A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac⁺ by prophage precise excision with a relatively high frequency (about 1×10⁻⁶). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced β-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.     

A novel illegitimate recombination event: precise excision and reintegration with the Mu gem mutant prophage

The bacteriophage Mu is known to insert its DNA more or less randomly within the Escherichia coli chromosome, as do transposable elements, but unlike the latter, precise excision of the prophage, thereby restoring the original sequence, is not observed with wild-type Mu, although it has been reported with certain defective mutants. We show here that the mutant prophage Mu gem2ts can excise precisely from at least three separate loci —malT, Iac and thyA (selected as Mal⁺, Lac⁺ and Thy⁺, respectively). This excision occurs under permissive conditions for phage development, is observed in fully immune (c⁺) lysogens, and is independent of RecA and of Mu transposase. Mu gemts2 excision is invariably accompanied by reintegration of a Mu gem2ts prophage elsewhere in the chromosome, in the case of Mal⁺ revertants, this prophage is systematically located at 94min on the E. coli chromosome. Mu gem2ts excision therefore sheds some light on the long-standing paradox of the lack of precise Mu excisio.     

Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

Carboxyl-terminal mutants of phage Mu transposase

A study of the properties of deletion mutants at the 3’ end ofA, the gene encoding the transposase protein of phage Mu, shows that the mutants are defective in the high-frequency non-replicative transposition observed early after Mu infection as well as the high-frequency replicative transposition observed during Mu lytic growth. They show near-normal levels of lysogenization, low frequency transposition and precise excision. The mutants behave as if they are “blind” to the presence of Mu B, a protein whose function is essential for the high frequency of both replicative and non-replicative Mudna transposition. We have sequenced these deletion mutants as well as the amber mutant A 7110 which is known to be defective in replicative transposition.A 7110 maps at the 3’ end of geneA. We suggest that the carboxyl-terminal region of the A-protein is involved in protein-protein interactions, especially with the B-protein. We also show in this study that mutations upstream of the Shine-Dalgarno sequence of geneA and within the preceding genener, perturb the synthesis of A-protein and that higher levels of A-protein cause an inhibition ofA activity.     

Mini-muduction: A new mode of gene transfer mediated by mini-Mu

Summary We compared the transducing properties of Mucts62 and Mucts62/mini-Mu lysates, using Mu immune and non immune Rec⁺ and recA recipient strains. The Mu/mini-Mu lysates transduced all bacterial markers tested 10 times more efficiently than the Mucts62 lysates in Rec⁺ recipients. Most of the transductants obtained after infection with the Mu/mini-Mu lysates result from the substitution of the mutated gene of the recipient by the wild type allele from the donor, most probably carried on the gigantic variable end linked to the mini-Mu genome.Moreover the Mu/mini-Mu lysates gave a new type of Rec-independent transduction that we called mini-muduction. Mini-muduction requires the activity of Mu gene A and provides transductants which carry the transduced marker surrounded by two mini-Mu genomes similarly oriented, and inserted at random location in the recipient chromosome. The mini-Mu/transduced DNA/mini-Mu structures are able to transpose spontaneously, for instance into a transmissible plasmid, in the presence of Mu gene A product.     

Fate of plasmids containing Mu DNA: chromosome association and mobilization

Summary The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially all of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase-and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.     

Mu transposable elements are structurally diverse and distributed throughout the genusZea

Summary The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of theMu family of transposable elements. All characterizedMu elements contain similar 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20–100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to theMu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously clonedMu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termedMu4 andMu5, have an organization characteristic of transposable elements and possess 200-bpMu terminal inverted repeats that flank internal DNA, which is unrelated to other clonedMu elements.Mu4 andMu5 are both flanked by 9-bp direct repeats as has been observed for otherMu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions ofMu4 andMu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally fromMu-similar termini. The distribution of these elements in maize lines and related species suggests thatMu elements are an ancient component of the maize genome. Moreover, the structure of theMu termini and the fact thatMu termini are found flanking different internal sequences leads us to speculate thatMu termini once may have been capable of transposing as independent entities.     

Synchronous division induced in Escherichia coli K12 by gemts mutants of phage Mu

Summary Infection with the bacteriophage mutant Mu c ⁺ gemts2 at 42° C induces synchrony in cell division in cultures of Escherichia coli K12. This synchrony may last for several cycles and is not only due to selection since synchronization is observed even when bacterial survival to the infection is over 80% as in lysogens for Mu c ⁺ gemts2. The mechanism by which sycnhrony is induced is not known, but since the product of Mu gene gem (previously called lig) has been shown to interact with the enzymatic system in the bacteria controlling the degree of DNA supercoiling, the phenomenon could be a consequence of this interaction.     

