Neighboring plasmid sequences can affect Mini-Mu DNA transposition in the absence of expression of the bacteriophage Mu semi-essential early region

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa.

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

The products of gene A of the related phages Mu and D108 differ in their specificities

By recombination between different mutants of mutator phages Mu and D108, we isolated a set of viable hybrids. The structure of the hybrids was analyzed by digestion with different restriction enzymes. Genetic studies show that hybrids which carry the left end of the Mu genome complement a mini-Mu deleted from within the A gene as well as Mu while hybrids with the left end of the D108 genome or D108 do not. Vice versa, hybrids with the left end of the D108 genome or D108, but not hybrids with the left end of the Mu genome or Mu complement a mini-D108 deleted from within the A gene. The nucleotide sequence of the A gene of Mu and its equivalent on D108 are mainly similar except on their left end. These observations demonstrate that the two pA products, although only partially different, have different specificities.     

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.     

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.     

A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein

We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.     

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.     

Analysis of the ends of bacteriophage Mu using site-directed mutagenesis

We showed previously that two regions at the left end (L1 and L3) and one at the right end (R2) of bacteriophage Mu are essential for transposition. These regions all contain a 22 base-pair sequence with the consensus YGtTTCAYtNNAARYRCGAAAR, where Y and R represent any pyrimidine and purine, respectively. The Mu A protein binds to these regions in vitro, and weakly to sequences between nucleotides 1 and 30 of the right end (R1) and between nucleotides 110 and 135 of the left end (L2). These weak A binding sites contain the sequence AARYRCGAAAR. Here we show, using site-directed mutagenesis, that the weak A binding sites are essential for transposition. Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base-pairs in the stronger A binding sites, located adjacent to these weak A binding sites. We confirm the importance of several of the conserved base-pairs in the consensus sequence YGtTTCAYtNNAARYRCGAAAR. The base-pairs in the A binding sites that are shown to be essential for transposition are all conserved in the ends of the related bacteriophage D108. Furthermore, it is shown that the distance of 90 base-pairs between the two regions at the left end (L1 and L2L3) is essential.     

Organization of chimeras between filamentous bacteriophage f1 and plasmid pSC101

Two recombinants formed in vivo between the filamentous phage f1 and the tetracycline-resistance-conferring plasmid pSC101 are capable of transducing sensitive cells to Tet^r. These chimeric filamentous phage, VO-1 and VO-2, were previously shown to contain the entire f1 and pSC101 genomes (Vovis et al., 1977; Ohsumi et al., 1978). The genomes of VO-1 and VO-2 are unstable in vivo; VO-1 breaks down to yield a molecule similar to pSC101 and an f1-like species, f1′. f1′ was previously shown to differ from f1 by the presence of 209 additional nucleotides inserted in the carboxy-terminal portion of gene IV (Ravetch et al., 1979). We have found by hybridization analysis and direct DNA sequencing that this 209-nucleotide segment is present in one copy in pSC101, and that it has properties similar to known transposable elements. Therefore, we have called this sequence IS101. We have characterized the structures of both VO-1 and VO-2 in greater detail by restriction mapping and DNA sequence analysis. Both chimeras contain two copies of IS101, which are present as direct repeats and form the junctions between the f1 and pSC101 genomes. The IS101 elements in VO-1 and VO-2 are flanked by a five-base direct repeat of f1 sequence that is not repeated in wild-type f1. The junction between f1 and pSC101 in VO-1 is located at the same point as the IS101 element in f1′, while in VO-2 the junction between the two genomes is at a point in f1 located between the promoter and ribosome binding site for gene VIII. The pSC101-like molecules derived from the breakdown of VO-1 in vivo are identical to the original pSC101 in the region of IS101. The IS101 elements in the original and derived pSC101 plasmids are not flanked by any repeated sequence. Attempts to regenerate VO-1 from f1′ and pSC101, both of which contain one IS101 element, indicate that the breakdown of VO-1 is irreversible. These results are discussed in terms of current models for transposition, which postulate structures similar to VO-1 and VO-2 as intermediates in transposition.     

Site-specific deletions in the recombinant plasmid pSC101 containing the redB-ori region of phage lambda

Large deletions occur in the hybrid plasmid formed by pSC101 and the EcoRI fragment f2 of phage lambda (redB-ori region) under well defined growth conditions (Bernardi and Bernardi, 1980). We have sequenced the novel joints of the four deletions so obtained and shown that they have one endpoint in pSC101, identical in all four cases, the other endpoint being located in four different lambda sequences. Furthermore, the nucleotide sequences of the novel joints show homologies between the conserved pSC101 sequence and the lambda sequences both conserved and deleted. The presence of an IS-type element in pSC101 is postulated; however, this element is unrelated to the 200 bp element already described in pSC101 (Ravetch et al., 1976).     

Mini-Mu transduction: cis-inhibition of the insertion of Mud transposons

Mud (mini-Mu) transposons are defective phage Mu genomes that conserve the Mu ends. The transduction of Mud transposons is strictly dependent on Mu complementation, inefficient, and affected by modifications in the Mud internal sequences. The transduction of Mud transposons depends on transposition, which appears to be low, relative to wild-type Mu. Insertions of Mud into a plasmid can be frequently recovered among transductants; new Mud insertions into plasmids that already have both Mu ends, or just one, are rarely found. This suggests that the presence of Mu ends "immunizes" the plasmid against further insertion. This phenomenon may be similar to the transposition immunity of Tn3.     

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.     

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.     

Complete sequence of an IS element present in pSC101

Recently a new insertion element (IS102)b ha been described in plasmid pSC101. We have determined its complete sequence: it consists of 1057 bp; 338 bp at one end are identical to those already determined for the kanamycin resistance transposon Tn903. It is not flanked by any direct repeat. Its coding capabilities are discussed, and compared to those of IS903.     

Mutator bacteriophage D108 and its DNA: an electron microscopic characterization

Three types of phage particles were observed on CsCl step gradients when D108 was purified from lysates prepared by induction of a prophage. These particle types were identified to be the mature phage, tailless DNA-filled heads, and a form of nucleoprotein aggregates. The nucleoprotein aggregates banded at a density (rho) of greater than 1.6. DNA molecules isolated from mature phage particles were (38.305 +/- 1.226) kilobases (kb) in length. Denaturation and renaturation of D108 DNA resulted in the formation of linear double-stranded molecules with variable-length single-stranded tails at one end. About 30% of the annealed molecules also carried an internal nonhomology, which was shown to be the region called the G-loop in Mu and P1 DNAs. Following the notation used for different regions of denatured, annealed Mu DNA, we measured the lengths of the equivalent D108 DNA regions to be as alpha-D108 = (32.178 +/- 1.370) kb; G-D108 = (3.07 +/- 0.382) kb; beta-D108 = (2.291 +/- 0.306) kb; SE-D108 = (0.966 +/- 0.433) kb. Formation of D108; Mu heteroduplexes disclosed the presence of five nonhomologies, two of which were partial. One of the partial heterologies was in the G-loop region. The largest nonhomology, (1.393 +/- 0.185) kb in size, was near the c end (immunity region) and probably spans the c and the ner genes of Mu. beta-D108 was shown to carry a (0.556 +/- 0.097)-kb insertion close to its right end. A short 100-base-pair region appeared to have been conserved at the ends of D108 and Mu. Occasionally, a 50-to 100-base-pair-long unpaired region was also observed at the left end of D108: Mu heteroduplexes. These sequences were presumably of bacterial DNA. Taken together, our results complement and extend our earlier genetic studies which established that D108 was a mutator phage heteroimmune to Mu with a host range different from Mu's.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.