Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element.A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the Rprime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates our mutagenesis scheme. Mutants arising from Tn7 insertions outside of the genetic map have been isolated. Light harvesting II mutations have been isolated; one mutant lacks only the 14,000 M_r, polypeptide.     

Conferral of transposable properties to a chromosomal gene in Escherichia coli

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK⁺-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.     

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI

The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo, mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A.Proteins made in cells infected with the fin mutants were labelled with [³⁵S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique.gpA overproducers can also be isolated by selecting for λAam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called Aop and maps in the Nu1-A region. Aop is also a fin mutation, since its presence in λFI^? enables it to plate on non-permissive hosts.Therefore, it seems that one condition sufficient for normal growth of FI^? phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.     

Genetic evidence that GTP is required for transposition of IS903 and Tn552 in Escherichia coli

Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.     

Lambda CIN-1, a New Mutation Which Enhances Lysogenization by Bacteriophage Lambda, and the Genetic Structure of the Lambda CY Region

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm⁴³⁴ region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3–5 nucleotide pairs. Lying approximately 11–21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that λimm⁴³⁴ cin-1 cy2001 lysogenizes efficiently, but not λimm⁴³⁴ cin-1 cy2001 cII68 nor any other λimm⁴³⁴ cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.     

Isolation and properties of Tn10 insertions in the rac locus of Escherichia coli

Summary Two Tn10 insertions that are in the rac locus of the chromosome of Escherichia coli have been isolated and characterized. These insertions are located at min 29.7 and min 30.0. The insertions are stable when an F123 rac::Tn10 episome is transferred to an F^- rac ⁺ recipient, but they are lost at a high frequency when transferred to an F^- rac ^- recipient. This latter condition has been previously, demonstrated to cause the excision of the rac locus. The Tn10 insertions are also lost at a high frequency when strains containing them are lysogenized with reverse. If the lysogens that have lost the Tn10 insertion are subsequently cured of reverse, the cells no longer contain sequences homologous with rac locus DNA. These strains were rac ^- when tested for recombination activation (Low 1973), and this procedure consequently provides a simple means to make isogenic rac ^- and rac ^- strains.     

Control of lysogenization by phage P22 I. The P22 cro gene

P22 cro^? mutants were isolated as one class of phage P22 mutants (cly mutants) that have a very high frequeney of lysogeny relative to wild-type P22. These mutants: (1) do not form plaques and over-lysogenize relative to wild-type P22 after infection of a wild-type Salmonella host; (2) are defective in anti-immunity; and (3) fail to turn off high-level synthesis of P22 c2-repressor after infection.P22 cro^? mutations are recessive and map between the P22 c2 and c1 genes. P22 cro^? mutations are suppressed by clear-plaque mutations in the c1 gene, one of which is simultaneously cy^?. They are also suppressed, but incompletely, by mutations in the c2 (repressor) gene, especially those that do not completely abolish c2 gene function.Salmonella host mutants have been isolated that are permissive for the lytic growth of the P22 cro^? mutants.     

Bacteriophage Lambda as a Delivery Vector for Tn10-Derived Transposons in Xenorhabdus bovienii

Xenorhabdus bovienii wild-type strains lack a functional receptor protein (LamB) in the outer membrane and as a result are unable to adsorb coliphage lambda (λ). Introduction of plasmids encoding lamB into X. bovienii T228 results in constitutive expression of LamB in the outer membrane of this organism. LamB-expressing strains of X. bovienii adsorb lambda bacteriophage particles and can be used as hosts for lambda::Tn constructs. A Tn10-derived transposon, element 9 (J. C. Way, D. Davis, D. Morisato, D. E. Roberts, and N. Kleckner, Gene 32:369-379, 1984) was used to construct a variety of insertion mutants of X. bovienii. Mutants that had altered expression of protease, lipase, DNase, dye-binding capability, and hemolytic activity, in addition to a series of auxotrophic mutants, were isolated.     

Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Summary The transposition of Tn10 from the E. coli chromosome to pDU202 (a Tc^S deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency of 10^-7 in both rec ⁺ and recA hosts. Seventeen transfer deficient mutants of pDU202 caused by the insertion of Tn10 into the R factor's transfer genes were analysed. Insertion of Tn10 occurred at at least eight sites, with some apparent clustering in the second part of the transfer operon, and with equal numbers in each orientation. Both strongly and weakly polar insertions were observed, and the strength of the polarity was correlated with the Tn10 orientation. The map order of the second part of the transfer operon was confirmed as traC traF traH traG traS traD, analogous to that of F. The strongly polar insertion mutants still expressed traI at wild-type levels, providing further evidence that this gene does not belong to the transfer operon of R100.     

Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra⁺) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10^?8 indicating adequate stability of Tn10 mutations.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

A genetic analysis system of Burkholderia cepacia: construction of mobilizable transposons and a cloning vector

A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest. To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying Km^R and Tp^R genes. The plasmids have the origin of ColE1 replication and the mobilization gene of RP4. Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec). Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1. Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome. Thirteen protease-deficient mutants were also lipase deficient. An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, Ap^R and Cm^R genes, and several unique restriction sites for cloning. Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production.     

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa

Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp¹). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tp^r transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tp^r transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.     

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.     

Tn602: A naturally occurring relative of Tn903 with direct repeats

We report the characterization of Tn602, a transposon encoding resistance to kanamycin and related aminoglycosides present on the R-plasmid pGD10. Tn602 is highly homologous to the previously characterized Tn903, present on the R-plasmid R6, in that it consists of a gene for aminoglycoside-phosphotransferase-3'-I (homologous to that of Tn903) flanked by copies of an IS-element homologous to IS903. Tn602 differs from Tn903 in the following respects: the flanking IS-elements (IS602) are in direct rather than inverted orientation as in Tn903; the fusion points between the IS-elements and the central region are different from those in Tn903; and several sequence changes, detected by the loss and acquisition of restriction sites, show the two repeats of IS602 to be nonidentical and different from IS903, IS102, and IS903.B. These structural details suggest that Tn602 and Tn903 evolved separately from related modules.