Versatile modulators for laser-based FEL seeding at SwissFEL.

The Paul Scherrer Institute is implementing laser-based seeding in the soft X-ray beamline (Athos) of its free-electron laser, SwissFEL, to enhance the temporal and spectral properties of the delivered photon pulses. This technique requires, among other components, two identical modulators for coupling the electron beam with an external laser with a wavelength range between 260 and 1600 nm. The design, magnetic measurements results, alignment, operation and also details of the novel and exotic magnetic configuration of the prototype are described.

Demonstration of Large Bandwidth Hard X-Ray Free-Electron Laser Pulses at SwissFEL.

We have produced hard x-ray free-electron laser (FEL) radiation with unprecedented large bandwidth tunable up to 2%. The experiments have been carried out at SwissFEL, the x-ray FEL facility at the Paul Scherrer Institute in Switzerland. The bandwidth is enhanced by maximizing the energy chirp of the electron beam, which is accomplished by optimizing the compression setup. We demonstrate continuous tunability of the bandwidth with a simple method only requiring a quadrupole magnet. The generation of such broadband FEL pulses will improve the efficiency of many techniques such as x-ray crystallography and spectroscopy, opening the door to significant progress in photon science. It has already been demonstrated that the broadband pulses of SwissFEL are beneficial to enhance the performance of crystallography, and further SwissFEL users plan to exploit this large bandwidth radiation to improve the efficiency of their measurement techniques.

The SwissFEL soft X-ray free-electron laser beamline: Athos.

The SwissFEL soft X-ray free-electron laser (FEL) beamline Athos will be ready for user operation in 2021. Its design includes a novel layout of alternating magnetic chicanes and short undulator segments. Together with the APPLE X architecture of undulators, the Athos branch can be operated in different modes producing FEL beams with unique characteristics ranging from attosecond pulse length to high-power modes. Further space has been reserved for upgrades including modulators and an external seeding laser for better timing control. All of these schemes rely on state-of-the-art technologies described in this overview. The optical transport line distributing the FEL beam to the experimental stations was designed with the whole range of beam parameters in mind. Currently two experimental stations, one for condensed matter and quantum materials research and a second one for atomic, molecular and optical physics, chemical sciences and ultrafast single-particle imaging, are being laid out such that they can profit from the unique soft X-ray pulses produced in the Athos branch in an optimal way.

Ultrafast Structural Dynamics along the ß-γ Phase Transition Path in MnAs.

We investigate the orthorhombic distortion and the structural dynamics of epitaxial MnAs layers on GaAs(001) using static and time-resolved x-ray diffraction. Laser-induced intensity oscillations of Bragg reflections allow us to identify the optical phonon associated with orthorhombic distortion and to follow its softening along the path towards an undistorted phase of hexagonal symmetry. The frequency of this mode falls in the THz range, in agreement with recent calculations. Incomplete softening suggests that the ß-γ transformation deviates from a purely second-order displacive transition.

Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis.

UNLABELLED: AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients. IMPORTANCE: The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.

Interactive regulation by the Bacillus subtilis global regulators CodY and ScoC.

CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding.

PRESTO, the on-line photon energy spectrometer at FERMI: design, features and commissioning results.

Measurement of the emission wavelength and the spectral content of the photon radiation is essential information for both machine and experimental physicists at a free-electron laser (FEL) user facility. Knowledge of the photon beam spectral properties is needed during the machine optimization and for performing machine studies (i.e. monitoring the change of the FEL output as a function of the machine parameters). The experimentalists, on the other hand, need to know the photon beam spectral distribution of the source, shot to shot, to discriminate the acquired data. Consequently, the main requirement for the instrument, supposed to obtain this information, is the capability of working on-line and shot-to-shot, with minimal perturbation of the beam delivered to the experimental stations. Starting from the grating fundamental equations, the conceptual design of the FERMI Pulse-Resolved Energy Spectrometer: Transparent and On-line (PRESTO) is presented, explaining the optical design in detail. The performance of PRESTO, in terms of resolving power, efficiency and spectral response, is also discussed. Finally, some useful features beyond the usual measurement of the energy spectrum are reported, as they have been routinely used by both machine and experimental physicists.

Generation of Phase-Locked Pulses from a Seeded Free-Electron Laser.

