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| J Bacteriol, 1987 May, 169(5), 1972 - 8 2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi; Condemine G et al.; In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates . We studied the biochemical properties of this transport system . The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate . The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7) . 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively . The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity . kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli . After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene . We introduced a kdgT-lac fusion into the E . chrysanthemi chromosome by marker exchange recombination and studied its regulation . kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate . Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied . Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression. Mol Gen Mikrobiol Virusol, 1987 May, (5), 22 - 5 {Expression of the pectate lyase gene from Erwinia chrysanthemi ENA49 in cells of other Erwinia species}; Evtushenkov AN et al.; 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. Ann Inst Pasteur Microbiol, 1987 May-Jun, 138(3), 289 - 96 Amber suppressors of Erwinia chrysanthemi; Schoonejans E et al.; Mutations trp1 and thyA1, both of a polyauxotrophic derivative of the Erwinia chrysanthemi strain B374, were characterized as amber mutations with an Escherichia coli suppressor, supA1P2, which inserts a glutamine in response to UAG . Simultaneous reversion of both mutations allowed us to isolate amber suppressor mutants of E . chrysanthemi . These suppressors were tested with a set of amber mutants of bacteriophage Mu which had been previously characterized on E . coli . The two independently isolated suppressors behaved as supD and supE mutants, respectively, of E . coli. J Bacteriol, 1987 May, 169(5), 1899 - 904 A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species; Leemans R et al.; We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon . The vector carries the strong leftward promoter (pL) of coliphage lambda as well as the cI857 allele, which codes for a thermolabile repressor protein . The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter . The plasmid was introduced into Erwinia and Serratia species by means of mobilization . Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera . As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form. Eur J Biochem, 1987 Jan 15, 162(2), 311 - 6 Characterization of a new endoglucanase from Erwinia chrysanthemi; Boyer MH et al.; The structural gene coding for a new endo-beta-1,4-glucanase of Erwinia chrysanthemi strain 3665, previously identified in a cosmid library, was subcloned into pUC18 . The gene is expressed from a 1.9 X 10(3)-base-pair insert and its direction of transcription was determined . The properties of the gene product purified from cell-free extracts of Escherichia coli have been studied . The purified protein has an endoglucanase activity but is significantly different from the major endoglucanase Z secreted by E . chrysanthemi strain 3665 . The new enzyme was designated as endoglucanase Y and the related gene celY . In E . coli, most of the endoglucanase activity was found in the periplasmic space. J Basic Microbiol, 1987, 27(3), 147 - 53 Hemagglutinating activity in phytopathogenic bacteria surface compounds; Serra MT et al.; Extracellular components of plant pathogenic bacteria were obtained from their culture medium as well as from the whole cells by using NaCl 1 M, pH 6.0; 20% sucrose dissolved in 0.03 M Tris buffer, pH 8.0; or 0.05 M Na2EDTA . All the extracts from Erwinia carotovora subsp . carotovora, Xanthomonas campestris pv . campestris, Pseudomonas syringae pv . phaseolicola, Xanthomonas campestris pv . phaseoli, Pseudomonas solanacearum, and Erwinia carotovora subsp . atroseptica, were assayed for hemagglutinating activity on sheep, rabbit and chicken red blood cells (RBCs) . The only active extracts were those obtained by NaCl treatment . They agglutinated sheep and rabbit erythrocytes . Extracts from E . carotovora subsp . atroseptica gave rise to the high agglutination titer on rabbit RBCs . These extracts had the lowest polysaccharide/protein ratio . E . carotovora subsp . carotovora extracts showed only a low titer (18.5 units) . The agglutinating activity present in NaCl extracts of the bacteria tested was inhibited by different carbohydrates to various extent . Extracts from E . carotovora subsp . atroseptica appeared to be the most sensitive ones while those of E . carotovora subsp . carotovora least sensitive to the presence of sugar . It is suggested that hemagglutinins observed in plant pathogenic bacteria and those in plant host are similar and that both may, in some way, be involved in the plant-parasite relationship. Gene, 1987, 55(1), 125 - 33 Molecular cloning of an Erwinia chrysanthemi oligogalacturonate lyase gene involved in pectin degradation; Reverchon S et al.; Mutants of Erwinia chrysanthemi 3937 deficient in the pectin catabolic enzyme oligogalacturonate lyase were isolated by chemical and phage Mud(Aplac) insertion mutagenesis . The ogl mutation was biochemically characterized and localized near the trp his markers on the E . chrysanthemi chromosomal map . Analysis of Mud(Aplac) insertions, which generate polar mutations, revealed that oligogalacturonate lyase was the only affected enzyme in the pectin catabolic pathway, indicating that the ogl gene probably forms a separate transcriptional unit . Out of the two Mud(Aplac) insertions obtained, neither was an ogl-lac fusion . We cloned the ogl gene by complementing the mutation using the RP4::miniMu plasmid pULB113 . pR'ogl plasmids were analyzed for the presence of other unselected genes of strain 3937 . One of them, called pROU2, also carried the kduD and kdgR genes encoding 2-keto-3-deoxygluconate oxidoreductase, an enzyme of the pectin catabolic pathway, and the KdgR repressor, governing the expression of several genes of pectin degradation, respectively . The plasmid pROU2 harbored a chromosomal DNA insert of about 35 kb indicating that ogl, kduD and kdgR are very closely linked . Structural analysis of the ogl gene was carried out in subcloning experiments . This gene was localized on a 3.5-kb PstI fragment. Gene, 1987, 57(2-3), 239 - 46 An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis; Ried JL et al.; A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi . The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E . chrysanthemi . The cartridge was inserted into a Sau3A site in a cloned E . chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination . The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient . The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion . The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance . The resulting E . chrysanthemi mutant was Kms and PLc-deficient . The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E . chrysanthemi. J Bacteriol, 1986 Nov, 168(2), 619 - 23 Molecular cloning of virulence genes from Erwinia stewartii; Coplin DL et al.; A library of Erwinia stewartii DNA was constructed in cosmid pVK100 and used to complement spontaneous and Mu pf7701-induced (designated by the prefix MU) avirulent mutants . Plasmid pES4507 restored water-soaking ability and extracellular polysaccharide (EPS) synthesis to mutants MU14110 and MU2B70 (group I); pES1044 restored water-soaking ability to MU43, MU51, MU136, MU141, and RDF6011 (group II); and pES2144 complemented four spontaneous EPS- mutants (group III) . Hybridization of labeled plasmid DNA to Southern blots of genomic DNA from the mutants revealed that a Mu pf7701 insertion was associated with the respective