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Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora.

ABSTRACT: 1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s(0) (20,w) 7.43S and a sedimentation-equilibrium molecular weight of 135000+/-10000 give a frictional ratio (f/f(0)) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2+/-0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5-8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate-polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500-38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m'](233)=-3522+/-74 degrees and [m'](200)=9096+/-1700 degrees . These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

SUBMITTER: Cammack KA

PROVIDER: S-EPMC1178385 | biostudies-other | 1972 Jan

REPOSITORIES: biostudies-other

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Project description:Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.

Project description:BACKGROUND: Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella. RESULTS: B. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX. CONCLUSION: B. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this type of cell death in vascular plants, our results suggest that E.c. carotovora CFSCC1 containing HrpN and B. cinerea could also induce this type of cell death in Physcomitrella. Our studies thus establish Physcomitrella as an experimental host for investigation of plant-pathogen interactions and B. cinerea and elicitors of E.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant.

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Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora.

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