In a coherent control experiment, light pulses are used to guide the real-time evolution of a quantum system. This requires the coherence and the control of the pulses' electric-field carrier waves. In this work, we use frequency-domain interferometry to demonstrate the mutual coherence of time-delayed pulses generated by an extreme ultraviolet seeded free-electron laser. Furthermore, we use the driving seed laser to lock and precisely control the relative phase between the two free-electron laser pulses. This new capability opens the way to a multitude of coherent control experiments, which will take advantage of the high intensity, short wavelength, and short duration of the pulses generated by seeded free-electron lasers.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

UNLABELLED: CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE: CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

Spectrotemporal shaping of seeded free-electron laser pulses.

We demonstrate the ability to control and shape the spectrotemporal content of extreme-ultraviolet (XUV) pulses produced by a seeded free-electron laser (FEL). The control over the spectrotemporal properties of XUV light was achieved by precisely manipulating the linear frequency chirp of the seed laser. Our results agree with existing theory, which allows us to retrieve the temporal properties (amplitude and phase) of the FEL pulse from measurements of the spectra as a function of the FEL operating parameters. Furthermore, we show the first direct evidence of the full temporal coherence of FEL light and generate Fourier limited pulses by fine-tuning the FEL temporal phase. The possibility of tailoring the spectrotemporal content of intense short-wavelength pulses represents the first step towards efficient nonlinear optics in the XUV to x-ray spectral region and will enable precise manipulation of core-electron excitations using the methods of coherent quantum control.

Two-colour generation in a chirped seeded free-electron laser: a close look.

We present the experimental demonstration of a method for generating two spectrally and temporally separated pulses by an externally seeded, single-pass free-electron laser operating in the extreme-ultraviolet spectral range. Our results, collected on the FERMI@Elettra facility and confirmed by numerical simulations, demonstrate the possibility of controlling both the spectral and temporal features of the generated pulses. A free-electron laser operated in this mode becomes a suitable light source for jitter-free, two-colour pump-probe experiments.

An X-ray free-electron laser with a highly configurable undulator and integrated chicanes for tailored pulse properties.

X-ray free-electron lasers (FELs) are state-of-the-art scientific tools capable to study matter on the scale of atomic processes. Since the initial operation of X-ray FELs more than a decade ago, several facilities with upgraded performance have been put in operation. Here we present the first lasing results of Athos, the soft X-ray FEL beamline of SwissFEL at the Paul Scherrer Institute in Switzerland. Athos features an undulator layout based on short APPLE-X modules providing full polarisation control, interleaved with small magnetic chicanes. This versatile configuration allows for many operational modes, giving control over many FEL properties. We show, for example, a 35% reduction of the required undulator length to achieve FEL saturation with respect to standard undulator configurations. We also demonstrate the generation of more powerful pulses than the ones obtained in typical undulators. Athos represents a fundamental step forward in the design of FEL facilities, creating opportunities in FEL-based sciences.

Identification of a Novel Brevibacillus laterosporus Strain With Insecticidal Activity Against Aedes albopictus Larvae.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.

The twin-arginine signal peptide of Bacillus subtilis YwbN can direct either Tat- or Sec-dependent secretion of different cargo proteins: secretion of active subtilisin via the B. subtilis Tat pathway.

Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis alpha-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.

Widely tunable two-colour seeded free-electron laser source for resonant-pump resonant-probe magnetic scattering.

The advent of free-electron laser (FEL) sources delivering two synchronized pulses of different wavelengths (or colours) has made available a whole range of novel pump-probe experiments. This communication describes a major step forward using a new configuration of the FERMI FEL-seeded source to deliver two pulses with different wavelengths, each tunable independently over a broad spectral range with adjustable time delay. The FEL scheme makes use of two seed laser beams of different wavelengths and of a split radiator section to generate two extreme ultraviolet pulses from distinct portions of the same electron bunch. The tunability range of this new two-colour source meets the requirements of double-resonant FEL pump/FEL probe time-resolved studies. We demonstrate its performance in a proof-of-principle magnetic scattering experiment in Fe-Ni compounds, by tuning the FEL wavelengths to the Fe and Ni 3p resonances.

Chirped pulse amplification in an extreme-ultraviolet free-electron laser.

Chirped pulse amplification in optical lasers is a revolutionary technique, which allows the generation of extremely powerful femtosecond pulses in the infrared and visible spectral ranges. Such pulses are nowadays an indispensable tool for a myriad of applications, both in fundamental and applied research. In recent years, a strong need emerged for light sources producing ultra-short and intense laser-like X-ray pulses, to be used for experiments in a variety of disciplines, ranging from physics and chemistry to biology and material sciences. This demand was satisfied by the advent of short-wavelength free-electron lasers. However, for any given free-electron laser setup, a limit presently exists in the generation of ultra-short pulses carrying substantial energy. Here we present the experimental implementation of chirped pulse amplification on a seeded free-electron laser in the extreme-ultraviolet, paving the way to the generation of fully coherent sub-femtosecond gigawatt pulses in the water window (2.3-4.4 nm).

