Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.     

Phase variation: genetic analysis of switching mutants

Site-specific inversion of a controlling element is responsible for flagellar phase transition in Salmonella. When a 900 bp DNA sequence is in one configuration, it allows the expression of the H2 gene, a structural gene which codes for the flagellar antigen. When it is in the opposite configuration, the H2 gene is not expressed. A hybrid λ phage containing the invertible control region and the adjacent H2 gene was constructed, and expression of the H2 gene was shown to be regulated by the orientation of the inversion region. Transposon Tn5 insertion derivatives of this hybrid phage were isolated and λH2::Tn5 mutants defective for inversion (H2 switching) were selected and characterized. Two classes of switching phenotypes were observed among the mutants—those which had slightly reduced frequencies of transition from expression of the H2 gene (H2 on) to nonexpression (H2 off) (intermediate class) and those in which the frequency of transition was reduced at least three orders of magnitude (null class). Physical mapping of the Tn5 insertion sites revealed that in all mutants the insertion was located inside the inversion region. Tn5 insertion sites in the null class of mutants defined a region of DNA including approximately 500 bp which was necessary for inversion. Genetic complementation tests showed that these λH2::Tn5 mutants could invert the H2 gene control element if the 500 bp region was introduced in the trans configuration. It is concluded that a gene is located inside the inversion segment and codes for a protein which is required for the inversion event. Furthermore, the two sites at which the crossover event occurred functioned in a cis configuration and were required for inversion. The presence of a gene which is involved in controlling site-specific recombination events may be a general feature of transposon-like elements.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

Mapping by Transposons of the Inversion Termini in Escherichia Coli K-12 Strain 1485in

Strain 1485IN carries a chromosomal inversion which corresponds to 35% of the chromosome and includes proC, trp and his genes. The termini of the inversion lie between the lac and proC loci and between his and cdd of the normal strain. Using Tn10 and Tn5 in transduction crosses between the normal and inversion strains, the termini were mapped to sites located approximately 0.25 min and 1.6 min away from proC and his, respectively within a region of roughly 4 kb long. The crosses where the normal strains carrying Tn10 near the terminus are donors and the inversion strain is a recipient, yielded unusual Tetr His- recombinants, which arose from illegitimate recombination leading to the replacement of a chromosomal his+ region with a transducing fragment carrying proC. Another rearrangement was detected between the normal and inversion strains in a region outside the inverted segment near the cdd locus.     

Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12

We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.     

Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses.

A mutant defective in aciduricity, GS5Tn1, was constructed following mutagenesis of Streptococcus mutans GS5 with the conjugative transposon Tn916. The mutant grew poorly at acidic pH levels and was sensitive to high osmolarity and elevated temperatures. These properties resulted from a single insertion of Tn916 into the GS5 chromosome, and the DNA fragment harboring the transposon was isolated into the cosmid vector, charomid 9-20. Spontaneous excision of Tn916 from the cosmid revealed that Tn916 inserted into a 8.6-kb EcoRI fragment. On the basis of the restriction analyses of insert fragments, it was found that Tn916 inserted into a 0.9-kb EcoRI-XbaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames, ORF1 and ORF2. By using a marker rescue strategy, a 6.0-kb HindIII fragment including the target site for Tn916 insertion and the 5' end of ORF1 was isolated and sequenced. The deduced amino acid sequences of ORF1 and ORF2 showed significant homology with the diacylglycerol kinase and Era proteins, respectively, from Escherichia coli. Nucleotide sequence analysis of the Tn916 insertion junction region in the GS5Tn1 chromosome revealed that the transposon inserted near the 3' terminus of ORF1. Restoration of ORF1 to its original sequence in mutant GS5Tn1 was carried out following transformation with integration vector pVA891 containing an intact ORF1. The resultant transformant showed wild-type levels of aciduricity as well as resistance to elevated temperatures and high osmolarity. These results suggest that the S. mutans homolog of diacylglycerol kinase is important for adaptation of the organism to several environmental stress signals.     

