Guy Duval-Valentin

Requirement of IS911 replication before integration defines a new bacterial transposition pathway

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut‐and‐paste and cointegrate formation (paste‐and‐copy). Cut‐and‐paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy‐and‐paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure‐eight) in which both ends are joined by a single‐strand bridge after a first‐strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure‐eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host‐specific replication pathway.

Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, pIRL, in the left end (IRL). Circle junction formation creates a strong promoter, pjunc, with a −35 sequence located in the right end and the −10 sequence in the left. pjunc assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in pjunc disassembly and a return to the low pIRL‐ driven transposase levels. We demonstrate that pjunc plays an important role in regulating IS911 transposition. Inactivation of pjunc strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess pjunc equivalents, underlining that the regulatory mechanisms adopted to fine‐tune transposition may be different.

Cotranslational Control of DNA Transposition: A Window of Opportunity

Transposable elements are important in genome dynamics and evolution. Bacterial insertion sequences (IS) constitute a major group in number and impact. Understanding their role in shaping genomes requires knowledge of how their transposition activity is regulated and interfaced with the host cell. One IS regulatory phenomenon is a preference of their transposases (Tpases) for action on the element from which they are expressed (cis) rather than on other copies of the same element (trans). Using IS911, we show in vivo that activity in cis was ~200 fold higher than in trans. We also demonstrate that a translational frameshifting pause signal influences cis preference presumably by facilitating sequential folding and cotranslational binding of the Tpase. In vitro, IS911 Tpase bound IS ends during translation but not after complete translation. Cotranslational binding of nascent Tpase permits tight control of IS proliferation providing a mechanistic explanation for cis regulation of transposition involving an unexpected partner, the ribosome.

Copy-out–Paste-in Transposition of IS911: A Major Transposition Pathway

IS911 has provided a powerful model for studying the transposition of members of a large class of transposable element: the IS3 family of bacterial Insertion Sequences (IS). These transpose by a Copy-out-Paste-in mechanism in which a double-strand IS circle transposition intermediate is generated from the donor site by replication and proceeds to integrate into a suitable double strand DNA target. This is perhaps one of the most common transposition mechanisms known to date. Copy-out-Paste-in transposition has been adopted by members of at least eight large IS families. This chapter details the different steps of the Copy-out-Paste-in mechanism involved in IS911 transposition. At a more biological level it also describes various aspects of regulation of the transposition process. These include transposase production by programmed translational frameshifting, transposase expression from the circular intermediate using a specialized promoter assembled at the circle junction and binding of the nascent transposase while it remains attached to the ribosome during translation (co-translational binding). This co-translational binding of the transposase to neighboring IS ends provides an explanation for the longstanding observation that transposases show a cis-preference for their activities.

Truncated forms of IS911 transposase downregulate transposition

IS911 naturally produces transposase (OrfAB) derivatives truncated at the C‐terminal end (OrfAB‐CTF) and devoid of the catalytic domain. A majority species, OrfAB*, was produced at higher levels at 42°C than at 30°C suggesting that it is at least partly responsible for the innate reduction in IS911 transposition activity at higher temperatures. An engineered equivalent of similar length, OrfAB[1–149], inhibited transposition activity in vivo or in vitro when produced along with full‐length transposase. We isolated several point mutants showing higher activity than the wild‐type IS911 at 42°C. These fall into two regions of the transposase. One, located in the N‐terminal segment of OrfAB, lies between or within two regions involved in protein multimerization. The other is located within the C‐terminal catalytic domain. The N‐terminal mutations resulted in reduced levels of OrfAB* while the C‐terminal mutation alone appeared not to affect OrfAB* levels. Combination of N‐ and C‐terminal mutations greatly reduced OrfAB* levels and transposition was concomitantly high even at 42°C. The mechanism by which truncated transposase species are generated and how they intervene to reduce transposition activity is discussed. While transposition activity of these multiply mutated derivatives in vivo was resistant to temperature, the purified OrfAB derivatives retained an inherent temperature‐sensitive phenotype in vitro. This clearly demonstrates that temperature sensitivity of IS911 transposition is a complex phenomenon with several mechanistic components. These results have important implications for the several other transposons and insertion sequences whose transposition has also been shown to be temperature‐sensitive.