Single-shot spectro-temporal characterization of XUV pulses from a seeded free-electron laser.

Intense ultrashort X-ray pulses produced by modern free-electron lasers (FELs) allow one to probe biological systems, inorganic materials and molecular reaction dynamics with nanoscale spatial and femtoscale temporal resolution. These experiments require the knowledge, and possibly the control, of the spectro-temporal content of individual pulses. FELs relying on seeding have the potential to produce spatially and temporally fully coherent pulses. Here we propose and implement an interferometric method, which allows us to carry out the first complete single-shot spectro-temporal characterization of the pulses, generated by an FEL in the extreme ultraviolet spectral range. Moreover, we provide the first direct evidence of the temporal coherence of a seeded FEL working in the extreme ultraviolet spectral range and show the way to control the light generation process to produce Fourier-limited pulses. Experiments are carried out at the FERMI FEL in Trieste.

The fate of extracellular proteins tagged by the SsrA system of Bacillus subtilis.

In bacteria, SsrA, a highly conserved RNA molecule, functions in a mechanism meant to rescue stalled ribosomes. In this process, a peptide tag encoded by SsrA is cotranslationally added to truncated polypeptides, thereby targeting these molecules for proteolytic degradation, at least when they stay inside the cell. This study examined the fate of two extracellular proteins that were tagged by the SsrA system of Bacillus subtilis. Gene constructs encoding human interleukin-3 (hIL-3) fused to a signal peptide and B. subtilis alpha-amylase, both lacking an in-frame stop codon, were used as models to achieve ribosome stalling and activation of the SsrA system. Introduction of these gene constructs into B. subtilis led to tagging of the gene products by SsrA RNA. The tagged protein products bound to antibodies that were raised against the proteolysis tag encoded by B. subtilis SsrA [(A)GKTNSFNQNVALAA]. The apolar C-terminal SsrA-tag does not function as a specific signal for proteolytic degradation of SsrA-tagged amylase directly after trans-translation or during the secretion process. Also, SsrA-tagged amylase appeared to be very stable once outside the cell. In contrast, hIL-3 molecules tagged with the native, apolar SsrA-tag were considerably less stable than hIL-3 molecules that received a negatively charged control tag. Not one specific protease, but several non-specific proteases seem to play a role in the rapid degradation of SsrA-tagged hIL-3. The polarity of the C-terminus of heterologous hIL-3 protein proved to be an important determinant for protein stability when produced by B. subtilis. As observed previously in Escherichia coli and B. subtilis, SsrA tagging also occurs frequently in normally growing Gram-positive bacilli and it appears that intracellular proteins are the predominant natural substrates of SsrA.

A novel member of the subtilisin-like protease family from Bacillus subtilis.

aprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins. Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase. The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes. aprX is not essential for either growth or sporulation.

Quantitative proteome profiling during the fermentation process of pleiotropic Bacillus subtilis mutants.

Using a combined quantitative proteomic and bioinformatic approach, we monitored the cytoplasmic proteome profile of the Gram-positive bacterium Bacillus subtilis during a fermentation process in complex medium. Proteome signatures were applied to elucidate the physiological changes occurring in the gene expression profile during growth. Furthermore, we determined the significance level of quantitative proteome changes, identified relative to the threshold of scatter in replicated samples and developed a statistically rigorous method that allowed us to determine significant fold-changes at 95% confidence between different proteomes. Different functional groups of proteins were clustered according to their pattern of significant expression changes. The largest group is induced by the exhaustion of glucose and the presence of alternative carbon and nitrogen sources. Furthermore, depletion of glucose caused the induction of the trichloroacetic acid (TCA) cycle enzymes and the downregulation of glycolytic enzymes. The onset of the transition phase may be provoked by amino acid starvation, resulting in the RelA-dependent repression of proteins involved in the translation process and in the induction of amino acid biosynthetic pathways. Comparisons between the parental strain and two subtilisin-hypersecreting strains revealed only small cytoplasmic differences in the main metabolic pathways. Instead, the overproduction of degradative enzymes in both of these mutants was reflected in the extracellular proteome.