Inversions and deletions of the Salmonella chromosome generated by the translocatable tetracycline resistance element Tn10

Spontaneous tetraoyoline-sensitive derivatives of a Tn10 insertion in the hisG gene of Salmonella typhimurium were isolated and subjected to genetic analysis. All 123 of the drug-sensitive derivatives characterized have undergone stable alterations in the Tn10 element itself; over half of the derivatives have also undergone major alterations of neighboring regions of the Salmonella chromosome. These chromosomal rearrangements are of two types: inversions and deletions. Any single inversion or deletion affects a contiguous stretch of chromosomal material extending from the site of the original Tn10 element either leftward or rightward.The genetic properties of deletion and inversion derivatives suggest that these chromosomal alterations are promoted by the Tn10 element itself. The role of translocatable elements in promoting chromosomal deletions is well documented; the ability of an element to promote inversions of chromosomal material has not previously been reported. Possible analogies between the 1400-base-pair inverted repetition at the end of Tn10 and the small insertion sequence IS1 predict particular structures for Tn10-promoted deletions. A structural explanation or model for Tn10-promoted inversions is presented. The observation that Tn10 promotes the formation of inversions suggests that such elements could play a previously unanticipated role in promoting chromosomal inversions during evolution of prokaryotic organisms. Generally applicable genetic methods for the identification and characterization of chromosomal inversions are described.     

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.     

Characterization of the staphylococcal beta-lactamase transposon Tn552.

The staphylococcal beta-lactamase transposon Tn552 is a member of a novel group of transposable elements. The organization of genes in Tn552 resembles that of members of the Tn21 sub-group of Tn3 family transposons, which transpose replicatively by cointegrate formation and resolution. Thus, a possible resolution site ('resL') and a resolvase gene (tnpR or 'binL') have been identified. However, consistent with the fact that Tn552 generates 6 bp (rather than 5 bp) flanking direct repeats of target DNA, neither the putative transposase protein, nor the terminal inverted repeats of Tn552 are homologous to those of Tn3 elements. Tn552, like phage Mu and retroelements, is defined by the terminal dinucleotides 5' TG .. CA 3'. A naturally occurring staphylococcal plasmid, pI9789, contains a Tn552-derived resolution system ('resR-binR') that acts as a 'hotspot' for Tn552 transposition; insertion creates a segment of DNA flanked by inversely repeated resolution sites, one (resR) on pI9789 and the other (resL) on Tn552. The putative Tn552 resolvase, the most closely related of known resolvases to the homologous DNA invertases, initially was identified as a DNA invertase ('Bin') as a result of its ability to mediate efficient inversion of this segment in vivo.     

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.     

A high-throughput assay for Tn5 Tnp-induced DNA cleavage

Transposition causes genomic instability by mobilizing DNA elements. This phenomenon is mechanistically related to other DNA rearrangements, such as V(D)J recombination and retroviral DNA integration. A conserved active site architecture within the transposase/integrase superfamily catalyzes these distinct phenomena. The Tn5 transposase (Tnp) falls within this protein class, and many intermediates of the Tn5 transposition reaction have been characterized. Here, we describe a method for the rapid identification of Tn5 Tnp small molecule effectors. This high-throughput screening strategy will aid in the identification of compounds that perturb Tnp-induced DNA cleavage. This method is advantageous, since it identifies effectors that specifically inhibit catalysis without inhibiting Tnp-DNA binding interactions. Effectors identified using this method will serve as a valuable aid both in the isolation and characterization of metal-bound reaction intermediates and in co-crystallization studies involving the effector, Tnp and DNA, to identify the structural basis of the interaction. Furthermore, since Tn5 Tnp shares a similar active site architecture to other transposase/integrase superfamily members, this strategy and any effectors identified using this method will be readily applicable to these other systems.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.     

Absence of direct repeats flanking transposons resulting from intramolecular transposition

Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3. The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660. In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation. In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.     

Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.