Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid

A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.     

Isolation and characterization of lambda pleu bacteriophages.

In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage lambda into cistron leuA. The orientation of lambda in the chromosome is ara leuDCB lambda JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., lambda pleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., lambda pleu 13) transduces leuA strains to prototrophy. lambda pleu 13 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 lambda pleu13). A phage carrying the entire leucine operon (lambda K2) was constructed by a cross between lambda pleu9 and lambda pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with lambdaK2 indicated that leuO and leuP are present and functional in lambda K2. leu-specific messenger ribonucleic acid from E. coli hybridizes to the heavy (r) strand of lambdaK2. The leucine operon of lambda G4 pleuABCD (an S7 derivative of lambda K2) exists intact on a 7.3 x 10(6)-dalton fragment (lambdaG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between lambda G4 and lambda show a 5.4 x 10(6)-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 10(6)-dalton piece of lambda DNA starting at 0.46 fractional unit on the map of lambda. Fragment lambda G4EcoRI-B has about 0.6 x 10(6) daltons of lambda DNA from the b2 region at one end and about 1.4 x 10(6) daltons of lambda DNA from the int region at the other end.     

Molecular structures of packageable ColE1 hybrids and the generation of deletion mutants from one of the hybrids

ColE1 derivatives carrying cohesive end sites of lambda phage genome (= cos lambda) can be packaged within lambda phage particles. The DNA structure of the prototype ColE1-cos lambda derivative named pKY2257 was studied because of its potential usefulness in various fields in molecular biology. pKY2257, which carries an intact galactose operon of E. coli, is a convenient replicon to detect Tn3 translocation. It was found that one of the PK2257::Tn3 derivatives, pKY2113, generated various small plasmids in E. coli. The molecular structures of some of these deletion mutants were compared with each other and with those of parental plasmid DNAs by heteroduplex analysis and restriction enzyme digestion. A possible mechanism, which seems to be unique to this kind of deletions, is discussed on the basis of the present results.     

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.     

Deletion analysis of the DNA sequence required for the in vitro initiation of replication of bacteriophage lambda

Supercoiled DNA containing the replication origin of bacteriophage lambda can be replicated in vitro. This reaction requires purified lambda O and P replication proteins and a partially purified mixture of Escherichia coli proteins (Tsurimoto, T., and Matsubara, K. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7639-7643; Wold, M. S., Mallory, J.B., Roberts, J. D., LeBowitz, J. H., and McMacken, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6176-6180). The lambda origin region has four repeats of a 19-base pair sequence to which O protein binds. To the right of these sites on the lambda map is a 40-base pair region that is rich in adenine and thymine, followed by a 28-base pair palindromic sequence. To define more precisely the boundaries of the lambda origin, we cloned a 358-base pair piece of lambda DNA containing the origin region into M13mp8 in both orientations. In vitro replication of RF I DNAs prepared from cells infected with these two M13 ori lambda phage was dependent on lambda O and P proteins and a crude protein fraction from uninfected E. coli; with these conditions there was no replication of M13mp8 RF I DNA. We made deletions from the left and the right ends of the lambda origin DNA and determined the deletion end points by DNA sequencing. We have tested RF I DNAs prepared from cells infected with phage carrying ori lambda deletions for their ability to function as templates for O- and P-dependent replication in vitro. Our results show that lambda DNA between nucleotide positions 39072 and 39160 is required for efficient O- and P-dependent replication. This 89-base pair piece of DNA includes only two of the four 19-base pair O protein-binding sites (the two right-most) and the adjoining adenine- and thymine-rich region to the right of the O-binding sites.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions

Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.     

Lambda bacteriophage-mediated transduction of ColE1 deoxyribonucleic acid having a lambda bacteriophage-cohesive end site: selection of packageable-length deoxyribonucleic acid.

An in vitro recombinant ColE1-cos lambda deoxyribonucleic acid (DNA) molecule, pKY96, has 70% of the length of lambda phage DNA. The process of lambda phage-mediated transduction of pKY96 generated a small amount of transducing phage particles containing ColE1-cos lambda DNA molecules of 80 or 101% of the length of lambda phage DNA, in addition to those containing original pKY96 DNA molecules. The newly isolated larger plasmid DNAs were transduced 100 times more efficiently than pKY96 DNA. Their structures were compared with that of a prototype pKY96 DNA, and the mechanism of the formation of these molecules is discussed.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.     

Mechanism of conjugation and recombination in bacteria. XVIII. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization.

Polarity of donor DNA strand transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H (lambdat11) as a donor and thermosensitive F- CR34 dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 of lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

Superinfection Exclusion by P22 Prophage and the Replication Complex

Wild-type sie(+) P22 prophage converted Salmonella typhimurium lysogens to exclude deoxyribonucleic acid (DNA) injected by superinfecting phage. DNA from a P22 superinfecting virulent phage associated with the replication complex in a sie(-) lysogen but not in a sie(+) lysogen.     

Functional domains of bacteriophage Mu transposase: properties of C-terminal deletions

We have generated a series of 3' deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the lambda PI promoter, were analysed in vivo for their capacity to complement a super-infecting MuAam phage, both for lytic growth and lysogeny, and for their effect on growth of wild-type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.     

Structure of a hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome

The structure of a repeated DNA sequence located on the short arm of the human Y chromosome is described. Genomic mapping and cloning in lambda or cosmid vectors show that the repeated sequence consists of units 20.3 x 10(3) base-pairs long that contain the three previously described DNA sequences: Y-156, Y-190 and Y-223a. Analysis of male genomic DNA by pulsed-field gel electrophoresis shows that the units are tandemly arranged and are organized into two blocks. The major block is hypervariable in size and alleles in the range approximately 540 x 10(3) to 800 x 10(3) base-pairs were detected. The minor block is not variable in size and is approximately 60 x 10(3) base-pairs long. Analysis of rearranged Y chromosomes shows that both blocks are located on the short arm of the chromosome. Most commonly, the major block is distal to the minor block, but the opposite arrangement is also found.