Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'-N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

Magnetic Breakdown and Topology in the Kagome Superconductor CsV_{3}Sb_{5} under High Magnetic Field.

The recently discovered layered kagome metals of composition AV_{3}Sb_{5} (A=K, Rb, Cs) exhibit a complex interplay among superconductivity, charge density wave order, topologically nontrivial electronic band structure and geometrical frustration. Here, we probe the electronic band structure underlying these exotic correlated electronic states in CsV_{3}Sb_{5} with quantum oscillation measurements in pulsed fields up to 86 T. The high-field data reveal a sequence of magnetic breakdown orbits that allows the construction of a model for the folded Fermi surface of CsV_{3}Sb_{5}. The dominant features are large triangular Fermi surface sheets that cover almost half the folded Brillouin zone. These sheets have not yet been detected in angle resolved photoemission spectroscopy and display pronounced nesting. The Berry phases of the electron orbits have been deduced from Landau level fan diagrams near the quantum limit without the need for extrapolations, thereby unambiguously establishing the nontrivial topological character of several electron bands in this kagome lattice superconductor.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.

Escherichia coli K-12 has two distinguishable PriA-PriB replication restart pathways.

In Escherichia coli, PriA, PriB, PriC, and DnaT proteins mediate three pathways for Replication Restart called PriA-PriB, PriA-PriC, and PriC. PriA is crucial for two of the three pathways. Its absence leads to slow growth, high basal levels of SOS expression, poorly partitioning nucleoids, UV sensitivity, and recombination deficiency. PriA has ATPase and helicase activities and interacts with PriB, DnaT, and single-stranded DNA-binding protein (SSB). priA300 (K230R) and priA301 (C479Y) have no phenotype as single mutants, but each phenocopy a priA-null mutant combined with ∆priB. This suggested that the two priA mutations affected the helicase activity that is required for the PriA-PriC pathway. To further test this, the biochemical activities of purified PriA300 and PriA301 were examined. As expected, PriA300 lacks ATPase and helicase activities but retains the ability to interact with PriB. PriA301, however, retains significant PriB-stimulated helicase activity even though PriA301 interactions with PriB and DNA are weakened. A PriA300,301 variant retains only the ability to interact with DNA in vitro and phenocopies the priA-null phenotype in vivo. This suggests that there are two biochemically and genetically distinct PriA-PriB pathways. One uses PriB-stimulated helicase activity to free a region of ssDNA and the other uses helicase-independent remodeling activity.

Dynamics of Proteins and Macromolecular Machines in Escherichia coli.

Proteins are major contributors to the composition and the functions in the cell. They often assemble into larger structures, macromolecular machines, to carry out intricate essential functions. Although huge progress in understanding how macromolecular machines function has been made by reconstituting them in vitro, the role of the intracellular environment is still emerging. The development of fluorescence microscopy techniques in the last 2 decades has allowed us to obtain an increased understanding of proteins and macromolecular machines in cells. Here, we describe how proteins move by diffusion, how they search for their targets, and how they are affected by the intracellular environment. We also describe how proteins assemble into macromolecular machines and provide examples of how frequent subunit turnover is used for them to function and to respond to changes in the intracellular conditions. This review emphasizes the constant movement of molecules in cells, the stochastic nature of reactions, and the dynamic nature of macromolecular machines.

Disorder raises the critical temperature of a cuprate superconductor.

With the discovery of charge-density waves (CDWs) in most members of the cuprate high-temperature superconductors, the interplay between superconductivity and CDWs has become a key point in the debate on the origin of high-temperature superconductivity. Some experiments in cuprates point toward a CDW state competing with superconductivity, but others raise the possibility of a CDW-superconductivity intertwined order or more elusive pair-density waves (PDWs). Here, we have used proton irradiation to induce disorder in crystals of [Formula: see text] and observed a striking 50% increase of [Formula: see text], accompanied by a suppression of the CDWs. This is in sharp contrast with the behavior expected of a d-wave superconductor, for which both magnetic and nonmagnetic defects should suppress [Formula: see text] Our results thus make an unambiguous case for the strong detrimental effect of the CDW on bulk superconductivity in [Formula: see text] Using tunnel diode oscillator (TDO) measurements, we find indications for potential dynamic layer decoupling in a PDW phase. Our results establish irradiation-induced disorder as a particularly relevant tuning parameter for the many families of superconductors with coexisting density waves, which we demonstrate on superconductors such as the dichalcogenides and [Formula: see text].

Function of a strand-separation pin element in the PriA DNA replication restart helicase.

DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a ß-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the ß-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA ß-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted ß-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.

Skyrmion Lattice Topological Hall Effect near Room Temperature.

