A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

The effect of R-plasmids on the reproduction of Pseudomonas aeruginosa phage SM

The inheritance of plasmids Rms163 and R74 by Pseudomonas aeruginosa strain PAO hs been shown to effect the reproduction of a temperature bacteriophage SM. The decrease in plating efficiency of bacteriophage on Pseudomonas aeruginosa PAO (rms163) lawn is explained by the high degree of cell lysogenization by bacteriophage. Plasmid R74 inhibits bacteriophage SM propagation ultimately, evidently due to interruption of definite stages in vegetative development of bacteriophage by the products of plasmid specific genes.     

Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.     

IS222, a new insertion element associated with the genome of Pseudomonas aeruginosa.

A new insertion element, IS222, was identified to be associated with the DNA of a mutant strain of the converting Pseudomonas aeruginosa bacteriophage D3. The insertion sequence was 1,350 base pairs in size and possessed terminal inverted repeats. The nucleotide sequence contained single cleavage sites for EcoRI and PvuI but none for BamHI, PstI, HindIII, SmaI, or SalI. By Southern hybridization analysis, no homology was found with genomic DNA from P. aeruginosa PAT or Escherichia coli. Genomic DNA from the phage host, P. aeruginosa PAO, contained two sequences homologous to IS222.     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.     

Characterization and construction of molecular cloning vehicles within Staphylococcus aureus.

Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.     

Physical map of the DNA of bacteriophage tf of Pseudomonas putida

A physical map has been constructed for P. putida bacteriophage tf DNA containing single-strand breaks (nicks). Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined. Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples. The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.     

Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments

A plasmid cloning vector, pHSG664, has been constructed which is suitable for the direct-selection of transformed cells with recombinant DNAs. The plasmid contains the replicon and ApR-gene region of pUC9 ligated to the strA+ (rpsL+) gene derived from pNO1523 [Dean, Gene 15 (1981) 99-102]. The vector contains ten unique restriction sites: EcoRI, HpaI, PvuII, SphI, PstI, SmaI(XmaI), BamHI, SalI(AccI, HincII), XbaI and HindIII. Five sites (bold-face lettering) are located within the coding region of the strA+ gene. Any insertion at the five bold-faced sites or any nucleotide replacement involving the strA+ gene region and the other unique sites can be selected by Ap and Sm double selection in a strA- (SmR) strain such as E. coli HB101. Thus, this vector is useful for cDNA cloning at either the SphI or the PstI site with the G:C-tailing procedure, as well as for the cloning of double-digest DNA segments.     

Isolation and analysis of Pseudomonas aeruginosa PAO mutants resistant to nonlysogenic bacteriophages

Nonlysogenizing Pseudomonas aeruginosa PAO bacteriophages were studied. According to morphology of the plaques, they were distributed into three groups: phi k, phi m and phi mn. The mutants of P. aeruginosa PAO resistant to these bacteriophages were selected. On the basis of cris-cross resistance analysis of the mutants, a formal scheme of the receptor sites on the P. aeruginosa PAO bacterial cell surface is drawn. It is shown that bacteriophages phi k and phi m use different receptors for their adsorption. The receptors of phi m and phi mn phages are specifically interconnected. Thus, the receptor for phi k phages is connected with the receptor for phage phi 11. It appears that the receptor for bacteriophage E79 is identical to those of phi m phages. The phi m receptor is of a composite structure: it includes two different receptors used by phi mn phages.     

Construction of a restriction map of bacteriophage T3 DNA

A restriction endonuclease cleavage map of bacteriophage T3 DNA has been constructed. The enzymes used and, within parentheses, the number of their cleavage sites on T3 DNA are: HindIII (1), XbaI (1), BglII (1), KpnI (2), MboI (9), and HpaI (17). The size and the relative location of each fragment have been established, defining an accurate physical map of T3 DNA.     

Plasmids of the cyanobacterium Synechocystis sp. 6803

Three cryptic plasmids have been isolated from cyanobacterium Synechocystis sp. 6803::pSS2 (1.4 Md), pSS3 (36 Md), pSS4 (60 Md). Plasmid DNA was isolated in Cs-Cl-EB density gradient and analyzed by gel electrophoresis and electron microscopy by gel electrophoresis and electron microscopy techniques. The restriction map is constructed for plasmid pSS2 having the cleavage sites for Sau3a, HincII, HindIII, MspI restriction endonucleases. The plasmid may be used to construct the recombinant vector DNAs capable of autonomous replication in cyanobacterium Synechocystis sp. 6803. cells.     

Transfecting activity of Pseudomonas aeruginosa bacteriophage SM

Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.     

A small mobilizable IncP group plasmid vector packageable into bacteriophage lambda capsids in vitro

A mobilizable cosmid derivative of an IncP group plasmid was constructed by cloning the oriT region of RK2, a wide host-range plasmid, and the minimal DNA sequence of bacteriophage lambda required for efficient packaging in vitro. This cosmid is 13 kb in size and has unique restriction sites for EcoRI, XhoI, HindIII, and SalI. The XhoI and HindIII sites are within the kanamycin-resistance gene and the SalI site is in the tetracycline-resistance gene. This plasmid was mobilizable from an Escherichia coli donor to a number of diverse gram-negative bacteria at a frequency of 0.8 to 10 per 100 donors. This vector is one of the smallest of all wide host-range cosmids described in the literature. As part of this study, another mobilizable IncP group plasmid vector has also been constructed which, in addition to the sites listed above, has a unique BglII site, but which lacks the packager sequence.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.