Philippe Rousseau

Conformational changes induced by phosphorylation of the FixJ receiver domain.

BACKGROUND A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways. In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily. The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function. The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain. In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes. RESULTS The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 A and 2.4 A resolution, respectively. They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator. Phosphorylation of the conserved aspartate induces major structural changes in the beta 4-alpha 4 loop, and in the signaling surface alpha 4-beta 5 that mediates dimerization of the phosphorylated full-length response regulator. A site-directed mutant at this protein-protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold. CONCLUSIONS The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

Phosphorylation‐induced dimerization of the FixJ receiver domain

The ‘two‐component’ transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation‐induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second‐order reactions with respect to protein concentration. However, the second‐order phosphorylation constant is 44‐fold higher for FixJN than for FixJ. Therefore, the C‐terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity ≈ 40‐fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ~P dimerization interface involves Val‐91 and Lys‐95 in helix α4. Dimerization was required for high‐affinity binding to fixK promoter DNA.

Drift Removal Procedures in the Analysis of Electrochemical Noise

Abstract Removing direct current (DC) trends before calculating standard deviations and power spectral densities is a necessary operation, but the choice of method is probably one of the most diffi...

High-throughput single-molecule analysis of DNA–protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA–protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.

Frequency Analysis of Transients in Electrochemical Noise: Mathematical Relationships and Computer Simulations

Abstract The relationship between electrochemical noise (EN) time records and the power spectral densities (PSD) derived from them was examined. It was shown how the PSD of EN consisting of a serie...

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.

TPM analyses reveal that FtsK contributes both to the assembly and the activation of the XerCD-dif recombination synapse

Circular chromosomes can form dimers during replication and failure to resolve those into monomers prevents chromosome segregation, which leads to cell death. Dimer resolution is catalysed by a highly conserved site-specific recombination system, called XerCD-dif in Escherichia coli. Recombination is activated by the DNA translocase FtsK, which is associated with the division septum, and is thought to contribute to the assembly of the XerCD-dif synapse. In our study, direct observation of the assembly of the XerCD-dif synapse, which had previously eluded other methods, was made possible by the use of Tethered Particle Motion, a single molecule approach. We show that XerC, XerD and two dif sites suffice for the assembly of XerCD-dif synapses in absence of FtsK, but lead to inactive XerCD-dif synapses. We also show that the presence of the γ domain of FtsK increases the rate of synapse formation and convert them into active synapses where recombination occurs. Our results represent the first direct observation of the formation of the XerCD-dif recombination synapse and its activation by FtsK.

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.

Control of IS911 target selection: how OrfA may ensure IS dispersion

IS911 transposition involves a closed circular insertion sequence intermediate (IS‐circle) and two IS‐encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N‐terminal region with OrfA. This includes a helix–turn–helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non‐specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.