Persistence of plant DNA sequences in the blood of dairy cows fed with genetically modified (Bt176) and conventional corn silage.

To determine whether plant sequences, including transgenic sequences, are present in animal blood, we tested blood samples from Holstein cows fed with either Bt176 genetically modified corn or conventional corn. We used previously described sensitive real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence), a monocopy maize-specific sequence (ADH promoter), and two multicopy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The presence of Cry1A(b) protein in bovine blood samples was also tested using a sandwich ELISA kit. Our study shows the ability of plant nuclear and/or chloroplast DNA fragments to enter bovine blood circulation. However, maize nuclear DNA, both mono- and multicopy sequences, was less detected than chloroplast DNA, probably because the higher number of chloroplast copies and also possibly because nuclear DNA might be less protected by the nuclear membrane. Despite our data confirm the ability of small (ca.150 bp) plant DNA fragments to cross the intestinal barrier, we were unable to demonstrate clearly the presence of transgenic DNA or proteins in bovine blood. No sample tested positive with the two real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence). Only faint punctual positive results occurred randomly and were probably due to postsample collection or laboratory contamination or can be considered as artifact as they have never been confirmed. Our data highlight the difficulties to detect transgenic sequences in blood of dairy cows fed genetically modified corn (Bt176) silage. Those results show that in order to meet the consumers' demand of animals fed with GM products there is currently no cost-effective analytical procedure to replace documentary traceability.

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies.

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subspp. atroseptica.

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpson's index of diversity (Simpson's D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0.46 (RFLP), 0. 39 (ERIC), 0.83 (phage typing), 0.82 (RAPD) and 0.26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes.

Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining.

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.

Metabolism of polychlorinated phenols by Pseudomonas cepacia AC1100: determination of the first two steps and specific inhibitory effect of methimazole.

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 metabolize both dichlorophenols, such as 2,4-dichlorophenol, 2,6-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol, and more highly substituted phenols, such as 2,4,6-trichlorophenol and pentachlorophenol, to the corresponding chlorohydroquinones. The first hydroxylation occurs in the para position of the phenol regardless of whether this position is replaced by a chlorine substituent. The first evidence leading to the characterization of para-hydroxylase as a flavin-containing enzyme is provided by the inhibitory effect of methimazole, an alternate substrate for this monooxygenase, on the degradative ability of the strain. In a second step, with tetrachlorohydroquinone, trichlorohydroxyquinone was isolated and completely characterized. Trichlorohydroxyquinone was also obtained from tetrachloroquinone. Incubation of the cells in the presence of an external source of NADPH prevents the further degradation of tetrachlorohydroquinone, suggesting that the quinone derived from the two-electron oxidation of the hydroquinone is more likely the substrate for the second hydroxylation.

Analysis of linear oligogalacturonic acids by negative-ion electrospray ionization mass spectrometry.

Linear oligogalacturonic acids (1,4-linked alpha-D-galacturonic acid oligomers), obtained by partial acid hydrolysis of orange polygalacturonides, were studied by negative-ion electrospray ionization mass spectrometry, without prior sample derivatization. After preparative separation using high-resolution anion-exchange chromatography, some fractions enriched in uronic acids were desalted, transferred into a methanol+water solution adjusted to pH 10, and directly submitted to electrospray ionization mass spectrometry in the negative-ion recording mode. Clear molecular mass assignments of the oligomers, covering a degree of polymerization between 4 and 7, were obtained.

Ribotyping of Erwinia chrysanthemi Strains in Relation to Their Pathogenic and Geographic Distribution.

16S and 23S rRNAs from Escherichia coli were used to study the relationship among a representative collection of strains of Erwinia chrysanthemi differing in their original host and geographical origin. Phenetic analysis of restriction fragment length polymorphisms allowed the distribution of the studied strains into seven clusters. These clusters were similar to those obtained by cladistic methods and appeared to correlate well with the established pathovars and biovars but to a lesser extent with geographical distribution. Except for two groups of strains defined as tropical and temperate isolates (clusters 3 and 4, respectively), our clustering correlated well with botanical classifications of host plants. However, the rRNA groupings were shown to be more discriminative than biovar analysis. To assess the relationship between rRNA clusters and pathogenicity, 12 representative strains from different clusters were tested for pathogenicity on different plants. The two typical symptoms, maceration and wilting, were observed for these strains. The occurrence of the tobacco hypersensitivity reaction for a subset of these strains is discussed in light of recent results concerning the presence of an hrp gene. Considering symptom expression only, rather than the capacity for plant infection, strains from the same cluster were shown to induce similar symptoms in test plants. Thus, since host specificity is still quite controversial, rRNA patterns may constitute a useful tool in taxonomic and epidemiological studies of Erwinia chrysanthemi species.

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed.

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.

Characterization and overexpression of the pem gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937.

The pem gene encoding the pectin methylesterase (PME) of Erwinia chrysanthemi strain 3937 was subcloned and its nucleotide sequence determined. The gene contains an open reading frame of 1098 bp and codes for a protein of 366 amino acids (aa). The mature 37-kDa form of the protein is 342 aa long and has a calculated isoelectric point of 9.64. A plasmid was constructed to overproduce PME: a DNA fragment carrying pem was amplified by the polymerase chain reaction and cloned downstream from the pL promoter of the lambda phage, in a high-copy-number plasmid. In an Escherichia coli strain transformed with this plasmid, an increase in PME production of more than 60-fold was obtained, compared with the wild-type Er. chrysanthemi strain. PME represents about 5% of the total protein content of the cells. Comparison of this PME sequence with six PMEs from prokaryotic or eukaryotic organisms showed six highly conserved segments whose possible role in enzyme activity are discussed.

Purification of the acidic pectate lyase and nucleotide sequence of the corresponding gene (pelA) of Erwinia chrysanthemi strain 3937.

The pelA gene from Erwinia chrysanthemi strain 3937, which encodes the acidic pectate lyase, PLa, has been sequenced and characterized. The structural gene consists of a 1179 bp open reading frame encoding a polypeptide of 41,555 Da, which includes an N-terminal signal peptide. The deduced amino acid sequence shows a protein very similar to some PLs already sequenced. Cloning of the pelA gene behind the lacZ promoter of the vector pTZ19R allowed overexpression of PLa into a derivative of strain 3937 deleted of the other pel genes. The mature protein was obtained in milligram amounts from the supernatant of this strain and at homogeneous purity after two purification steps. Its biochemical properties were similar to those of other PLs. Polyclonal antibodies raised against the purified PLa cross-reacted with the basic pectate lyase PLd, but not with PLe. The role of PLa in pathogenicity is discussed.

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album.

Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producing both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.

Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes.

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47-1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx-positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL-positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.

Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase.

Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.

Detection of depolymerase isoenzymes after electrophoresis or electrofocusing, or in titration curves.

The cup-plate technique makes it possible to detect enzyme activities after diffusion into buffered, substrate-containing agar gels. This technique has been used after nondenaturing blotting transfer in order to detect depolymerizing enzyme activities once analytical protein separation (e.g., by electrophoresis, electrofocusing, or titration curves) has been completed. This rapid and very sensitive method was successfully applied to the enzymes polygalacturonate lyase, polygalacturonate hydrolase, endoglucanase, and xylan hydrolase. Other possible applications are presented.