Magnetic skyrmions are stable nanosized spin structures that can be displaced at low electrical current densities. Because of these properties, they have been proposed as building blocks of future electronic devices with unprecedentedly high information density and low energy consumption. The electrical detection of an ordered skyrmion lattice via the Topological Hall Effect (THE) in a bulk crystal, has so far been demonstrated only at cryogenic temperatures in the MnSi family of compounds. Here, we report the observation of a skyrmion lattice Topological Hall Effect near room temperature (276 K) in a mesoscopic lamella carved from a bulk crystal of FeGe. This region coincides with the skyrmion lattice location revealed by neutron scattering. We provide clear evidence of a re-entrant helicoid magnetic phase adjacent to the skyrmion phase, and discuss the large THE amplitude (5 nΩ.cm) in view of the ordinary Hall Effect.

Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase.

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

A priA Mutant Expressed in Two Pieces Has Almost Full Activity in Escherichia coli K-12.

The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par-), UV sensitivity, and recombination deficiency (Rec-). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell.IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3' DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen.

Porous Coordination Polymer Based on Bipyridinium Carboxylate Linkers with High and Reversible Ammonia Uptake.

The zwitterionic bipyridinium carboxylate ligand 1,1'-bis(4-carboxyphenyl)-4,4'-bipyridinium (pc1) in the presence of cadmium chloride affords novel porous coordination polymers (PCPs): [Cd4(pc1)3Cl6]·CdCl4·guest (1) crystallizing in the P3̅1c space group. In the structure, [Cd4Cl6(CO2)6] building units are linked together by six pc1 ligands, leading to a 3D high-symmetrical network exhibiting hexagonal channels along the c axis. The walls of this PCP consist of cationic electron-acceptor bipyridinium units. The PCP 1 reversibly adsorbs H2O and CH3OH up to about 0.1 g/g at saturation showing the adsorption isotherms characteristic of a moderately hydrophilic sorbent. Adsorption of ammonia (NH3) follows a different pattern, reaching an exceptional uptake of 0.39 g/g (22.3 mmol/g) after the first adsorption cycle. Although the crystalline structure of 1 collapses after the first adsorption, the solid can be regenerated and maintains the capacity of 0.29 g/g (17 mmol/g) in the following cycles. We found that the high NH3 uptake is due to a combination of pore filling taking place below 150 h·Pa and chemisorption occurring at higher pressures. The latter process was shown to involve two phenomena: (i) coordination of NH3 molecules to Cd(2+) cations as follows from (113)Cd NMR and (ii) strong donor-acceptor interactions between NH3 molecules and pc1 ligands.

Structure and Function of the PriC DNA Replication Restart Protein.

Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks.

Toward Superconducting Critical Current by Design.

A new critical-current-by-design paradigm is presented. It aims at predicting the optimal defect landscape in superconductors for targeted applications by elucidating the vortex dynamics responsible for the bulk critical current. To this end, critical current measurements on commercial high-temperature superconductors are combined with large-scale time-dependent Ginzburg-Landau simulations of vortex dynamics.

Statistical distance as a measure of physiological dysregulation is largely robust to variation in its biomarker composition.

Physiological dysregulation may underlie aging and many chronic diseases, but is challenging to quantify because of the complexity of the underlying systems. Recently, we described a measure of physiological dysregulation, DM, that uses statistical distance to assess the degree to which an individual's biomarker profile is normal versus aberrant. However, the sensitivity of DM to details of the calculation method has not yet been systematically assessed. In particular, the number and choice of biomarkers and the definition of the reference population (RP, the population used to define a "normal" profile) may be important. Here, we address this question by validating the method on 44 common clinical biomarkers from three longitudinal cohort studies and one cross-sectional survey. DMs calculated on different biomarker subsets show that while the signal of physiological dysregulation increases with the number of biomarkers included, the value of additional markers diminishes as more are added and inclusion of 10-15 is generally sufficient. As long as enough markers are included, individual markers have little effect on the final metric, and even DMs calculated from mutually exclusive groups of markers correlate with each other at r~0.4-0.5. We also used data subsets to generate thousands of combinations of study populations and RPs to address sensitivity to differences in age range, sex, race, data set, sample size, and their interactions. Results were largely consistent (but not identical) regardless of the choice of RP; however, the signal was generally clearer with a younger and healthier RP, and RPs too different from the study population performed poorly. Accordingly, biomarker and RP choice are not particularly important in most cases, but caution should be used across very different populations or for fine-scale analyses. Biologically, the lack of sensitivity to marker choice and better performance of younger, healthier RPs confirm an interpretation of DM physiological dysregulation and as an emergent property of a complex system.

Detection of a novel, integrative aging process suggests complex physiological integration.