cloned region in all mutants except MU2B70 and MU223 . In these strains, the plasmid may be suppressing the avirulent phenotype rather than complementing the mutation. J Bacteriol, 1986 Nov, 168(2), 595 - 606 Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli; Keen NT et al.; The pelB and pelE genes from Erwinia chrysanthemi EC16, which encode different pectate lyase enzymes, were sequenced and expressed at a high level in Escherichia coli . The genes possessed little similarity to each other in 5' signal regions, signal peptide sequences, coding sequences, or 3' noncoding regions . Both genes contained their own promoters as well as sequences 3' to the coding regions with considerable secondary structure which may function as rho-independent transcriptional termination signals . High-level expression plasmids were constructed with both genes, which led to 20% or more of E . coli cellular protein . The pectate lyases were secreted efficiently to the periplasm and, to a lesser extent, the culture medium . The mature proteins in E . coli periplasmic fractions were obtained in milligram amounts and high purity with a single-column affinity purification method . E . coli cells which produced high amounts of the pelE protein macerated potato tuber tissue as efficiently as E . chrysanthemi EC16 cells but cells producing high amounts of the pelB protein were less effective . Thus, the pelE gene product is an important pathogenicity factor which solely enables E . coli to cause a soft-rot disease on potato tuber tissue under laboratory conditions. J Bacteriol, 1986 Nov, 168(2), 886 - 91 Lactose and melibiose metabolism in Erwinia chrysanthemi; Gray JS et al.; A Lac+ mutant of Erwinia chrysanthemi was isolated from the Lac- wild type on lactose agar . beta-Galactosidase was expressed independently of lactose transport in both the mutant and the wild type, and neither strain expressed thiogalactoside transacetylase . Lactose transport and alpha-galactosidase, constitutive in the Lac+ strain, were coordinately induced in the Lac- strain by melibiose and raffinose but not by isopropyl-beta-D-thiogalactopyranoside or thiomethyl-beta-D-galactopyranoside . Melibiose was a strong inhibitor of both the melibiose- and the raffinose-induced lactose permeases, whereas raffinose was a strong inhibitor of only the raffinose-induced lactose permease. Ann Inst Pasteur Microbiol, 1986 Sep-Oct, 137B(2), 145 - 53 {Preliminary characterization of a new system allowing maltose assimilation by Escherichia coli}; Bloch MA et al.; We showed that Klebsiella pneumoniae and Erwinia herbicola possessed a pathway for maltose metabolism which could function in parallel with that encoded by the classical maltose regulon . Indeed, specific DNA fragments isolated from these two bacteria allowed growth on maltose of any of the mal mutants of Escherichia coli when in a multicopy number . The preliminary characterization of the E . herbicola DNA fragment indicated that a 4-Kb region was sufficient to complement the mal mutations of E . coli, and that this region encoded at least one 50-Kd protein . This protein is probably bound to the cytoplasmic membrane and its synthesis is induced by maltose independently of malT, the positive regulator gene of the maltose regulon. Appl Environ Microbiol, 1986 Aug, 52(2), 305 - 10 Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp . carotovora, and Erwinia carotovora subsp . atroseptica; Ried JL et al.; 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) J Bacteriol, 1986 Aug, 167(2), 496 - 502 Release of cell-free ice nuclei by Erwinia herbicola; Phelps P et al.; Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium . Only E . herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C . These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C . Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy . Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents. J Bacteriol, 1986 Jul, 167(1), 400 - 3 Pectate lyase gene regulatory mutants of Erwinia chrysanthemi; Diolez A et al.; The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region . Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected . Such mutants produced other pectate lyase isoenzymes in the absence of the inducer. J Bacteriol, 1986 Jul, 167(1), 279 - 84 Requirement for two or more Erwinia carotovora subsp . carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli; Roberts DP et al.; Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp . carotovora EC14 . Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s) . Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices . Only one E . coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration . The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D . P . Roberts, P . M . Berman, C . Allen, V . K . Stromberg, G . H . Lacy, and M . S . Mount, Can . J . Plant Pathol . 8:17-27, 1986) and include two secreted endo-pectate lyases . The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase . These enzymes are similar in physicochemical properties to those produced by EC14 . Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration. Appl Biochem Biotechnol, 1986 Jun, 12(3), 229 - 47 Large-scale recovery and purification of L-asparaginase from Erwinia carotovora; Lee SM et al.; A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora . Cells were harvested from 150 L of fermentation broth and washed . A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer . After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover . The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B . After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product . The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility . The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison. Mol Gen Mikrobiol Virusol, 1986 Apr, (4), 19 - 24 {Cloning of pectate-lyase genes of Erwinia chrysanthemi in Escherichia coli cells}; Evtushenkov AN et al.; Erwinia chrysanthemi DNA fragment digested by restriction endonuclease EcoRI and carrying the gene EC16 determining the synthesis of pectatelyase with Rf 0.20 and mol . mass 40kD has been cloned in plasmid pUC 9 plasmid in Escherichia coli HB101 cells . Three genes for pectatelyases of Erwinia chrysanthemi ENA49 have been cloned in vector phage lambda 47.1 in Escherichia coli cells . Two genes determining the synthesis of pectatelyases with Rf 0.06 and 0.19 and mol . masses 40 kD and 39 kD have been cloned as a part of an 7 kb Eco RI-fragment, that suggested their close location on the chromosome of Erwinia chrysanthemi ENA49 . All of the cloned pectatelyase genes are expressed constitutively with pectatelyases accumulating in periplasm and being unable to secret into the cultural medium. J Bacteriol, 1986 Apr, 166(1), 346 - 8 Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli; Barras F et al.; Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin . In contrast, Nalr isogenic strains grew only on cellobiose . It is proposed that expression of cloned E . chrysanthemi clb genes is reduced by the E . coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters. J Pharm Pharmacol, 1986 Apr, 38(4), 264 - 71 Soluble asparaginase-dextran conjugates show increased circulatory persistence and lowered antigen reactivity; Wileman TE et al.; Oxidized dextrans of increasing molecular weight were bound covalently to Erwinia carotovora asparaginase . The resulting conjugates retained 50% of their enzyme activity and showed marked resistance to proteolysis by trypsin and chymotrypsin and inactivation by asparaginase-specific antibody . When tested in-vivo, the larger molecular weight conjugates showed prolonged circulatory survival in both immune and non-immune animals and failed to elicit full type III hypersensitivity or anaphylactic reactions when injected into sensitized guinea-pigs . Rabbits could tolerate multiple doses of the asparaginase conjugate without developing an immunity to the enzyme . A conjugate showing increased circulatory half-life and lowered antigen reactivity should have therapeutic potential. J Bacteriol, 1986 Mar, 165(3), 937 - 41 Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation; Condemine G et al.; Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis . A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source . Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon . Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression . In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism . Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate . 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation . 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage . These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis. J Infect, 1986 Jan, 12(1), 71 - 3 Erwinia herbicola as a cause of bacterial endocarditis; Williams AJ et al.; A case of endocarditis due to Erwinia herbicola is reported . Three years previously the patient had been fitted with a porcine xenograft. Gene, 1986, 49(2), 215 - 24 Organization of a pectate lyase gene family in Erwinia chrysanthemi; Reverchon S et al.; The pelA, pelD and pelE genes encode three of the five major pectate lyase (PL) isoenzymes (PLa, PLd and PLe) in Erwinia chrysanthemi strains B374 and 3937 . These genes were previously isolated from genomic libraries or by in vivo cloning as R' factors promoted by the pULB113 plasmid . They are clustered near purE on the chromosomal map of E . chrysanthemi B374 {Van Gijsegem et al., EMBO J . 4 (1985) 787-792} . Genes pelA, pelD and pelE were subcloned separately into pBR322 derivatives, to test their individuality . It then became possible to select specific mutations in each separated gene . Such mutations were obtained using the transposable bacteriophage MudI1734 that allows the construction of lacZ gene fusions . Subcloning experiments and analysis of MudI1734 insertions permitted us to determine the length of each gene, the transcriptional orientation and the location of the promoter . We concluded that the three genes constitute three independent transcriptional units . They are clustered on a 5-kb DNA fragment, in the order: pelD-pelE-pelA . Genes pelD and pelE are transcribed in the same direction, while the transcription of pelA seems to be divergent . Organization of the pel region was very similar in the two strains B374 and 3937 . Moreover, lacZ gene fusions were introduced by marker exchange into the chromosome of E . chrysanthemi B374, giving rise to three strains lacking PLa, PLd or PLe . These fusions allowed us to study the regulation of the mutagenized genes. Gene, 1986, 46(1), 25 - 35 Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene; Minton NP et al.; The complete nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 gene coding for the chemotherapeutic enzyme L-asparaginase has been determined . The structural gene consists of an open reading frame commencing with an ATG start codon of 1044 bp followed by a TGA stop codon . Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid (aa) sequence with that derived by N-terminal aa sequencing of the purified protein . The gene has been shown to code for a 21-aa signal peptide at its N terminus which closely resembles the signal peptides of other secreted proteins . In common with highly expressed Escherichia coli genes, little use is made of modulator codons . The predicted aa sequence of the enzyme exhibits 46% identity with the determined primary sequence of the E . coli L-asparaginase, although the predicted secondary structure of both proteins indicates more extensive homology . Downstream of the TGA stop codon is a G + C-rich region of dyad symmetry (delta G = -25.4 kcal) characteristic of E . coli Rho-independent transcription terminators . Upstream of the structural gene there are no sequences which bear a strong resemblance to the consensus -35 and -10 regions of E . coli promoters . A sequence is present (CTGGCTCTCCTCTTGAT), however, which exhibits strong homology to the nif promoter consensus sequence (CTGGCACN5TTGCA) . Upstream of this region is a sequence which strongly resembles the consensus sequence for promoter regions which are subject to catabolite repression. J Gen Microbiol, 1986 Jan, 132 ( Pt 1), 151 - 60 Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora; Gilbert HJ et al.; A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er . chrysanthemi asparaginase detected using purified anti-asparaginase IgG . The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30 . The position and orientation of the asparaginase structural gene was determined by subcloning . The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space . Purified asparaginase from E . coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity {660-700 units (mg protein)-1}, pI value (8.5), and subunit molecular weight (32 X 10(3} . Expression of the cloned gene was subject to glucose repression in E . coli but was not significantly repressed by glycerol . Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain). J Bacteriol, 1985 Dec, 164(3), 1110 - 6 Chromosomal mapping in Erwinia carotovora subsp . carotovora with the IncP plasmid R68::Mu; Forbes KJ et al.; Conjugational gene transfer was established in Erwinia carotovora subsp . carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients . It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca . 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold . Using this system it was possible to order 17 chromosomal mutations . The behavior of Mu in E . carotovora subsp . carotovora was also studied. J Biol Chem, 1985 Nov 25, 260(27), 14604 - 9 Bactericidal agents generated by the peroxidase-catalyzed oxidation of para-hydroquinones; Beckman JS et al.; For the three Gram-negative bacteria, Pseudomonas fluorescens, Escherichia coli, and Erwinia amylovora, p-benzoquinone was the principal bactericidal agent formed in vitro during the oxidation of hydroquinone by horseradish peroxidase, whereas no toxicity could be associated with either phenolic or oxygen-free radicals . Even the continuous generation of p-benzosemiquinone during the simultaneous reduction of p-benzoquinone by xanthine oxidase and reoxidation of hydroquinone by peroxidase was no more toxic than p-benzoquinone alone . Anaerobiosis had no effect on the toxicity of either p-benzoquinone or the peroxidase reaction and the generation of superoxide and hydroxyl radicals catalyzed by xanthine oxidase was not bactericidal . Substitutions on the p-benzoquinone ring decreased quinone toxicity in rough proportion to the decrease in quinone redox potential, suggesting that strong oxidizing potentials are important for such quinone toxicity. J Bacteriol, 1985 Nov, 164(2), 717 - 22 araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora; Lei SP et al.; The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium . The araB and araC genes in E . coli, E . carotovora, and S . typhimurium were transcribed in divergent directions . In E . carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E . coli and S . typhimurium they were separated by 147 base pairs . The nucleotide sequence of the E . carotovora araC gene was determined . The predicted sequence of AraC protein of E . carotovora was 18 and 29 amino acids longer than that of AraC protein of E . coli and S . typhimurium, respectively . The DNA sequence of the araC gene of E . carotovora was 58% homologous to that of E . coli and 59% homologous to that of S . typhimurium, with respect to the common region they share . The predicted amino acid sequence of AraC protein was 57% homologous to that of E . coli and 58% homologous