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.     

Adjacent insertion sequences IS2 and IS5 in bacteriophage Mu mutants and an IS5 in a lambda darg bacteriophage

Using electron microscopic heteroduplex analysis, we have demonstrated that an insertion found in a Mu prophage and in some infectious. Mu deletion-substitution mutants derived from it consists of bacterial insertion sequence IS2 linked directly to IS5. Other infectious Mu mutants derived from the same lysogen have only IS5 or a portion of IS2. In addition, we have found that an independent insertion in a transducing phage, lambda 13 dargB2, is IS5. The ends of IS5 are short, inverted duplications of each other. These observations support the notion that the DNA insertion previously designated IS5 on the basis of a single example in lambda KH100 is a bona fide bacterial insertion sequence.     

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.     

Virulent mutants of temperate phage Mu-1

Summary Virulent mutants of phage Mu have been isolated after mutagenesis. The virulent phenotype results from most probably 2 mutations located in the c-A region of the Mu genome.Vir mutants are trans-dominant; they induce the resident prophage upon infection in broth of any Mu lysogen. They however form plaques only on certain lysogens, that are monolysogenic for a mutant prophage. We further isolated secondary mutations in Mu Vir which suppress the virulent phenotype.     

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.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.     

Isolation and Characterization of Mutants of Colicin Plasmids E1 and E2 After Mu Bacteriophage Infection

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.     

IS2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage P1: non-random distribution of target sites

Insertion mutations arising spontaneously in the P1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (IS2). Filter hybridization identified 28 out of 44 independent insertions as IS2. Their target specificity is not random. A region that amounts to < 2% of the phage genome had trapped 15 of the 28 IS2 elements. However, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site. Rather, the nine IS2 are distributed over the whole target segment and IS2 are found in both orientations. Sequence data indicate that at least two sequence variants of IS2 participated in mutagenesis of the phage genome. The detectable transposition of IS2 from the host chromosome to the prophage occurs with a frequency of 3 x 10(-5) per cell per generation under the particular experimental conditions. It is concluded that IS2, a natural resident of Escherichia coli K12 strains, is an important agent for spontaneous mutagenesis and exerts this action non-randomly along the genome.     

Characterization of the types of mutational events that spontaneously occur in a plasmid system

The immunity region from a cI857 derivative of bacteriophage lambda has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA- E. coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed. All of the subcultures contained mutants that allowed growth at the restrictive temperature without showing a detectable change in plasmid size. 75% of the total mutants fell in this class. More than half of these mutations involved the lambda leftward promoter, pL, and such mutants were found in all 20 subcultures. The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures. 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the lambda kil gene. 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred. About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized. In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred. The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence. A mutant with both an IS1 insertion and an alteration involving pL has also been isolated.     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.     

Mu Element-Generated Gene Conversions in Maize Attenuate the Dominant Knotted Phenotype

The knotted1 gene was first defined by dominant mutations that affect leaf morphology. The original allele, Kn1-O, results from a 17-kb tandem duplication. Mutator (Mu) insertions near the junction of the two repeats suppress the leaf phenotype to different degrees depending on the position of the insertion. The Mu insertions also increase the frequency of recombination at Kn1-O to create derivative alleles in which the Mu element and one copy of the repeat are lost. These derivatives are normal in appearance. Here we describe two derivatives that retained the tandem duplication but gained insertions of 1.7 and 3 kb in length in place of the Mu element. In each case, the inserted DNA is a sequence that normally flanks the distal repeat unit. Thus, each derivative consists of a tandem duplication in which the repeat unit has been extended at its distal end by the length of the new insertion. The 1.7-kb insertion dampens the phenotype, as did the original Mu insertion, whereas the 3-kb insertion completely suppresses the knotted phenotype. We propose that gene conversion, stimulated by the double-strand break of the Mu excision, gave rise to these derivatives.     

Lethal Transposition of Mud Phages in Rec(-) Strains of Salmonella Typhimurium

Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Identification and Characterization of a Novel Flagellum-Dependent Salmonella-Infecting Bacteriophage,iEPS5

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for iEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria.     

General method for the isolation of conditional lethal mutants in any required region of the virus genome: its application to the 'semi-essential' region of phage Mu

A general method is presented that enables viral mutants to be isolated in any required region of their genome. An application of the method is reported relating to the isolation of conditional lethal mutants in the "semi-essential' region of phage Mu. Some properties are described of one mutant found in this way, Mucts62SEEam8 Apl, which is typical of a class of mutants that were isolated.     

The cell wall receptor for bacteriophage Mu G(−) in Erwinia and Escherichia coli C

Abstract Adsorption of bacteriophage Mu with its invertible DNA segment in the G(−) orientation requires a terminal glucose residue for binding to the core lipopolysaccharide (LPS) of Gram-negative bacteria. Analysis of a Mu-resistant mutant shows that the receptor for Mu G(−) in Erwinia B374 is a Glc-β1,6-Glc disaccharide. A spontaneously occurring host-range mutant, Mu G(−)h101, grows on Escherichia coli C. The loss of the terminal β1,3-linked glucose from the LPS of E. coli C leads to resistance to the phage Mu. These mutants are also resistant to phage P1 and D108 which have largely homologous G segments. This shows that Mu G(+) and G(−) phage particles differ with respect to their cell-wall receptors in the type of glycosidic linkage of a terminal glucose residue: α1, 2 for G(+) and β1,6 for G(−).