Interferometric quantum control (IQC) by fs/ns rotational coherent anti-Stokes Raman spectroscopy (RCARS).

A new rotational coherent anti-Stokes Raman spectroscopy (RCARS) concept based on interferometric quantum control (IQC) is demonstrated. Two wavepackets originating from pure rotational states are created by a femtosecond stimulated rotational Raman interaction. The two Raman responses are instantly probed by a single-mode ns pulse generating two interfering RCARS polarizations. The resulting signal is an IQC-RCARS spectrum detected by a streak camera. Here we demonstrate IQC-interferograms of N2 by varying the temporal separation between the two fs pulses within a full rotational revival period, as well as signal amplification and selective detection of nuclear-spin isomers at room conditions and inside a flame.

E. coli allantoinase is activated by the downstream metabolic enzyme, glycerate kinase, and stabilizes the putative allantoin transporter by direct binding.

Allantoin is a good source of ammonium for many organisms, and in Escherichia coli it is utilized under anaerobic conditions. We provide evidence that allantoinase (AllB) is allosterically activated by direct binding of the allantoin catabolic enzyme, glycerate 2-kinase (GlxK) in the presence of glyoxylate. Glyoxylate is known to be an effector of the AllR repressor which regulates the allantoin utilization operons in E. coli. AllB has low affinity for allantoin, but its activation by GlxK leads to increased affinity for its substrate. We also show that the predicted allantoin transporter YbbW (re-named AllW) has allantoin specificity and the protein-protein interaction with AllB. Our results show that the AllB-dependent allantoin degradative pathway is subject to previously unrecognized regulatory mechanisms involving direct protein-protein interactions.

Single-shot coherent control of molecular rotation by fs/ns rotational coherent anti-Stokes Raman spectroscopy.

We present a novel method, to our knowledge, to control the shape of the spectra using 2-beam hybrid femtosecond (fs)/nanosecond (ns) coherent anti-Stokes Raman scattering (RCARS). The method is demonstrated experimentally and theoretically by utilizing a species-selective excitation approach via a field-free molecular alignment as an illustrative example. Two non-resonant fs laser pulses with proper delay selectively create and then annihilate N2 resonances in a binary mixture with O2 molecules. The RCARS signal is simultaneously resolved in spectral and temporal domains within a single-shot acquisition. The method requires very low pulse energies for excitation, hence minimizing multiphoton ionization probability, allowing for coherent control at various temperatures and pressures, with spectroscopic applications in non-stationary and unpredictable reacting flows.

Auxotrophic and prototrophic conditional genetic networks reveal the rewiring of transcription factors in Escherichia coli.

Bacterial transcription factors (TFs) are widely studied in Escherichia coli. Yet it remains unclear how individual genes in the underlying pathways of TF machinery operate together during environmental challenge. Here, we address this by applying an unbiased, quantitative synthetic genetic interaction (GI) approach to measure pairwise GIs among all TF genes in E. coli under auxotrophic (rich medium) and prototrophic (minimal medium) static growth conditions. The resulting static and differential GI networks reveal condition-dependent GIs, widespread changes among TF genes in metabolism, and new roles for uncharacterized TFs (yjdC, yneJ, ydiP) as regulators of cell division, putrescine utilization pathway, and cold shock adaptation. Pan-bacterial conservation suggests TF genes with GIs are co-conserved in evolution. Together, our results illuminate the global organization of E. coli TFs, and remodeling of genetic backup systems for TFs under environmental change, which is essential for controlling the bacterial transcriptional regulatory circuits.

Quantitative Genetic Screens for Mapping Bacterial Pathways and Functional Networks.

Escherichia coli synthetic genetic array (eSGA) screening procedure enables high-throughput systematic mapping of pairwise genetic interactions in E. coli. The eSGA method exploits E. coli's rapid growth, its ease of genetic manipulation, and efficient genetic exchange via conjugation. Replica pinning is used to grow and mate arrayed sets of single gene mutant strains as well as to select double mutants en masse. Strain fitness, which is the eSGA readout, is determined by the digital imaging of the plates and subsequent colony size measurements. Comparing single and double mutant colony sizes then allows for identifying interacting genes. Using eSGA on a global or a smaller process-centric scale can help reveal gene functions and reconstruct genetic interaction networks with known and novel connections between genes and pathways.

Laser-induced thermal grating spectroscopy based on femtosecond laser multi-photon absorption.