Bacteriophage Mu DNA replication is stimulated by non-essential early functions

Summary The replication of a spontaneous Kil^– mutant of bacteriophage Mu has been investigated. The Kil^– mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions. The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene.Mucts62-13/4 phages form extremely small plaques on wildtype indicator strains. The replication of the insertion mutant as compared to Mucts62 is strongly reduced. Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions. Apparently, early genes other than A and B are involved in normal Mu DNA replication.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.     

The timing of mu activity in maize

The timing of mutator activity of Mu in maize was tested in three ways: (1) by allelism tests of phenotypically similar male-transmitted mutants, (2) by studying the clustering of phenotypically similar mutants as demonstrated by ear maps and the subsequent allelism tests of these mutants, and (3) by the induction of somatic sectors in Mu plants heterozygous for plant and endosperm marker genes. Allelism tests of phenotypically similar mutants in outcrosses of Mu plants as males established that 18.6% were allelic and that premeiotic mutants are induced. This conclusion was supported by ear maps of Mu-bearing plants, which revealed sectors of seeds that produced plants bearing phenotypically similar allelic mutants. The smallness of these sectors indicated that the premeiotic activity of Mu that gave rise to them occurred very late. The lack of visible sectors in mature sporophytic, endosperm and aleurone tissue in plants carrying Mu supports the conclusion that the mutator activity of Mu does not occur throughout the ontogeny of the plant and seems to be restricted to a time shortly before and/or during meiosis.     

Mutator Transposon in Maize and MULEs in the Plant Genome

Mutator (Mu) is by far the most mutagenic plant transposon. The high frequency of transposition and the tendency to insert into low copy sequences for such transposon have made it the primary means by which genes are mutagenized in maize (Zea mays L.). Mus like elements (MULEs) are widespread among angiosperms and multiple-diverged functional variants can be present in a single genome. MULEs often capture genetic sequences. These Pack-MuLEs can mobilize thousands of gene fragments, which may have had a significant impact on host genome evolution. There is also evidence that MULEs can move between reproductively isolated species. Here we present an overview of the discovery, features and utility of Mu transposon. Classification of Mu elements and future directions of related research are also discussed. Understanding Mu will help us elucidate the dynamic genome.     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse

Background

Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain.

Methods

We evaluated BCG vaccine efficacy against challenge with ∼3×10⁵ M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly.

Principal Findings

BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice.

Conclusions

The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.     

The generation of Mutator transposable element subfamilies in maize

The mobile DNAs of the Mutator system of maize (Zea mays) are exceptional both in structure and diversity. So far, six subfamilies of Mu elements have been discovered; all Mu elements share highly conserved terminal inverted repeats (TIRs), but each sub-family is defined by internal sequences that are apparently unrelated to the internal sequences of any other Mu subfamily. The Mu1/Mu2 subfamily of elements was created by the acquisition of a portion of a standard maize gene (termed MRS-A) within two Mu TIRs. Beside the unusually long (185–359 bp) and diverse TIRs found on all of these elements, other direct and inverted repeats are often found either within the central portion of a Mu element or within a TIR.Our computer analyses have shown that sequence duplications (mostly short direct repeats interrupted by a few base pairs) are common in non-autonomous members of the Mutator, Ac/Ds, and Spm(En) systems. These duplications are often tightly associated with the element-internal end of the TIRs. Comparisons of Mu element sequences have indicated that they share more terminal components than previously reported; all subfamilies have at least the most terminal 215 bp, at one end or the other, of the 359-bp Mu5 TIR. These data suggest that many Mu element subfamilies were generated from a parental element that had termini like those of Mu5. With the Mu5 TIRs as a standard, it was possible to determine that elements like Mu4 could have had their unusual TIRs created through a three-step process involving (1) addition of sequences to interrupt one TIR, (2) formation of a stem-loop structure by one strand of the element, and (3) a subsequent DNA repair/gene conversion event that duplicated the insertion(s) within the other TIR. A similar repair/conversion extending from a TIR stem into loop DNA could explain the additional inverted repeat sequences added to the internal ends of the Mu4 and Mu7 TIRs. This same basic mechanism was found to be capable of generating new Mu element subfamilies. After endonucleolytic attack of the loop within the stem-loop structure, repair/conversion of the gap could occur as an intermolecular event to generate novel internal sequences and, therefore, a new Mu element subfamily. Evidence supporting and expanding this model of new Mu element subfamily creation was identified in the sequence of MRS-A.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

Analysis of the killing effect of Mu ligts mutants on host cells

Infection of Escherichia coli with the mutant lig ts2 of bacteriophage Mu at a temperature nonpermissive for this mutant is lethal for the host cells. This effect is insensitive to phage immunity of the host cells, to inhibitors of protein synthesis and is not suppressed in trans in bacterial strains producing the Lig⁺ active protein. These data suggest that the killing effect of this mutant is different from the other kil functions identified in Mu [1].