The IS200/IS605 Family and "Peel and Paste" Single-strand Transposition Mechanism.

This chapter presents an analysis of the organization and distribution of the IS200/IS605 family of insertion sequences (IS). Members of this family are widespread in both bacteria and archaea. They are unusual because they use obligatory single-strand DNA intermediates, which distinguishes them from classical IS. We summarize studies of the experimental model systems IS608 (from Helicobacter pylori) and ISDra2 (from Deinococcus radiodurans) and present biochemical, genetic, and structural data that describe their transposition pathway and the way in which their transposase (an HuH rather than a DDE enzyme) catalyzes this process. The transposition of IS200/IS605 family members can be described as a "Peel-and-Paste" mechanism. We also address the probable domestication of IS200/IS605 family transposases as enzymes involved in multiplication of repeated extragenic palindromes and as potential homing endonucleases in intron-IS chimeras.

A target specificity switch in IS911 transposition: the role of the OrfA protein.

The role played by insertion sequence IS911 proteins, OrfA and OrfAB, in the choice of a target for insertion was studied. IS911 transposition occurs in several steps: synapsis of the two transposon ends (IRR and IRL); formation of a figure-of-eight intermediate where both ends are joined by a single-strand bridge; resolution into a circular form carrying an IRR-IRL junction; and insertion into a DNA target. In vivo, with OrfAB alone, an IS911-based transposon integrated with high probability next to an IS911 end located on the target plasmid. OrfA greatly reduced the proportion of these events. This was confirmed in vitro using a transposon with a preformed IRR-IRL junction to examine the final insertion step. Addition of OrfA resulted in a large increase in insertion frequency and greatly increased the proportion of non-targeted insertions. The intermolecular reaction leading to targeted insertion may resemble the intramolecular reaction involving figure-of-eight molecules, which leads to the formation of circles. OrfA could, therefore, be considered as a molecular switch modulating the site-specific recombination activity of OrfAB and facilitating dispersion of the insertion sequence (IS) to 'non-homologous' target sites.

Promoter-specific involvement of the FixJ receiver domain in transcriptional activation.

The "two-component" FixLJ system activates nitrogen fixation genes via nifA and fixK in Sinorhizobium meliloti. Like other response regulators, the FixJ protein can be decomposed into an N-terminal phosphorylatable "receiver" domain FixJN and a C-terminal transcriptional activator domain FixJC. The FixJN receiver domain was known to regulate activity of FixJC negatively at the nifA promoter. Here we show a different situation at the fixK promoter where FixJN also contributes positively to transcriptional activation. This promoter-specific effect was mapped by alanine-scanning mutagenesis to the beta2 strand of the receiver domain. This interaction with FixJN is required for the recruitment of RNA polymerase at the fixK promoter by phosphorylated FixJ. Altogether the FixJ receiver domain appears to carry at least four functions, some of which can be separated by mutation: (1) autophosphorylation; (2) inhibition of FixJC; (3) dimerization; (4) transcriptional activation at pfixK. This example illustrates the formidable functional plasticity of receiver domains.

The role of tandem IS dimers in IS911 transposition.

Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR-IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.

Integrating DNA: transposases and retroviral integrases.

Transposable elements appear quite disparate in their organization and in the types of genetic rearrangements they promote. In spite of this diversity, retroviruses and many transposons of both prokaryotes and eukaryotes show clear similarities in the chemical reactions involved in their transposition. This is reflected in the enzymes, integrases and transposases, that catalyze these reactions and that are essential for the mobility of the elements. In this chapter, we examine the structure-function relationships between these enzymes and the different ways in which the individual steps are assembled to produce a complete transposition cycle.

IS911 transposon circles give rise to linear forms that can undergo integration in vitro.

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single-strand cleavage at one 3' transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single-strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut-and-paste mechanism. However, the linear species appeared not to be derived directly from the plasmid-based copy by direct double-strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single-strand cleavages at the 3' transposon ends to generate a linear transposon form with a 3'-OH and a three-nucleotide 5' overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.

Multiple oligomerisation domains in the IS911 transposase: a leucine zipper motif is essential for activity.

Structure-function relationships involved in oligomerisation of the transposase OrfAB of the bacterial insertion sequence IS911 have been investigated. Site-directed mutagenesis and sequential deletion coupled with immunoprecipitation have led to the definition of three regions of the protein capable of promoting multimerisation. These include a region predicted to assume a coiled-coil conformation, which is shown to be essential for activity, promoting correct multimerisation of the N-terminal domain of OrfAB and sequence-specific binding to the IS911 terminal inverted repeats mediated by this domain. This region presents the structural and functional characteristics of the leucine zipper motif described in eukaryotic proteins. The two other regions are located further towards the C-terminal end of the protein, adjacent to the leucine zipper and in the region that carries the conserved catalytic DD(35)E motif.

Efficient transposition of IS911 circles in vitro.

An in vitro system has been developed which supports efficient integration of transposon circles derived from the bacterial insertion sequence IS911. Using relatively pure preparations of IS911-encoded proteins it has been demonstrated that integration into a suitable target required both the transposase, OrfAB, a fusion protein produced by translational frameshifting between two consecutive open reading frames, orfA and orfB, and OrfA, a protein synthesized independently from the upstream orfA. Intermolecular reaction products were identified in which one or both transposon ends were used. The reaction also generated various intramolecular transposition products including adjacent deletions and inversions. The circle junction, composed of abutted left and right IS ends, retained efficient integration activity when carried on a linear donor molecule, demonstrating that supercoiling in the donor molecule is not necessary for the reaction. Both two-ended integration and a lower level of single-ended insertions were observed under these conditions. The frequency of these events depended on the spacing between the transposon ends. Two-ended insertion was most efficient with a natural spacing of 3 bp. These results demonstrate that transposon circles can act as intermediates in IS911 transposition and provide evidence for collaboration between the two major IS911-encoded proteins, OrfA and OrfAB.

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition.

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.

Structural organization of the Streptococcus pneumoniae chromosome and relatedness of penicillin-sensitive and -resistant strains in type 9V.

Fragmentation of Streptococcus pneumoniae genomic DNA with low-frequency-cleavage restriction endonucleases and separation of the fragments by field-inversion gel electrophoresis (FIGE) provides a DNA-fingerprint of a strain. This method enables us to construct a physical and genetic map of the R6 laboratory strain what will be presented. The origin of replication containing several Dna boxes was located in the dnaA region. It was of interest to compare the profiles of subclones. Two clones of strain R36A (R6 and C13) were cultivated separately for more than 15,000 generations in two laboratories. FIGE profiles differed by only one band. Another R36A descendant, isolated in 1958 by Ravin, strain Rx was of interest since it was deficient in Dpn restriction enzymes and methylases and in the hex B function. Its origin was questionable; its profile is identical to others R6 descendants, demonstrating that Rx is derived from R36A. FIGE analysis was carried out on several penicillin-resistant strains of type 9V because penicillin-resistance in this type increased recently. The profiles of a collection of a number of these resistant isolates were very similar, showing that they result from a clone. The profiles of penicillin sensitive isolates of the same type are very similar to the resistant isolates. This suggests that the 9V type has spread recently from a clone, and the resistance genes have mutated and were selected when penicillin was extensively used.

IS911-mediated transpositional recombination in vitro.

A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.