to that of S . typhimurium . The 5' noncoding regions of the araB and araC genes of E . carotovora had little homology to either of the other two species. Genetika, 1985 Nov, 21(11), 1787 - 93 {The role of te transposition of Tn1000 from Flac+-plasmid into chromosome of Erwinia chrysanthemi in the formation of Hfr-type donors}; Prokulevich VA et al.; Based on the data of stability of the donor state of Hfr-like strain Erwinia chrysanthemi VY1-10 in RecA+ and RecA- cells, it can be suggested that the donor properties of the strain are mediated by the presence of the genetic homology region which occurred as a result of transposition of the Tn1000 from the Flac+ plasmid into the chromosome of E . chrysanthemi . Tn1000 may be transposed into several sites on the chromosome of E . chrysanthemi ENA49 . This leads to the appearance of donors transferring their chromosome from several fixed points oriT and in opposite directions . The location of these points and the direction of transfer are determined by Tn1000 insertion sites and their orientation. J Bacteriol, 1985 Nov, 164(2), 831 - 5 Evidence that polygalacturonase is a virulence determinant in Erwinia carotovora; Lei SP et al.; Polygalacturonase (PG) was purified from Erwinia carotovora EC . A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E . carotovora genomic library by an immunological screening method . A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices . In addition, the E . coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices . Our results suggest that PG plays an important role in soft-rot disease. J Bacteriol, 1985 Oct, 164(1), 390 - 6 recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp . carotovora; Zink RT et al.; Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp . carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light . To determine whether the recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E . carotovora subsp . carotovora recA+ gene, inactivated it by Tn5 insertion, and constructed an E . carotovora subsp . carotovora recA::Tn5 strain by gene replacement via homologous recombination . The RecA- strain was more sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent . The recA mutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the ability to cause soft rot of potato tubers . With this mutant, unlike with the RecA+ parent strain, PNL and CTV were not induced by mitomycin C or detected in potato tuber tissue . The RecA+ phenotype, including the inducibility of PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E . carotovora subsp . carotovora or Escherichia coli . We conclude that, in E . carotovora subsp . carotovora, the recA product is required in the induction of PNL and CTV. J Bacteriol, 1985 Oct, 164(1), 359 - 66 Cloning and expression of bacterial ice nucleation genes in Escherichia coli; Orser C et al.; Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants . We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria . The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P . syringae Cit7R1 . The cloned fragments imparted ice-nucleating activity in Escherichia coli . Substantial increases in the nucleating activity of both E . coli and P . syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors . Southern blot analysis showed substantial homology between the ice regions of P . syringae and E . herbicola, although individual restriction sites within the ice regions differed between the two species. Mol Gen Mikrobiol Virusol, 1985 Oct, (10), 34 - 9 {Characteristics of transduction in Erwinia by phage 59}; Romaniuk LV et al.; A temperate bacteriophage 59 from polylysogenic strain Erwinia carotovora 268 transduces the following genetic markers: arg+, ilv+, leu+, met+, thr+, thy+, trp+, ura+ . The transduction frequencies varied from 1 x 10(-8)- to 1 x 10(-6) and dependent on the multiplicity of infection, UV-irradiation of transducing bacteriophage, the nature of phage lysates . The characteristics of single transductants have been studied.Analysis of the obtained results suggests bacteriophage 59 to perform the generalized transduction. Appl Environ Microbiol, 1985 Oct, 50(4), 894 - 8 Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16; Thurn KK et al.; Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL) . By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL . Physical analysis revealed that single copies of Tn5 had inserted into the E . chrysanthemi chromosome, producing a similar export-defective (Out-) phenotype . The synthesis of PL, PG, and CL was not affected by the Tn5 insertions . These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space . Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition . The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria. J Bacteriol, 1985 Oct, 164(1), 473 - 6 Effects of a mutation that eliminates UDP glucose-pyrophosphorylase on the pathogenicity of Erwinia carotovora subsp . carotovora; Jayaswal RK et al.; A nonpathogenic mutant of Erwinia carotovora obtained by Mu d1 mutagenesis was defective in the ability to utilize several carbon sources . The basis of the mutation was analyzed biochemically and shown to be a defect in the ability to form UDP glucose-pyrophosphorylase . The nonpathogenic phenotype of the mutant was caused by its sensitivity to galactose. J Bacteriol, 1985 Sep, 163(3), 913 - 7 Mu-lac insertion-directed mutagenesis in a pectate lyase gene of Erwinia chrysanthemi; Diolez A et al.; 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. Antibiot Med Biotekhnol, 1985 Aug, 30(8), 588 - 91 {Adenosine transformation into adenosine-5'-monophosphate by intact Erwinia herbicola cells}; Popov IL et al.; Strain 47/3 was isolated from the natural sources of Erwinia herbicola . The cells of this strain contain nucleoside phosphate transferase responsible for specific phosphorylation of the adenosine 5'-hydroxyl group . With the use of the strain intact cells, conditions for preparation of AMP were determined . The cells were grown in the meat-peptone broth supplemented with yeast extract . p-Nitrophenylphosphate was used as a donor of phosphate groups . It was shown that the concentration of the reaction substrates, their ratio and PH of the reaction mixture were factors defining the cell activity . Under the optimal conditions (0.2 M acetate buffer, pH 4.5; concentrations of p-nitrophenylphosphate and adenosine 96 and 12 mg/ml respectively; concentration of the cells 1 per cent) the cells phosphorylated adenosine with a yield of 74 mol % . This indicates that the method may be used for production of AMP in preparative amounts. J Bacteriol, 1985 Jul, 163(1), 221 - 7 Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity; Expert D et al.; A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants . The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions . The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities. Anal Biochem, 1985 May 15, 147(1), 114 - 9 An improved DNA sequencing strategy; Lin HC et al.; A modification of Hong's systematic DNA sequencing strategy is described . The original procedure has been simplified and transfectant yield increased . After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel . After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site . The original intact DNA is linearized whereas the deleted subclone is not . The background is decreased to an undetectable level . This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora . A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol. Mikrobiologiia, 1985 May-Jun, 54(3), 410 - 3 {Dependence of the degree of antibacterial and antiphage action of ozone on cell and phage particle concentrations in nutrient media}; Grits NV et al.; The work was aimed at studying the inactivating effect of ozone on Escherichia coli K-12 AB1157, Pseudomonas aeruginosa PA01, Erwinia herbicola EH103 and their phages T4, SM and I4 . The degree of bacterial and phage inactivation was found to increase with a decrease in their initial concentration during the treatment . The effect depends on differences in the quantity of ozone per cell or per phage particle in the reaction medium . This conclusion is based on the fact that, irrespective of the suspension density, the amount of surviving bacteria and phages plotted versus O3 concentration and recalculated per one bacterial cell or phage particle is described graphically by one and the same curve typical of a strain under study . This technique for assessing the sensitivity of microbiological objects to ozone can be used in order to compare experimental data obtained in different laboratories. