Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions.

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.     

The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.     

Characteristics of transduction in Erwinia by phage 59

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.     

Restriction map of the temperate phage E105 from the polylysogenic culture of Erwinia carotovora 268

DNA structure of the temperate bacteriophage E105 from polylysogenic culture of Erwinia carotovora 268 has been studied. The viral 29.12-29.17 MD DNA has been shown to be linear and nonpermuted. The complete restriction map of the viral DNA has been constructed for MvaI and HpaI and partial for Eco31 restriction endonucleases based on the pair hydrolysis of the native DNA as well as its fragments. Altogether, 19 sites for restriction endonucleases have been localized on bacteriophage DNA.     

Interaction of Yersinia pestis bacteria with bacteriophage mu

Yersinia pestis cells are shown to be sensitive to bacteriophage Mu cts62 infection. Lysis of bacteria has been shown to be more efficient on solid nutrient medium than in LB broth. 10(-5) pfu per ml is the maximal concentration of bacteriophage particles yielded from the broth cultures of bacteria. Moi 0.1 has been used to obtain such yields of bacteriophage. Lysogenization of Yersinia pestis cells has not been achieved when the standard methods of bacteriophage infection have been used. It was accomplished by the conjugal transfer of plasmid RP4::MU cts62 to Yersinia pestis from Escherichia coli. The deficiency of Yersinia pestis in producing bacteriophage Mu cts62 mature particles during the lytic cycle of bacteriophage is discussed.     

Localization of prophage SM of Pseudomonas aeruginosa in the chromosome of host cells

Pseudomonas aeruginosa PAO SM-prophage was localized on the chromosome between thr-9001 and pur-66 locuses on 42-43 min of chromosomal genetic map. The location of prophage was identified on the basis of prophage linkage with the above-mentioned markers and confirmed by the purine, hypoxanthine and threonine deletions in course of thermoinduction of SM cts6 prophage from lysogens. The decrease for two orders in lysogenization frequency of thr mutants by SM bacteriophage suggests the integration of SM prophage in these cells into some other region of chromosome.     

Transfer of prophage Mu into methylotrophic bacteria in the plasmid RP4

Bacteriophage Mu genome has been transferred into the cells of Pseudomonas methanolica and Methylobacterium sp. SKF240, that are naturally resistant to the bacteriophage, as a fragment of a hybrid plasmid RP4::Mu cts62. Temperature induction of the bacteriophage results in host cell lysis. Plasmid RP::Mu cis62 is maintained in methylotrophic cells presenting a cointegrative structure.The genetic and electrophoretic, analyses of the DNA isolated from transconjugant cells have confirmed the conclusion. Bacteriophage Mu propagation has been shown to be restricted in methylotrophic cells.     

Isolation of transmissible cointegrates of Yersinia pestis plasmids pYV and pYT with the plasmid RP4::Mu cts 62, IncP1

The transmissible cointegrates of the Yersinia pestis plasmids pYV and pYT with the broad host range plasmid RP4::Mu cts62 of the incompatibility group IncP have been constructed by the in vivo recombination. The cointegrative plasmid pKR14 (pYV76 omega RP4::Mu cts62) conferred on the transconjugants the properties of Ca2(+)-dependence at 37 degrees C, V-antigen synthesis, RP4 plasmid markers (ApR, KmR, TcR), immunity to the lysis by the bacteriophage Mu cts62 and incompatibility with the homologous replicon pYV76. Cointegrates pKR103 and pKR106 (pYT omega RP4::Mu cts62) conferred on the transconjugant clones the ability to synthesize the "mouse" toxin and fraction I. The capability of Escherichia coli cells to synthesize the latter products has been demonstrated together with the deficiency of these cells to transport the synthesized fraction I to the cell surface.     

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.     

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.     

Insertion of a transposon for chloramphenicol resistance into bacteriophage Mu.

We have isolated mutants of bacteriophage Mu carrying the X mutations caused by the insertion of cam (Tn9), a transposon for chloramphenicol resistance. The Mu X cam mutants were obtained by selecting for heat-resistant survivors of a Mucts62, P1cam dilysogen. Like the previously described X mutants, Mu X cam mutants are defective prophages which can be excised from the host DNA at a frequency of 10(-5) to 10(-7) per cell. Tn9 insertions in Mu X cam mutants are located within 5000 base pairs of the left end of Mu DNA in a region that controls early replication functions of Mu. There is one EcoRI cleavage site in Tn9. The Tn9 transposon itself can be excised precisely from the Mu X cam mutants to generate wild type Mu. In most Mu X cam mutants, precise excision of Tn9 occurs at a low frequency (10(-6) per cell), whereas in some, the frequency is higher (10(-4) per cell). Mu X cam prophages can replicate after induction with the help of wild type Mu. The lysates containing Mu X cam particles, however, fail to transduce chloramphenicol resistance at a high frequency; Mu X cam mutants apparently have a cis dominant defect in integration.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Mapping of insertion mutations in gnd of Escherichia coli with deletions defining the ends of the gene.

A genetic map was prepared for gnd, the gene of Escherichia coli which encodes the metabolically regulated 6-phosphogluconate dehydrogenase. Direct selection methods were used for the isolation of mutants with deletions that define the respective ends of gnd. These selections depended on the availability of a defective lysogen in which gnd was present on a lambda h80 dgnd his prophage located at the att phi 80 region of the chromosome. Mutants with deletions entering gnd from the his-distal end were selected as Gnd- TonB- mutants. Mutants with his-proximal gnd deletions were selected as Gnd-, temperature-resistant mutants of a specially prepared stable lysogen. Gnd- mutants were also isolated after mutagenesis with bacteriophage Mu cts61, and genetic tests were used to determine which mutants carry a Mu cts61 prophage in gnd. The deletion mutations were mapped against each other and against the insertion mutations through the use of F' merodiploid strains. The insertion mutations mapped at seven distinct sites in gnd; three mapped under the deletions defining the his-proximal portion of the gene and three mapped with the his-distal deletions.     

High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

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).