteases of Erwinia chrysanthemi 3937

Erwinia chrysanthemi causes soft rot on various plants. The maceration of plant tissues is mainly due to the action of endopectate lyases. The E. chrysanthemi strain 3937 produces eight endopectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) that are secreted by the Out pathway. The necrotic response elicited by the wild-type E. chrysanthemi strain on tobacco leaves is due to an extracellular protein secreted by the Out machinery. Purification of the active factor revealed that it corresponds to a pectate lyase presenting immunological cross-reaction with PelI. Analysis of pelI and out mutants indicated that the necrosis-inducing pectate lyase results from a post-translational modification of PelI occurring extracellularly both in culture media and in planta . This modification consists of the cleavage of 97 N-terminal amino acids by the extracellular proteases of E. chrysanthemi . The enzymatic properties of the maturated form, PelI-3, are not, or only weakly, modified. However, this maturation gives rise to a small size and basic form that is active as a defence elicitor in plants.     

Expression of the pectate lyase gene from Erwinia chrysanthemi ENA49 in cells of other Erwinia species

The gene for a pectate lyase of E. chrysanthemi ENA49 cloned in a recombinant plasmid pPTL1 (a derivative of RSF1010) was transferred into E. carotovora. The pectate lyase determined by the cloned gene was secreted into the cultural medium from the cells of E. crysanthemi EC16. Partial secretion of the enzyme was registered for E. carotovora cells. The major part of EC1 E. chrysanthemi pectate lyase synthesized by E. carotovora cells is accumulated in periplasmic and cytoplasmic fractions. The obtained results suggest the different specificity or efficiency of pectate lyase secretion systems in the studied Erwinia strains.     

Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi.

The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis.     

Mu-lac insertion-directed mutagenesis in a pectate lyase gene of Erwinia chrysanthemi.

The pelC gene, which encodes one of the five major pectate lyase (PL) isoenzymes in Erwinia chrysanthemi 3937, designated PLc, was subcloned from a hybrid lambda phage into a pBR322 derivative and mutagenized with a mini-Mu-lacZ transposable element able to form fusions to the lacZ gene. One plasmid (pAD1) which had an inactivated pelC gene and a Lac+ phenotype was selected in Escherichia coli. This plasmid was introduced into Erwinia chrysanthemi, and the pelC::mini-Mu insertion was substituted for the chromosomal allele by homologous recombination. This strain lacks the PLc isoenzyme. This Erwinia chrysanthemi strain has a Lac+ phenotype that is inducible by polygalacturonate, as are the wild-type PL activities.     

Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16.

The four pectate lyases (EC 4.2.2.2) secreted by Erwinia chrysanthemi EC16 have been individually produced as recombinant enzymes in Escherichia coli. Oligogalacturonates formed from polygalacturonic acid during reactions catalyzed by each enzyme have been determined by high-performance liquid chromatography analysis. PLa catalyzes the formation of a series of oligomers ranging from dimer to dodecamer through a random endolytic depolarization mechanism. PLb and PLc are trimer- and tetramer-generating enzymes with an identical combination of endolytic and exolytic mechanisms. PLe catalyzes a nonrandom endolytic depolymerization with the formation of dimer as the predominant product. The pectate lyases secreted by E. chrysanthemi EC16 represent a battery of enzymes with three distinct approaches to the depolymerization of plant cell walls.     

In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi

Using an RP4 plasmid which carries a mini-Mu prophage which allows it to integrate spontaneously random pieces of its host chromosome, we cloned in vivo at least some of the pectate lyase and cellulase genes of the Erwinia chrysanthemi strain B374. The RP4-prime plasmids were used to localize the cloned genes on the B374 chromosome by co-transposition mapping and to subclone most of the genes in a classic high copy number plasmid vector.     

The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi

The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.     

An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi.

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.     

Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.     

2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

Expression in Myxococcus xanthus of foreign genes coding for secreted pectate lyases of Erwinia chrysanthemi

Structural genes for the five major pectate lyases of Erwinia chrysanthemi, pelA, pelB, pelC, pelD and pelE, have been transferred and expressed in Myxococcus xanthus. These proteins, which are secreted by their original host, are also produced, mainly into the medium, by Myxococcus. These results are discussed in the context of current knowledge about secretion in bacteria.