Many studies of aging examine biomarkers one at a time, but complex systems theory and network theory suggest that interpretations of individual markers may be context-dependent. Here, we attempted to detect underlying processes governing the levels of many biomarkers simultaneously by applying principal components analysis to 43 common clinical biomarkers measured longitudinally in 3694 humans from three longitudinal cohort studies on two continents (Women's Health and Aging I & II, InCHIANTI, and the Baltimore Longitudinal Study on Aging). The first axis was associated with anemia, inflammation, and low levels of calcium and albumin. The axis structure was precisely reproduced in all three populations and in all demographic sub-populations (by sex, race, etc.); we call the process represented by the axis "integrated albunemia." Integrated albunemia increases and accelerates with age in all populations, and predicts mortality and frailty--but not chronic disease--even after controlling for age. This suggests a role in the aging process, though causality is not yet clear. Integrated albunemia behaves more stably across populations than its component biomarkers, and thus appears to represent a higher-order physiological process emerging from the structure of underlying regulatory networks. If this is correct, detection of this process has substantial implications for physiological organization more generally.

Two-dimensional mineral [Pb2BiS3][AuTe2]: high-mobility charge carriers in single-atom-thick layers.

Two-dimensional (2D) electronic systems are of wide interest due to their richness in chemical and physical phenomena and potential for technological applications. Here we report that [Pb2BiS3][AuTe2], known as the naturally occurring mineral buckhornite, hosts 2D carriers in single-atom-thick layers. The structure is composed of stacking layers of weakly coupled [Pb2BiS3] and [AuTe2] sheets. The insulating [Pb2BiS3] sheet inhibits interlayer charge hopping and confines the carriers in the basal plane of the single-atom-thick [AuTe2] layer. Magneto-transport measurements on synthesized samples and theoretical calculations show that [Pb2BiS3][AuTe2] is a multiband semimetal with a compensated density of electrons and holes, which exhibits a high hole carrier mobility of ∼1360 cm(2)/(V s). This material possesses an extremely large anisotropy, Γ = ρ(c)/ρ(ab) ≈ 10(4), comparable to those of the benchmark 2D materials graphite and Bi2Sr2CaCu2O(6+δ). The electronic structure features linear band dispersion at the Fermi level and ultrahigh Fermi velocities of 10(6) m/s, which are virtually identical to those of graphene. The weak interlayer coupling gives rise to the highly cleavable property of the single crystal specimens. Our results provide a novel candidate for a monolayer platform to investigate emerging electronic properties.

The Xer/dif site-specific recombination system of Campylobacter jejuni.

Chromosome dimers, which form during the bacterial life cycle, represent a problem that must be solved by the bacterial cell machinery so that chromosome segregation can occur effectively. The Xer/dif site-specific recombination system, utilized by most bacteria, resolves chromosome dimers into monomers using two tyrosine recombinases, XerC and XerD, to perform the recombination reaction at the dif site which consists of 28-30 bp. However, single Xer recombinase systems have been recently discovered in several bacterial species. In Streptococci and Lactococci a single recombinase, XerS, is capable of completing the monomerisation reaction by acting at an atypical dif site called dif SL (31 bp). It was recently shown that a subgroup of ε-proteobacteria including Campylobacter spp. and Helicobacter spp. had a phylogenetically distinct Xer/dif recombination system with only one recombinase (XerH) and an atypical dif motif (difH). In order to biochemically characterize this system in greater detail, Campylobacter jejuni XerH was purified and its DNA-binding activity was characterized. The protein showed specific binding to the complete difH site and to both halves separately. It was also shown to form covalent complexes with difH suicide substrates. In addition, XerH was able to catalyse recombination between two difH sites located on a plasmid in Escherichia coli in vivo. This indicates that this XerH protein performs a similar function as the related XerS protein, but shows significantly different binding characteristics.

Characterization of the Streptococcus suis XerS recombinase and its unconventional cleavage of the difSL site.

XerC and XerD are members of the tyrosine recombinase family and mediate site-specific recombination that contributes to the stability of circular chromosomes in bacteria by resolving plasmid multimers and chromosome dimers to monomers prior to cell division. Homologues of xerC/xerD genes have been found in many bacteria, and in the lactococci and streptococci, a single recombinase called XerS can perform the functions of XerC and XerD. The xerS gene of Streptococcus suis was cloned, overexpressed and purified as a maltose-binding protein (MBP) fusion. The purified MBP-XerS fusion showed specific DNA-binding activity to both halves of the dif site of S. suis, and covalent protein-DNA complexes were also detected with dif site suicide substrates. These substrates were also cleaved in a specific fashion by MBP-XerS, generating cleavage products separated by an 11-bp spacer region, unlike the traditional 6-8-bp spacer observed in most tyrosine recombinases. Furthermore, xerS mutants of S. suis showed significant growth and morphological changes.