Plasmid recombination stimulated by restriction endonuclease EcoRI in vivo: formation of recombinant plasmids in recA+-cells of E. coli

The possible participation of restriction endonuclease EcoRI in recombination of compatible nonhomologous plasmids in E. coli cells has been studied. To study the process, plasmids RP4 and R245 have been transferred by conjugation into the recipient cells of E. coli harbouring one of isogenic plasmids, pSA14 and pSA25, different for the genes coding restriction endonuclease EcoRI. The genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant plasmids after compatibility of parent plasmids in E. coli cells. Recombination of plasmid RP4 with the plasmid pSA14, carrying EcoRI genes, has been registered in E. coli cells, producing the restriction endonuclease, while plasmid recombination has not been found in the cells harbouring plasmid pSA25, isogenic for all genes, except for EcoRI genes, with plasmid pSA14. Restriction endonuclease EcoRI is concluded to stimulate site specific recombination of nonhomologous compatible plasmids in vivo. EcoRI-mediated recombination of plasmid R245 with plasmid pSA14 is discussed.     

Characteristics of RecA-independent recombination of plasmids in E. coli cells producing restriction endonuclease EcoRI

The restriction endonuclease EcoRI dependent recombination of compatible plasmids has been studied in RecA cells of Escherichia coli. Plasmid RP4 and the isogenic ColE1 type plasmids pSA14 or pSA25, differing in restriction-modification RM EcoRI genes, have been used to study this type of recombination. EcoRI dependent recombination of plasmids is demonstrated in RecA cells and, thus, is independent of general system of homologous recombination. The classes of recombinant plasmids isolated from RecA cells differ from the classes isolated from wild type cells. Levels of tetracycline resistance conferred by plasmid RP4 are shown to be dependent on the alleles of RecA+ gene, being extremely low in RecA cells. This property is demonstrated to be useful for obtaining the multicopy recombinant plasmids resulting from EcoRI dependent recombination in RecA cells of Escherichia coli.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.     

Plasmid localization and cloning of restriction modification genes from Citrobacter freundii 4111 strain]

Over 60 producing strains of restriction endonucleases type II have been found among 500 different strains, mostly Enterobacteriaceae. The strain Citrobacter freundii 4111 produces restriction endonuclease CfrBI, a new isoschisomer of StyI. The genes of the restriction-modification system CfrBI were located on the multicopy plasmid pZE8 containing the Co1E1-type replicon and cloned to E. coli K802. The deletion variant of 3.2-kb pZE8 which contains intact restriction-modification and a DNA fragment responsible for autonomous plasmid replication was selected among the recombinant plasmids. The strain with higher R. CfrBI production (at least 10,000,000 U/g cells, which is 500-fold higher than the wild strain) was constructed.     

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.     

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning.

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.     

Mapping of colicin E2 and colicin E3 plasmid deoxyribonucleic acid EcoR-1-sensitive sites.

Colicin plasmids E2 and E3 (Col E2 and Col E3) deoxyribonucleic acid (DNA) has been shown to contain, respectively, two and three EcoR1 restriction endonuclease-sensitive sites. This was determined by measuring the DNA fragments generated after EcoR1 endonuclease treatment by agarose gel electrophoresis and electron microscopy. The structure of heteroduplex Col E2-col E3 DNA molecules formed from EcoR1-generated fragments permitted a localization of the EcoR1-sensitive sites on the plasmid chromosomes.     

Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.     

Characterization and restriction analysis of the P sex factor and the cryptic plasmid of Vibrio cholerae strain V58

The P plasmid of Vibrio cholerae is a derepressed sex factor restricted to V. cholerae and has been shown to express surface exclusion. We have isolated the plasmids of strain V58 and have found that in addition to P, two further cryptic plasmids are also present. P has a size of 68 kb as determined by both electron microscopy and restriction endonuclease analysis. These other plasmids are 34 and 4.7 kb in size. Restriction maps of P and the larger cryptic plasmid have been determined. It has been demonstrated that P differs from the standard Inc group test plasmids and also expresses a surface exclusion system. The ability of the type Inc plasmids to be transferred to V. cholerae by either liquid or filter matings and the stability of these plasmids in V. cholerae have also been examined.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli.

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.     

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iord?nescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.     

The sequence arrangement of Drosophila melanogaster 5s DNA cloned in recombinant plasmids.

Segments of Drosophila melanogaster DNA containing 5S rRNA genes have been propagated in recombinant plasmids using E. coli as a host and Col E1 as a vector. Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of these plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. The insert in one plasmid (pCIT9) consists of 32 contiguous repeats. Restriction enzyme-gel electrophoresis analysis shows that all these repeats have the same length within ± 5 nucleotides. This repeat length is estimated as 370 ± 20 ntp by gel electrophoresis and 390 ± 40 ntp by partial denaturation mapping. A second plasmid (pCIT19) contains three complete genes, two complete spacers and incomplete flanking spacer sequences. The two complete repeat units released by suitable restriction endonuclease digestions differ in length by 20 ± 5 ntp, with estimated lengths of 370 and 390 ntp. The positions and spacings of the genes on this plasmid have been observed directly by ferritin labeling and by partial denaturation mapping. The A+T content of the 5S DNA spacer region is calculated to be 68%. By in situ hybridization, cRNA transcribed from one plasmid hybridizes to polytene chromosomes only at band 56F, the known locus of the 5S rRNA genes. Spontaneous excision of some of the tandem repeat units from the recombinant plasmids occurs during growth in E. coli; the frequency of excision does not depend upon the recA character of the host, but is greatly increased by chloramphenicol treatment.     

以报告基因分析霍乱弧菌噬菌体VP1的预测启动子

VP1为感染并裂解霍乱弧菌的噬菌体,全基因组为环状双链DNA。通过测定该基因组序列,预测出15个可能的启动子区,利用报告基因质粒转化及全噬菌体共感染的策略分析了这些推测启动子在霍乱弧菌中的活性,将预测的启动子区分别克隆到启动子探测lacZ融合质粒载体pRS1274,在转化于大肠埃希氏菌受体菌株JM109中时,所有克隆子均呈现兰斑。同时将质粒电击到缺失了lacZ基因的霍乱弧菌菌株7743△Z,然后用噬菌体VP1感染转化菌株。在转化成功的13个含预测启动子片段的重组质粒中,通过检测β_半乳糖苷酶活性表达随感染后时间的变化,提示P17为早期启动子,P2、P3、P9等为中期启动子,P18为晚期启动子。     

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.