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)     

Survival of soft rot coliforms, Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica in soil in Scotland

An enrichment method was used to monitor Erwinia carotovora in soil or the rhizosphere of different crops and weeds in 17 fields with different cropping histories on three farms. The bacteria were detected in all fields not cropped with potatoes, although not consistently, and the mean annual frequency of detection was generally low (< 10%). Fields in which potatoes were grown were extensively contaminated after harvest in September but contamination declined over the winter to very low levels by early summer in the following year. Contamination level tended to rise in some fields without potatoes regardless of their cropping history but for only a short time during autumn and winter. The bacteria were no more frequent in rhizosphere soil of any of the weeds or crops examined, with the exception of brassicas, than in bare soil. In fields where more than 16 months had elapsed since cropping with potatoes, 91% of erwinia isolates obtained were E. carotovora subsp. carotovora , the remainder being E. carotovora subsp. atroseptica. The bacteria were shortlived in soil and in the rhizospheres of inoculated field and pot grown crop and weed plants. Longevity was greater in dry (10% moisture) than in wet (21% moisture) soil and decreased as temperatures rose, particularly above 25°C. Survival was best in association with brassica plants, moderate on grasses and cereals, and least on potatoes and weeds. E.c. carotovora survived better than E.c. atroseptica. Because survival of the bacteria in soil is apparently restricted, their presence in fields could be attributed to recurrent introductions from different sources.     

Survival of the phytopathogen Erwinia carotovora subsp. carotovora in sterile and non-sterile soil, sand and their admixture

Survival of phytopathogens in irrigation water and irrigated soil is of major concern to the agricultural community. In the present study, an Erwinia carotovora subsp. carotovora strain was tested for survival capability in three non-sterile and heat-sterilized soil matrices (soil, sand and soil + sand) over 35 d. In all non-sterile soil matrices, Erw. carotovora subsp. carotovora numbers declined below the detection limit over 35 d, the main variance being the decline rate related to nutrients available in the different matrices (soil, soil + sand and sand respectively). In heat-sterilized soil and soil + sand matrices Erw. carotovora subsp. carotovora revealed a regrowth, while in sterile sand matrix its decline was lower over the same time period. In previous published reports, when soil was sterilized by irradiation, such a regrowth was not observed. Application of an initial single load of sodium nitrate solution (70 mg l⁻¹) was found to extend bacteria survival rate in non-sterile and sterile soil columns. In sterile soil columns supplemented with sodium nitrate, Erw. carotovora subsp. carotovora did survive well for up to 60 d, with a major regrowth over the first 12 d and decline up to day 60, reaching initial loading numbers. The information on the potential survival of Erw. carotovora subsp. carotovora in soil for up to 35 d and regrowth in sterile soil should be of concern, especially when irrigation is performed with poor quality water.     

Potato Protoplast-derived Callus Tissue Challenged with Erwinia carotovora subsp. carotovora: Survival,Growth and Identification of Resistant Callus Lines*

Abstract Potato (cv. Crystal) protoplast-derived callus tissue was evaluated for survival and growth when exposed to Erwinia carotovora subsp. carotovora (strain Ecc71). Calli were either directly exposed to the pathogen by inoculation or to metabolites produced by the pathogen via a bilayer medium. Individual calli were inoculated with 0.5 μl of bacterial suspensions at 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ and 10⁹ cfu/ml. The bilayer consistedof 10 ml of callus proliferation medium supplemented with pectin (2 g/l) and contained bacteria at 10², 10³, 10⁴, 10⁵ and 10⁶ cfu/ml. This medium was overlaid with 10 ml of bacteria free callus induction medium. Mean callus diameter of the inoculated treatments increased for 24 h, then declined. Over 90% of the inoculated calli were killed within 5 days but some survived as long as 14 days. Calli grown on the bilayer medium containing 10⁶, 10⁵ and 10⁴ cfu/ml also decreased in size. Most were killed within 9 days but some survived 20 days. Calli exposed to 10³ and 10² cfu/ml experienced limited growth with 20% and 7%, respectively, surviving after 27 days. Reactions to the pathogen varied considerably within the callus populations and individual calli with extended survival were identified in both experiments.     

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.     

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.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, km^R marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strains strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.     

Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42,639, and a mature protein of molecular weight 42,200. A putative KdgR-binding site was identified in the region 5' to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.     

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.     

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.     

Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora

Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.     

Survival and Impact of Genetically Engineered Pseudomonas putida Harboring Mercury Resistance Gene in Aquatic Microcosms

The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms. We used lake, river, and spring water samples. The density of genetically engineered and wild-type P. putida decreased rapidly within 5 days (population change rate k -0.87 ~ -1.00 day^?1), then moderately after 5 to 28 days (-0.10~, -0.14 day^?1). The population change rates of genetically engineered and wild-type P. putida were not significantly different. We studied the important factors affecting the survival of genetically engineered and wild-type P. putida introduced in aquatic microcosms. Visible light exerted an adverse effect on the survival of the two strains. The densities of genetically engineered and wild-type P. putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-µm membrane filter, or treated with cycloheximide to inhibit the growth of protozoa. These results suggested that protozoan predation was one of the most important factors for the survival of two strains. We examined the impact of the addition of genetically engineered and wild-type P. putida on indigenous bacteria and protozoa. Inoculation of genetically engineered or wild-type P. putida had no apparent effect on the density of indigenous bacteria. The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P. putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms.     

Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora

A clone producing a polygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in PUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402-amino-acid (aa) polypeptide with an Mr of 42,849. In E. carotovora the polygalacturonase was synthesized with a 26-aa cleavable signal peptide. The mature 376-aa PehA had a calculated Mr of 40,064 and a pl of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35-45 degrees C. Analysis of the reaction products of polygalacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of erwinias. However, substantial similarity was detected within the C-terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic polygalacturonases may have a common origin.