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1985 May, 18(2), 115 - 22 Transformation of Erwinia chrysanthemi by Escherichia coli plasmids DNA; Wang J et al.; We have demonstrated in this study that CaCl2-MnCl2 treatment is effective in the preparation of competent cells of Erwinia chrysanthemi for transformation . Plasmids pMB9 and pBR322, which are of Escherichia coli origins, were transformed into E . chrysanthemi at frequencies of 1.5 X 10(-7) and 4.5 X 10(-7) per recipient, respectively . The frequencies were 60- to 80-fold lower than those in the well established rec- E . coli; however, the procedure is practically useful in transformation experiments . The plasmids were maintained rather stable in the cells if antibiotics were included in the media . When transformed by pECl and pEC6, which were chimeric plasmids consisting of pBR322 and an E . chrysanthemi chromosomal DNA fragment of 2.4 and 3.5 Mdal, respectively, the transformation frequencies of E . chrysanthemi were at the same range of that by pBR322 . Neither colony morphology nor the polypectate degradation ability was changed after transformation by the plasmids . In conclusion, we have established a system of molecular cloning in E . chrysanthemi SR120A exploiting pBR322 as a vector. J Bacteriol, 1985 May, 162(2), 845 - 8 Inducible xylitol dehydrogenases in enteric bacteria; Doten RC et al.; Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp . strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium . The xylitol dehydrogenases were partially purified from the four strains, and those from M . morganii ATCC 25829, P . stuartii ATCC 25827, and S . marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose . These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecular weights ranging from 130,000 to 155,000 . In contrast, the xylitol dehydrogenase from Erwinia sp . strain 4D2P oxidized xylitol at the C-4 position to produce L-xylulose, had a Km for xylitol of 72 mM, and had a molecular weight of 102,000. J Bacteriol, 1985 May, 162(2), 708 - 14 Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase; Chatterjee AK et al.; Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI . Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate . Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes . In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains . Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates . The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did . In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s) . We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E . chrysanthemi. Appl Environ Microbiol, 1985 Apr, 49(4), 818 - 21 Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti; Handelsman J et al.; Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested . Some of these E . herbicola strains affected nodulation by certain strains of Rhizobium meliloti . In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R . meliloti 102F51 . In the absence of E . herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain . The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E . herbicola . All of the previously reported slower-nodulating strains derived from R . meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E . herbicola isolates. J Bacteriol, 1985 Mar, 161(3), 1226 - 7 Structure of the core oligosaccharide from lipopolysaccharide of Erwinia carotovora; Sandulache R et al.; The lipopolysaccharide of Erwinia carotovora was analyzed by quantitative sugar analysis, methylation analysis, and chromic oxide oxidation . This led to the following structure of the core oligosaccharide: (Formula; see text). Appl Environ Microbiol, 1985 Mar, 49(3), 714 - 7 Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp . carotovora; Zink RT et al.; Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp . carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79 . The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue. J Bacteriol, 1985 Feb, 161(2), 786 - 8 Efficient transformation of Erwinia carotovora subsp . carotovora and E . carotovora subsp . atroseptica; Hinton JC et al.; We used a modified version of the method of Hanahan (D . Hanahan, J . Mol . Biol . 166:557-580, 1983) to transform Erwinia carotovora subsp . carotovora and E . carotovora subsp . atroseptica with the plasmids pBR322, pBR325, and pAT153 . The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA . The nature of these transformants was confirmed by plasmid analysis . ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species. J Bacteriol, 1985 Feb, 161(2), 702 - 8 Isolation and characterization of Erwinia chrysanthemi mutants defective in degradation of hexuronates; van Gijsegem F et al.; Spontaneous and Tn9-induced mutants of Erwinia chrysanthemi were isolated which affect the degradative pathway of galacturonate and ketodeoxygluconate . The mutations were characterized both biochemically and functionally by complementation analysis and localized in the E . chrysanthemi chromosome . The kdgK gene mapped very close to ile, the kdgA gene was between trp and his, and the exuT-uxaC-uxaB-uxaA cluster was linked to thy . The different types of mutants obtained were consistent with an organization of the exu-uxa cluster into two transcription units, one containing the exuT gene, and the other containing the three uxa genes, with the transcription going from uxaC to uxaA. J Bacteriol, 1985 Feb, 161(2), 529 - 33 Characterization of xylitol-utilizing mutants of Erwinia uredovora; Doten RC et al.; Of the four pentitols ribitol, xylitol, D-arabitol, and L-arabitol, Erwinia uredovora was able to utilize only D-arabitol as a carbon and energy source . Although attempts to isolate ribitol- or L-arabitol-utilizing mutants were unsuccessful, mutants able to grow on xylitol were isolated at a frequency of 9 X 10(-8) . Xylitol-positive mutants constitutively synthesized both a novel NAD-dependent xylitol-4-dehydrogenase, which oxidized xylitol to L-xylulose, and an L-xylulokinase . The xylitol dehydrogenase had a Km for xylitol of 48 mM and showed best activity with xylitol and D-threitol as substrates . However, D-threitol was not a growth substrate for E . uredovora, and its presence did not induce either dehydrogenase or kinase activity . Attempts to determine the origin of the xylitol catabolic enzymes were unsuccessful; neither enzyme was induced on any growth substrate or in the presence of any polyol tested . Analysis of xylitol-negative mutants isolated after Tn5 mutagenesis suggested that the xylitol dehydrogenase and the L-xylulokinase structural genes were components of two separate operons but were under common regulatory control. Eur J Biochem, 1985 Jan 15, 146(2), 383 - 9 Purification of RNA polymerase and transcription-termination factor Rho from Erwinia carotovora; Nwankwo DO et al.; Erwinia carotovora RNA polymerase consists of the holoenzyme structure sigma 2 beta beta' sigma as found in Escherichia coli and other bacteria . E . carotovora RNA polymerase can synthesize RNA using lambda, T7 of T4 DNA as templates; however, it is two times less active on these templates and is more temperature-sensitive than the E . coli enzyme . The alpha subunit of the E. . carotovora enzyme is lower in molecular mass than its E . coli counterpart . The sigma factors from E . coli and E . carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA . An additional protein of 115 000 Da molecular mass, termed gamma, is found associated with E . carotovora RNA polymerase . The gamma protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl . It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio-Rex 70 and DEAE-cellulose columns . The gamma-protein, when present in excess over the sigma subunit, inhibits holoenzyme activity on T7 DNA but not on poly{d(A-T)}and may thus interfere with sigma activity . The gamma protein by itself cannot transcribe T7 DNA or poly{d(A-T)}, nor does it stimulate core enzyme activity on T7 DNA . E . carotovora rho has a subunit molecular mass of 48 000 Da and exhibits RNA-dependent phosphohydrolysis of adenosine ribonucleoside triphosphate . E . coli and E . carotovora rho are indistinguishable immunologically, as total fusion of precipitin bands is observed . E . carotovora rho elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E . coli rho . The poly(C)-dependent ATPase activity of E . carotovora rho is more-temperature sensitive and is six to ten times less active than that of E . coli rho . E . carotovora rho is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using lambda or T7 DNA as template and E . carotovora or E . coli polymerase as the transcribing-enzyme. Ann Allergy, 1985 Jan, 54(1), 65 - 8 Hypersensitivity pneumonitis in grain farmers due to sensitization to Erwinia herbicola; Dutkiewicz J et al.; Two cases of hypersensitivity pneumonitis in grain workers are described . Both cases had evidence of sensitization to the gram-negative bacteria Erwinia herbicola as judged by skin tests, detection of serum precipitins, and inhalation challenge . E . herbicola is frequently found in the microflora of grains. Gene, 1985, 35(1-2), 63 - 70 Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli; Lei SP et al.; A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method . A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E . carotovora culture supernatants . The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE . None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space . Plant tissue was macerated by the PLs made in E . coli. Appl Environ Microbiol, 1985 Jan, 49(1), 158 - 62 Production of D- and L-xylulose by mutants of Klebsiella pneumoniae and Erwinia uredovora; Doten RC et al.; D-Xylulose and L-xylulose were produced biologically by the oxidation of a corresponding pentitol . A Klebsiella pneumoniae mutant was constructed for the oxidation of D-arabitol to D-xylulose . This mutant constitutively synthesized the D-arabitol permease system and D-arabitol dehydrogenase but was unable to produce the D-xylulokinase of the D-arabitol pathway or the D-xylose isomerase and D-xylulokinase of the D-xylose pathway . An Erwinia uredovora mutant which constitutively synthesized a novel xylitol-4-dehydrogenase but could not synthesize L-xylulokinase was used for the oxidation of xylitol to L-xylulose . Washed cell suspensions of either mutant incubated with 0.5% pentitol would oxidize 60 to 65% of the pentitol to the corresponding ketopentose in 18 h and excrete the ketopentose into the medium . Ketopentoses were rapidly purified from the remaining pentitol by hydroxyl affinity chromatography. Histochemistry, 1985, 83(5), 397 - 9 Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al; Van Laere O et al.; For the identification of the plant pathogenic bacterium Erwinia amylovora, the immuno gold staining (IGS) and immuno gold silver staining (IGSS) techniques are tested . The IGS and IGSS methods are at least as sensitive an indirect immunofluorescence and require less primary antiserum . Moreover they have the advantage that the preparations can be conserved permanently and unchanged . The preparation of the IGS can be observed with transmitted light or--with considerable better result--using epipolarization microscopy . The IGSS method deserves special attention because of its high contrast in normal brigth field microscopy with transmitted light. J Bacteriol, 1984 Dec, 160(3), 1199 - 203 Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase; Andro T et al.; 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. J Antibiot (Tokyo), 1984 Oct, 37(10), 1198 - 203 Isolation of L-cycloserine from Erwinia uredovora; Shoji J et al.; An antibiotic which seems to be a cell wall synthesis-inhibitor was isolated from a bacteria strain identified as Erwinia uredovora . The antibiotic was identified with L-cycloserine from its physico-chemical properties . This is the first example for isolation of L-cycloserine as a microbial product. J Antibiot (Tokyo), 1984 Oct, 37(10), 1217 - 23 Enzymatic synthesis of phenoxymethylpenicillin using Erwinia aroideae enzyme; Nam DH et al.; Enzymatic synthesis of phenoxymethylpenicillin from 6-aminopenicillanic acid and phenoxyacetic acid methyl ester was attempted by using partially purified alpha-acylamino-beta-lactam acylhydrolase I (ALAHase I) enzyme from Erwinia aroideae NRRL B-138 . The reaction rates were carefully followed by determination of 6-aminopenicillanic acid (6-APA), phenoxymethylpenicillin (PNV), phenoxyacetic acid (POA), phenoxyacetic acid methyl ester (POM), and phenoxyacetylglycine (POG) using high performance liquid chromatography . Among the acyl donors tested, POM gave the highest yield (12.2% based on 6-APA) . The overall conversion increased almost linearly with an increase in molar ratio of POM to 6-APA up to 4:1 . The effects of organic solvents on the overall yield were also evaluated . Some improvement of PNV yield was observed when ethanol, 2-propanol, and acetone were used . ALAHase I was found to carry out three reactions simultaneously: transfer of acyl group to acyl acceptor to form semisynthetic beta-lactam antibiotic; hydrolysis of acyl donor in amide or ester bond, and hydrolysis of semisynthetic beta-lactam antibiotic which was produced by the enzyme . It was also observed that the hydrolysis reactions of POM and PNV were irreversible in this reaction system . The optimal pH for the three reactions was different . They were: pH 9.0 for POM hydrolysis, 6.8 for the transfer of phenoxyacetyl group to 6-APA, and 6.0 for the PNV hydrolysis . The apparent Km values for POM, 6-APA and PNV were estimated as 33, 25 and 31 mM, respectively. J Bacteriol, 1984 Oct, 160(1), 153 - 60 Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r; Keener SL et al.; 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. J Bacteriol, 1984 Sep, 159(3), 825 - 31 Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli; Keen NT et al.; A genomic library of Erwinia chrysanthemi EC16 was constructed in plasmid pHC79, and seven putative pectate lyase (PL) clones in Escherichia coli were selected on pectate agar . Six of the recombinant cosmids contained a common PstI fragment of ca . 