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.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Cloning, expression and characterisation of Erwinia carotovora L-asparaginase

Bacterial L-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. L-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised L-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of V(max) and V(max)/K(m) were analyzed. The pH-dependence of V(max) shows one transition in the acidic pH range with pK(a)=5.4, and the pH-dependence of V(max)/K(m) exhibits two transitions with pK(a)=5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pK(a) at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pK(a) observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.     

胡萝卜软腐欧文氏菌中信号分子合成酶expI基因的克隆、表达及其分析

用PCR方法从胡萝卜软腐欧文氏菌的基因组DNA中扩增出信号分子合成酶expI基因,将其克隆到大肠杆菌表达载体pET-28α(+)上,转化大肠杆菌BL21(DE3),获得高效表达expI基因的重组大肠杆菌BL21(pET28α-expI).重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为24.8kD,与预期分子量相符.经薄层层析和高效液相色谱分析发现该重组菌产生的信号分子种类为N-3-羰基己酰高丝氨酸内酯和N-己酰高丝氨酸内酯与胡萝卜软腐欧文氏菌产生的一致.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.     

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.     

Isolation and preliminary characterization of cryptic plasmids from Erwinia carotovora]

Of the fifty-two Erwinia carotovora strains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovora strains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovora strains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, 47.7-kbp pCA 6-2. Three E. carotovora subsp. carotovora strains and one E. carotovora subsp. atroseptica strain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

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

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.     

Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an ₂₂ tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.     

Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families

Pectate lyase (Pel) plays a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have characterized the four Pel isoenzymes of Erwinia carotovora subspecies carotovora strain SCRI193. In this paper we concentrate on two isoenzymes which have different locations in SCRI193: PLb is periplasmic and PLc is extracellular. Comparison of the gene products and nucleotide sequences of pelB and pelC allowed us to assign them to different gene families. In addition, we have identified a number of conserved amino acid residues that are common to all extracellular Pel isoenzymes.