Isolation and genetic study of Erwinia mutants devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system

Mutants of bacteria belonging the genus Erwinia (Erwinia chrysanthemi and Erwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI+ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that the ptsI and ptsH genes of E. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min. Sequencing of a portion of the E. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.     

Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35

The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PeIL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40 decreased C.     

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.     

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.     

Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B.

Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.     

Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB , codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB -like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.     

Structural and functional insights into Erwinia carotovora L-asparaginase

Bacterial L-asparaginases are enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral-asparaginase in the presence of L-Asp was determined at 2.5 A resolution and refined to an R cryst of 19.2 (R free = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.     

Genetic Transfer of Episomic Elements Among Erwinia Species and Other Enterobacteria: F′lac+

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.     

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.     

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.     

Cloning of a galacturonic acid uptake gene from Erwinia chrysanthemi EC16

Abstract A 3.4 kb fragment of Erwinia chrysanthemi EC16 DNA capable of complementing galacturonic acid uptake mutants ( exuT ) was identified and cloned into a multicopy vector. In E. chrysanthemi B374 exuT mutants, the cloned DNA provided for growth of the mutant strains on galacturonic acid by complementing the galacturonic acid uptake defect. Alkaline phosphatase ( phoA ) gene fusions with the cloned DNA suggested that most of the cloned DNA was necessary for complementation of exuT mutant strains. Using anti-alkaline phosphatase antibody, a hybrid ExuT-PhoA protein was localized to the membrane fraction of the bacterium.     

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3A-digested E. chrysanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the pEW325 cosmid with a 35-kb insert of Erwinia DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrysanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PL10, PL20 and PL130 with isoelectric points of about 9.3, 9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb HindIII fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPL01 was constructed. Starting from the pPL01, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPL04) and then in a 2.7-kb fragment (pPL03). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.     

recA-genes of Erwinia chrysanthemi ENA49

The recA gene of Erwinia chrysanthemi ENA49 has been cloned in vivo in Escherichia coli K12, recA13 cells using the plasmid pULB113. On the basis or DNA repair and recombination deficiencies complementation, of restoration of the inducible "SOS"-response functions the functional identity of the cloned gene with the recA gene was concluded. The recA gene was localized in the 18th min region of the chromosomal genetical map of Erwinia chrysanthemi ENA49 between the genes proA and pheA.     

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.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.