Laser-induced grating spectroscopy (LIGS) is for the first time explored in a configuration based on the crossing of two focused femtosecond (fs) laser pulses (800-nm wavelength) and a focused continuous-wave (cw) laser beam (532-nm wavelength). A thermal grating was formed by multi-photon absorption of the fs-laser pulses by [Formula: see text] with a pulse energy around 700 [Formula: see text]J ([Formula: see text] 45 TW/[Formula: see text]). The feasibility of this LIGS configuration was investigated for thermometry in heated nitrogen gas flows. The temperature was varied from room temperature up to 750 K, producing strong single-shot LIGS signals. A model based on the solution of the linearized hydrodynamic equations was used to extract temperature information from single-shot experimental data, and the results show excellent agreement with the thermocouple measurements. Furthermore, the fluorescence produced by the fs-laser pulses was investigated. This study indicates an 8-photon absorption pathway for [Formula: see text] in order to reach the [Formula: see text] state from the ground state, and 8 + 5 photon excitation to reach the [Formula: see text] state of the [Formula: see text] ion. At pulse energies higher than 1 mJ, the LIGS signal was disturbed due to the generation of plasma. Additionally, measurements in argon gas and air were performed, where the LIGS signal for argon shows lower intensity compared to air and [Formula: see text].

ZapG (YhcB/DUF1043), a novel cell division protein in gamma-proteobacteria linking the Z-ring to septal peptidoglycan synthesis.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

A Gaussian process-based definition reveals new and bona fide genetic interactions compared to a multiplicative model in the Gram-negative Escherichia coli.

MOTIVATION: A digenic genetic interaction (GI) is observed when mutations in two genes within the same organism yield a phenotype that is different from the expected, given each mutation's individual effects. While multiplicative scoring is widely applied to define GIs, revealing underlying gene functions, it remains unclear if it is the most suitable choice for scoring GIs in Escherichia coli. Here, we assess many different definitions, including the multiplicative model, for mapping functional links between genes and pathways in E.coli. RESULTS: Using our published E.coli GI datasets, we show computationally that a machine learning Gaussian process (GP)-based definition better identifies functional associations among genes than a multiplicative model, which we have experimentally confirmed on a set of gene pairs. Overall, the GP definition improves the detection of GIs, biological reasoning of epistatic connectivity, as well as the quality of GI maps in E.coli, and, potentially, other microbes. AVAILABILITY AND IMPLEMENTATION: The source code and parameters used to generate the machine learning models in WEKA software were provided in the Supplementary information. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chemical-genetic profiling reveals limited cross-resistance between antimicrobial peptides with different modes of action.

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.

The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC.

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the Khalf of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

Diode laser-based thermometry using two-line atomic fluorescence of indium and gallium.

A robust and relatively compact calibration-free thermometric technique using diode lasers two-line atomic fluorescence (TLAF) for reactive flows at atmospheric pressures is investigated. TLAF temperature measurements were conducted using indium and, for the first time, gallium atoms as temperature markers. The temperature was measured in a multi-jet burner running methane/air flames providing variable temperatures ranging from 1600 to 2000 K. Indium and gallium were found to provide a similar accuracy of ~ 2.7% and precision of ~ 1% over the measured temperature range. The reliability of the TLAF thermometry was further tested by performing simultaneous rotational CARS measurements in the same experiments.

Raman linewidth measurements using time-resolved hybrid picosecond/nanosecond rotational CARS.

We report an innovative approach for time-domain measurements of S-branch Raman linewidths using hybrid picosecond/nanosecond pure-rotational coherent anti-Stokes Raman spectroscopy (RCARS). The Raman coherences are created by two picosecond excitation pulses and are probed using a narrow-band nanosecond pulse at 532 nm. The generated RCARS signal contains the entire coherence decay in a single pulse. By extracting the decay times of the individual transitions, the J-dependent Raman linewidths can be calculated. Self-broadened S-branch linewidths for nitrogen and oxygen at 293 K and ambient pressure are in good agreement with previous time-domain measurements. Experimental considerations of the approach are discussed along with its merits and limitations. The approach can be extended to a wide range of pressures and temperatures and has potential for simultaneous single-shot thermometry and linewidth determination.

A comparative study of absorption in vertically and laterally oriented InP core-shell nanowire photovoltaic devices.

We have compared the absorption in InP core-shell nanowire p-i-n junctions in lateral and vertical orientation. Arrays of vertical core-shell nanowires with 400 nm pitch and 280 nm diameter, as well as corresponding lateral single core-shell nanowires, were configured as photovoltaic devices. The photovoltaic characteristics of the samples, measured under 1 sun illumination, showed a higher absorption in lateral single nanowires compared to that in individual vertical nanowires, arranged in arrays with 400 nm pitch. Electromagnetic modeling of the structures confirmed the experimental observations and showed that the absorption in a vertical nanowire in an array depends strongly on the array pitch. The modeling demonstrated that, depending on the array pitch, absorption in a vertical nanowire can be lower or higher than that in a lateral nanowire with equal absorption predicted at a pitch of 510 nm for our nanowire geometry. The technology described in this Letter facilitates quantitative comparison of absorption in laterally and vertically oriented core-shell nanowire p-i-n junctions and can aid in the design, optimization, and performance evaluation of nanowire-based core-shell photovoltaic devices.