8.2 kilobases (kb) . Subcloning of this fragment in either orientation into the PstI site of plasmid pBR329 resulted in E . coli transformants that produced a PL of pI 9.8 which was indistinguishable from one of two PLs produced by strain EC16 . A 6.6-kilobase PstI fragment from the remaining cosmid clone caused production of an Erwinia PL of pI 8.8 when the fragment was subcloned in either orientation into plasmid pBR329 and transformed into E . coli . Selected pBR329 subclones for the 8.2- and 6.6-kilobase PstI fragments showed no similarity in their restriction maps and did not cross-hybridize . All of the E . coli cosmid clones that produced large amounts of PL also caused soft-rot of potato tubers and tuber slices, thus confirming the role of the enzymes in plant tissue maceration . The E . coli cosmid clones and plasmid pBR329 subclones produced the PLs constitutively, unlike Erwinia chrysanthemi, which made the enzymes inducibly . However, catabolite repression appeared to function in the E . coli clones, and almost all of the PL activity occurred in the periplasm and culture fluids . Thus, the Erwinia PL clones appear to contain signal peptide sequences, transcription and translation signals, and a recognition sequence for the catabolite activator protein, all of which function efficiently in E . coli. Biochem J, 1984 May 15, 220(1), 213 - 20 The extraction and mechanism of a novel isomaltulose-synthesizing enzyme from Erwinia rhapontici; Cheetham PS; The single enzyme that mediates the bioconversion is demonstrated to be located in the cells' periplasmic space, a site that facilitates its use as an industrial biocatalyst, and to be a previously undescribed hexosyltransferase with four novel features . The enzyme is sucrose-specific, and has an intramolecular mechanism in which both glucose and fructose residues appear to be enzyme-bound . Thirdly, it is reaction-non-selective, forming simultaneously isomaltulose and a second hitherto uncharacterized alpha-(1----1)-linked disaccharide (trehalulose), by hydrolysis of sucrose followed by reaction of glucose with the C-6 and C-1 positions of the fructofuranose respectively . Finally, on extended incubation an unusual recycling mechanism caused the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum concentration and then fall, and the concentration of trehalulose, the thermodynamically favoured product, to rise slowly. Allergol Immunopathol (Madr), 1984 May-Jun, 12(3), 207 - 11 Relationship between skin tests and bronchial provocation tests with allergens found in organic dusts in patients with bronchial asthma; Durda M et al.; A group of 153 patients with bronchial asthma were subjected to bronchial provocation and intracutaneous tests with allergen from the gram negative bacterium Erwinia herbicola which commonly occurs in organic dusts . Of this group, 80 persons were also examined with both tests with the allergen of Aspergillus fumigatus and 96 persons with the house dust extract . The frequency of positive skin reactions to the tested allergens was much greater than that of the positive bronchial reactions . No significant correlation was found to exist between the frequency and intensity of the bronchial and skin reactions except for delayed and late reactions to Erwinia herbicola . The authors conclude that bronchial provocation tests represent a much more specific and reliable method in the assessment of the clinical status of patients with extrinsic asthma than do skin tests. J Bacteriol, 1984 May, 158(2), 764 - 6 Mutagenesis of Erwinia carotovora subsp . carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions; Jayaswal RK et al.; The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp . carotovora . Among ampicillin-resistant transductants . 1.4% were auxotrophs . The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains. J Bacteriol, 1984 Mar, 157(3), 809 - 14 Transposon Tn5 mutagenesis in Erwinia carotovora subsp . carotovora and E . carotovora subsp . atroseptica; Zink RT et al.; In matings between Escherichia coli 2492(pJB4JI) and Erwinia carotovora subsp . carotovora Ecc71 and E . carotovora subsp . atroseptica Eca12, Kmr Gms transconjugants were obtained at high frequencies, indicating instability of the Mu-containing plasmid pJB4JI and transposition of Tn5 into the recipient genome . This was verified by Southern blot hybridization with pRZ102 DNA containing Tn5 as the 32P-labeled probe . Examination of Kmr Gms transconjugants of Ecc71 and Eca12 disclosed that a proportion (2 to 3%) were either auxotrophic or defective in catabolism of specific carbohydrates . Spontaneous prototrophic revertants were obtained for all markers with the exception of ilv, tyr, and suc . Genetic and physical data indicate that scattered insertions of Tn5 from pJb4JI into the chromosome of Ecc71 and Eca12 produced a variety of altered phenotypes due mostly to single insertions of Tn5 not accompanied by Mu DNA. Mol Gen Genet, 1984, 197(3), 486 - 90 Cellobiose metabolism in Erwinia: genetic study; Barras F et al.; The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism . In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb- specific mutants . When introduced into wild-type E . coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes . From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three beta-glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mol Gen Genet, 1984, 198(1), 125 - 7 Conjugal transfer of E . coli F'lac from Erwinia chrysanthemi to Pseudomonas syringae pv . glycinea and the apparent stable incorporation of the plasmid into the pv . glycinea chromosome; Leary JV et al.; The E . coli F'lac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv . glycinea recipient . Transfer occurred at a frequency of approximately 10(-5)/donor . Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the F'lac plasmid in detectable frequency to other pv . glycinea or E . coli recipients . The plasmid DNA was shown to be integrated into the pv . glycinea chromosome (Fig . 1). Biochim Biophys Acta, 1983 Dec 12, 749(2), 133 - 42 The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora; Hellman K et al.; L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Erwinia carotovora undergoes extensive dissociation from active tetramer to inactive monomers when freeze-dried . The monomeric state is stabilized by reconstitution of the freeze-dried enzyme with buffers of high pH and high ionic strength . Some compounds, particularly sugars and sugar derivatives, prevent dissociation on freeze-drying, whereas others, such as urea and chaotropic ions, increase dissociation . The effects of additives are not related to water retention . The dissociation is completely reversible on reconstitution at neutral pH, but the alkali-stabilized monomer only partially reassociates when the pH is brought back to neutrality. J Virol, 1983 Jun, 46(3), 1018 - 21 Structure of Erwinia carotovora temperate bacteriophage 59 and its DNA; Kishko YG et al.; The temperate phage 59 from Erwinia carotovora and its DNA were studied . The phage particles have an icosahedral head and a long noncontractile tail with a base plate . The virus DNA makes up 50% of the total virus and exists as a linear molecule (molecular weight, 2.6 X 10(7)) . A model of virus structural organization is presented. J Bacteriol, 1983 Jun, 154(3), 1227 - 35 In vivo cloning of Erwinia carotovora genes involved in the catabolism of hexuronates; Van Gijsegem F et al.; Using the RP4::mini-Mu pULB113 plasmid, an RP4 derivative carrying a deleted Mu prophage which allows the plasmid to pick up any chromosomal DNA segment to form R' plasmids, we cloned all of the genes of Erwinia carotovora involved in the catabolism of the hexuronates and in the transport of these substrates . With the R' plasmids we isolated, we performed complementation analysis and found that, in the Erwinia carotovora strain we used, the genes involved in the catabolism of the hexuronates are clustered in four regions of the chromosome . This genetic organization is compared with that of Escherichia coli K-12. J Bacteriol, 1983 Jun, 154(3), 1489 - 92 Utilization of plasmid pULB113 (RP4::mini-Mu) to construct a linkage map of Erwinia carotovora subsp . chrysanthemi; Schoonejans E et al.; We report experimental evidence that pULB113, an RP4::mini-Mu plasmid, mediates chromosome transfer in a strain of Erwinia