A mutant of Escherichia coli which accumulates large amounts of coproporphyrin.

A mutant of Escherichia coli which accumulates a large amount of coproporphyrin, presumably because of a block in heme biosynthesis, has been isolated after nitrosoguanidine mutagenesis. On rich media, the mutant forms colonies which give bright orange fluorescence when illuminated with ultraviolet light. The mutant appears to be similar to a Salmonella typhimurium mutant, deficient in uroporphyrinogen III cosynthase, described by Sasarman and Desrochers ((1976) J. Bacteriol. 128, 717--721). A striking property of the mutant is that coproporphyrin is retained within the cells in rich media but is almost totally excreted out of cells in minimal glucose medium.

Infecting bacteriophage mu DNA forms a circular DNA-protein complex.

Upon superinfection of immune (lysogenic) cells with bacteriophage Mu, a form of Mu DNA accumulates that sediments about twice as fast as the linear phage DNA marker in neutral sucrose gradients. This form is also detected upon infection of sensitive cells with Mu. We have purified it and examined its physical nature. Under the electron microscope it appears circular and supertwisted. Upon treatment with Pronase, phenol or sodium dodecyl sulfate, however, it is converted to a linear Mu-length form, indicating that the circle is not covalently closed. The linear DNA still has heterogeneous host sequences at its termini. The circular DNA is resistant to the action of Escherichia coli exonuclease III and T7 exonuclease, but becomes sensitive to these nucleases after treatment with Pronase showing the presence of a protein that binds non-covalently to the ends of the DNA to circularize it as well as protect it from digestion with exonucleases. The complex is resistant to high salt (up to 6 M-NaCl) but can undergo transitions between forms that are partially open, open circular, linear and circular dimers and trimers. Examination of DNA from mature phage particles reveals that a circular DNA species is present in at least 0.1 to 1% of the population. The purified complex is extremely efficient in transfection of E. coli spheroplasts. We estimate the molecular weight of the protein in this DNA-protein complex to be approximately 64,000, and suggest that this complex might represent the integrative precursor of infecting Mu DNA.

DNA rearrangements associated with reversion of bacteriophage Mu-induced mutations.

Excision of transposable genetic elements from host DNA is different from the classical prophage lambda type of excision in that it occurs at low frequency and is mostly imprecise; only a minority of excision events restores the wild-type host sequences. In bacteriophage Mu, a highly efficient transposon, imprecise excision is 10-100 times more frequent than precise excision. We have examined a large number of these excision events by starting with mucts X mutants located in the Z gene of the lac operon of Escherichia coli. Mucts X mutants are defective prophages whose excision occurs at a measurable frequency. Imprecise excision was monitored by selecting for melibiose+ (Mel+) phenotype, which requires only a functioning lacY gene. Mel+ revertants exhibit an array of DNA rearrangements and fall in four main classes, the predominant one being comprised of revertants that have no detectable Mu DNA. Most of these revertants can further revert to Lac+. Perhaps 5 base-pair duplications, originally present at prophage-host junctions, are left in these lacZ-Y+ revertants, and they can be further repaired to lacZ+. Another class has, in addition to the loss of Mu DNA, deletions that extend generally, but not always, to only one side of the prophage. The other two classes of revertants, surprisingly, still have Mu DNA in the lacZ gene. One class has deletions in the Z gene, whereas, no deletions can be detected in the other. Many of the revertants in the last class can further revert to lacZ+, indicating that the lacY gene must have been turned on by a rearrangement within Mu DNA. Apparently, all of the detectable precise and most of the imprecise excision events require functioning of the Mu A gene. We suggest that a block in large-scale Mu replication allows the excision process to proceed.

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.

Heteroduplex electron microscopy of phage Mu mutants containing IS1 insertions and chloramphenicol resistance transposons.

We have examined by electron microscopy the DNA heteroduplexes of six bacteriophage Mu mutants, Mu X cam, generated by the insertion of the Tn9 transposon for chloramphenicol resistance. Tn9 was found to be 2.8 +/- 0.2 kilobases (kb) in length and to consist of a cam determinant flanked by two IS1 sequences arranged in a direct order. In two of the six Mu X cam mutants, the Tn9 insertion was at a fixed location, 3.9 kb from the left, or c, end. In the other four mutants, the position of the insertion varied, even though the lysogenic cultures induced were grown from single colonies. The insertion was located at either 3.3 kb, 3.9 kb, or, less frequently, at 4.4 kb from the left end of the DNA. Furthermore, at low frequencies, the insertions were found to be in an orientation opposite to what predominated in the preparation. Thus, Tn9 in the Mu X cam mutants examined could appear to undergo rapid rearrangements during Mu growth or over a few generations of cell growth. One of the Tn9 insertion sites was apparently the same as that for a 0.8 kb insertion found in a Mu X mutant. This latter insertion was identified as an IS1 sequence. The DNA molecules from all the Mu X cam mutant phage particles were found to be missing the bacterial DNA at the S (right) end, along with a variable amount of the adjoining Mu DNA in the beta region. This observation supports the headful packaging model for Mu DNA.

Expression of the gin and mom genes of bacteriophage Mu.

The gin and mom genes are located in the rightmost 1.6-kb segment, designated the beta segment, of bacteriophage Mu DNA. The gin gene is responsible for the inversion of the G segment of Mu, whereas the mom gene is involved in an unusual modification of the DNA. We have analyzed recombinant plasmids carrying one or both ends of Mu DNA for the expression of the Gin and Mom functions. The Gin protein and the presumptive Mom protein are not always detected in minicells, even though the plasmids being tested have the gin- and mom-containing segment of Mu DNA. However, some plasmids, in which the right end segment of Mu DNA is confined to the 1.6-kb beta segment, do give rise to these gene products in minicells. It seems that synthesis of the Gin and Mom proteins is inhibited in minicells, but this inhibition is lifted if most of the DNA to the left of the beta segment is eliminated from the plasmids. The most prominent Mu product detected in minicells is a 23-25-kDal polypeptide, termed here the zeta (zeta) protein. The function of the zeta protein remains unknown. In vitro transcription of Mu DNA with purified Escherichia coli RNA polymerase is limited to only two regions of the genome. The early region of Mu DNA is transcribed at a relatively high efficiency, whereas the beta region is transcribed at a low efficiency. This low-efficiency transcription appears to be specific for the gin gene; the mom gene transcript cannot be detected.

Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1.

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.

Comparison of left-end DNA sequences of bacteriophages Mu and D108.

The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined. The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200. The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence. Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Genetic analysis of heterogeneous DNA circles formed after prophage Mu induction.

Induction of a Mu prophage in Escherichia coli Hfr strains lyosgenic for Mu cts62 leads to the generation of F' episomes. Each episome thus formed carries at least one copy of the Mu genome. These results suggest that integration of Mu is mandatory for the formation of the heterogeneous circles during the lytic cycle. The circles may be precursors for phage maturation.

Events following prophage Mu induction.

Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.

Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.

Temperature-sensitive mutants of bacteriophage mu.

Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated. These mutants fall into two groups. Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants. All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.

A mechanism of DNA transposition.

Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element. We describe here a mechanism by which such integrative replication may take place. We hve examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the transposition process, (i) Association. A protein-mediated association is brought about between the transposable element and the target DNA. (ii) Attachment. One end of the element is nicked and attached to a site that undergoes a double-stranded cleavage. (iii) Roll-in replication. While one strand of the target DNA is linked to the nicked strand of the element, the complementary strand of the target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roll-in termination. When the distal end of the element arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats--i.e., amplification of circular DNA sequences.