carotovora subsp . chrysanthemi which does not accept the F episome . This allowed us to construct a genetic map of that strain by measuring the frequencies of cotransfer of different markers (thy, leu, pro, {his, trp}, thyA, rpsL, ile). J Bacteriol, 1983 May, 154(2), 663 - 8 Aldohexuronate transport system in Erwinia carotovora; Hugouvieux-Cotte-Pattat N et al.; The biochemical and physiological aspects of hexuronate transport in Erwinia carotovora were studied to approach the genetic regulation of the hexuronate degradative pathway in this bacterial species . An active transport system for glucuronate and galacturonate uptake exists in E . carotovora . The glucuronate entry reaction displayed saturation kinetics with an apparent Km of 0.05 mM (at 25 degrees C; pH 7) . Galacturonate appeared to be a competitive inhibitor of glucuronate uptake with a Ki of 0.1 mM . Glucuronate permeation was not induced by glucuronate itself in wild-type strains . Galacturonate induced the uptake of glucuronate (about fivefold) . The induced synthesis of the transport system was sensitive to catabolite repression by glucose . Mutants able to grow on glucuronate as the sole carbon source showed constitutive synthesis of the hexuronate transport system. J Bacteriol, 1983 Jan, 153(1), 222 - 31 Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola; Kozloff LM et al.; Chemical and biological properties of the ice nucleating sites of Pseudomonas syringae, strain C-9, and Erwinia herbicola have been characterized . The ice nucleating activity (INA) for both bacteria was unchanged in buffers ranging from pH 5.0 to 9.2, suggesting that there were no essential groups for which a change in charge in this range was critical . The INA of both bacteria was also unaffected by the addition of metal chelating compounds . Borate compounds and certain lectins markedly inhibited the INA of both types of bacterial cells . Butyl borate was not an inhibitor, but borate, phenyl borate, and m-nitrophenyl borate were, in order, increasingly potent inhibitors . These compounds have a similar order of affinity for cis hydroxyls, particularly for those found on sugars . Lentil lectin and fava bean lectin, which have binding sites for mannose or glucose, inhibited the INA of both bacteria . All other lectins examined had no effect . The inhibition of INA by these two types of reagents indicate that sugar-like groups are at or near the ice nucleating site . Sulfhydryl reagents were potent inhibitors of the INA of both bacteria . When treated with N-ethylmaleimide, p-hydroxymercuribenzoate, or iodoacetamide, the INA was irreversibly inhibited by 99% . The kinetics of inactivation with N-ethylmaleimide suggested that E . herbicola cells have at least two separate ice nucleating sites, whereas P . syringae cells have possibly four or more separate sites . The effect of infection with a virulent phage (Erh 1) on the INA of E . herbicola was examined . After multiple infection of a bacterial culture the INA was unchanged until 40 to 45 min, which was midway through the 95-min latent period . At that time, the INA activity began falling and 99% of the INA was lost by 55 min after infection, well before any cells had lysed . This decrease in INA before lysis is attributed to phage-induced changes in the cell wall. Z Allg Mikrobiol, 1983, 23(5), 297 - 301 Fluoride-induced filaments of Erwinia carotovora; Gashe BA et al.; Sodium fluoride induces filamentous growth of Erwinia carotovora when it is grown in liquid media containing aspartic acid only as the sole source of nitrogen . It is proposed that a stable complex of F(-)-Mg2+-enzyme and PO2(4) resulting in Mg2+ deficiency and consequent inability of E . carotovora cells to oxidize aspartic acid normally, is responsible for the formation of filaments in the presence of fluoride ions. Genetika, 1982 Nov, 18(11), 1806 - 10 {Conjugational transfer of chromosomal markers in the Erwinia chrysanthemi bacterial system . II . Characteristics of the donor strain of Erwinia chrysanthemi VY1-10}; Prokulevich VA et al.; Some properties of the donor Erwinia chrysanthemi VY1-10 strain are similar to those of Hfr donors of Escherichia coli . These are stable inheritance of the Flac plasmid and the capacity for linear and polarized transfer of the chromosome from one oriT site in the following order: O .. . thr, leu .. . pro .. . his . In addition to the ability to transfer its own chromosomal markers, the donor E . chrysanthemi VY1-10 also transfers lac+ marker during initial conjugational steps . The lac+ marker can be detected in recombinants as a structure similar to E . coli K-12 Flac plasmid which was used to obtain the donor strain . It is supposed that such properties are determined either by unusual integration of Flac plasmid or by the unstable Hfr state. Cancer Res, 1982 Oct, 42(10), 4068 - 71 Immunological and pharmacological characterization of poly-DL-alanyl-modified Erwinia carotovora L-asparaginase; Uren JR et al.; The covalent attachment of poly-DL-alanine peptides to lysyl residues on the surface of Erwinia carotovora L-asparaginase has produced a modified enzyme which is much less immunogenic in mice and demonstrates 100-fold longer plasma half-life in the rhesus monkey . Immunogenic responses towards both the immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody subclasses were evaluated in C57BL x DBA/2 F1 mice exposed to 250 rads of whole-body irradiation 4 hr prior to immunization with 5-diazo-4-oxynorvaline-inactivated native and modified L-asparaginase in complete Freund's adjuvant . Under these immunologically stressful conditions, the native enzyme evoked an IgE and IgG response which could be further amplified by a secondary immunization, whereas the modified enzyme evoked no IgE or IgG response even after a tertiary immunization . In experiments mimicking an intensive therapeutic schedule, whereby two groups of mice were given weekly injections of 5 to 10 units of either native or modified asparaginase for up to 14 weeks, neither enzyme form evoked a significant IgE response, and only the mice given injections of the native enzyme produced an IgG response . In a preliminary patient study, skin testing of a child who had shown an allergic reaction to the native enzyme resulted in a negative response after an intradermal injection of the modified enzyme, whereas a wheal and flare reaction was observed to both the native enzyme and a histamine control . All of these results suggest that the modified enzyme should show a definite reduction in immunological reactions associated with L-asparaginase treatment of childhood leukemia. J Bacteriol, 1982 Sep, 151(3), 1627 - 9 Plasmid-borne determinants of pigmentation and thiamine prototrophy in Erwinia herbicola; Gantotti BV et al.; Strains of Erwinia herbicola lost yellow pigmentation and thiamine prototrophy at high frequency when grown at elevated temperature (38 degrees C) or in the presence of sodium dodecyl sulfate . All pigmentless, thiamine-auxotrophic variants had lost a large plasmid (ca . 350 megadaltons) . Conversely, all pigmented, thiamine-prototrophic strains contained the large plasmid . The evidence presented indicates that pigmentation and thiamine prototrophy are specified or controlled by genes carried on the 350-megadalton plasmid. J Bacteriol, 1982 Sep, 151(3), 1595 - 7 Fructan from Erwinia herbicola; Blake JD et al.; Levan production by strains of Erwinia herbicola is common, and this property has some taxonomic significance for species differentiation within the "herbicola" group . The extracellular polysaccharide elaborated by strain 403 was characterized by nuclear magnetic resonance spectroscopy and methylation analysis . Results showed it to be a typical bacterial levan.
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