In vivo imaging of MmpL transporters reveals distinct subcellular locations for export of mycolic acids and non-essential trehalose polyphleates in the mycobacterial outer membrane.

The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms.

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated DNA partition.

In bacteria, most low-copy-number plasmid and chromosomally encoded partition systems belong to the tripartite ParABS partition machinery. Despite the importance in genetic inheritance, the mechanisms of ParABS-mediated genome partition are not well understood. Combining theory and experiment, we provided evidence that the ParABS system-DNA partitioning in vivo via the ParA-gradient-based Brownian ratcheting-operates near a transition point in parameter space (i.e., a critical point), across which the system displays qualitatively different motile behaviors. This near-critical-point operation adapts the segregation distance of replicated plasmids to the half length of the elongating nucleoid, ensuring both cell halves to inherit one copy of the plasmids. Further, we demonstrated that the plasmid localizes the cytoplasmic ParA to buffer the partition fidelity against the large cell-to-cell fluctuations in ParA level. The spatial control over the near-critical-point operation not only ensures both sensitive adaptation and robust execution of partitioning but also sheds light on the fundamental question in cell biology: how do cells faithfully measure cellular-scale distance by only using molecular-scale interactions?

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the ß-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a "clamping and sliding" mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although "clamping and sliding" can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

ATP-Driven Separation of Liquid Phase Condensates in Bacteria.

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.

Addressing the role of centromere sites in activation of ParB proteins for partition complex assembly.

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells.

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

Symmetry Properties of Mixed and Heat Photo-Assisted Noise in the Quantum Hall Regime.

We investigate the photo-assisted charge-heat mixed noise and the heat noise generated by periodic drives in Quantum Hall states belonging to the Laughlin sequence. Fluctuations of the charge and heat currents are due to weak backscattering induced in a quantum point contact geometry and are evaluated at the lowest order in the tunneling amplitude. Focusing on the cases of a cosine and Lorentzian periodic drive, we show that the different symmetries of the photo-assisted tunneling amplitudes strongly affect the overall profile of these quantities as a function of the AC and DC voltage contributions, which can be tuned independently in experiments.

A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids.

Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the F plasmid partition system. We found that "Nucleation & caging" is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) is a robust mechanism, (ii) does not directly involve ParA ATPase, (iii) results in a dynamic structure of discrete size independent of ParB concentration, and (iv) is not perturbed by active transcription but is by protein complexes. We refined the "Nucleation & caging" model and successfully applied it to the chromosomally encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

Bacterial partition complexes segregate within the volume of the nucleoid.

Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes.

Imaging centromere-based incompatibilities: Insights into the mechanism of incompatibility mediated by low-copy number plasmids.

In bacteria, low-copy number plasmids are faithfully segregated at cell division by active partition systems that rely on plasmid-specific centromere sequences. When an identical centromere is present on a second plasmid, faithful partition is impaired causing plasmid loss. Depending on the copy number of the co-resident replicon, several mechanisms have been proposed to account for this centromere-based plasmid incompatibility. To gain further insights into these mechanisms, we analyzed the positioning of the F plasmid in the presence of incompatible low- and high-copy number plasmids carrying the F centromere. Our data are fully compatible with the titration hypothesis when extra-centromeres are present on high-copy number plasmids. Interestingly, our plasmids' localization data revealed that the strong incompatibility phenotype, observed when extra centromeres are present on a partition defective low-copy number plasmid, does not directly result from a partition deficiency as previously proposed. We provide a new and simple hypothesis for explaining the strong incompatibility phenotype based on the timing of replication of low-copy number plasmids.

Stochastic Self-Assembly of ParB Proteins Builds the Bacterial DNA Segregation Apparatus.

Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS. This assembly model and approach to characterizing large-scale, dynamic interactions between macromolecules may be generalizable to many unrelated machineries that self-assemble in superstructures.

Interactions and charge fractionalization in an electronic Hong-Ou-Mandel interferometer.

We consider an electronic analog of the Hong-Ou-Mandel (HOM) interferometer, where two single electrons travel along opposite chiral edge states and collide at a quantum point contact. Studying the current noise, we show that because of interactions between copropagating edge states, the degree of indistinguishability between the two electron wave packets is dramatically reduced, leading to reduced contrast for the HOM signal. This decoherence phenomenon strongly depends on the energy resolution of the packets. Insofar as interactions cause charge fractionalization, we show that charge and neutral modes interfere with each other, leading to satellite dips or peaks in the current noise. Our calculations explain recent experimental results [E. Bocquillon Science 339 1054 (2013)] where an electronic HOM signal with reduced contrast was observed.

Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids.

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.

Insight into centromere-binding properties of ParB proteins: a secondary binding motif is essential for bacterial genome maintenance.

ParB proteins are one of the three essential components of partition systems that actively segregate bacterial chromosomes and plasmids. In binding to centromere sequences, ParB assembles as nucleoprotein structures called partition complexes. These assemblies are the substrates for the partitioning process that ensures DNA molecules are segregated to both sides of the cell. We recently identified the sopC centromere nucleotides required for binding to the ParB homologue of plasmid F, SopB. This analysis also suggested a role in sopC binding for an arginine residue, R219, located outside the helix-turn-helix (HTH) DNA-binding motif previously shown to be the only determinant for sopC-specific binding. Here, we demonstrated that the R219 residue is critical for SopB binding to sopC during partition. Mutating R219 to alanine or lysine abolished partition by preventing partition complex assembly. Thus, specificity of SopB binding relies on two distinct motifs, an HTH and an arginine residue, which define a split DNA-binding domain larger than previously thought. Bioinformatic analysis over a broad range of chromosomal ParBs generalized our findings with the identification of a non-HTH positively charged residue essential for partition and centromere binding, present in a newly identified highly conserved motif. We propose that ParB proteins possess two DNA-binding motifs that form an extended centromere-binding domain, providing high specificity.

Effect of measurement backaction on adiabatic coherent electron transport.

We study the backaction of a nearby measurement device on electrons undergoing coherent transfer via adiabatic passage (CTAP) in a triple-well system. The measurement is provided by a quantum point contact capacitively coupled to the middle well. We account for this continuous measurement by treating the whole {triple-well+detector} as a closed quantum system. This leads to a set of coupled differential equations for the density matrix of the enlarged system which we solve numerically. This approach allows us to study a single realization of the measurement process while keeping track of the detector output, which is especially relevant for experiments. In particular, we find the emergence of a new peak in the distribution of electrons inside the detector, accompanied by a drop in the fidelity of the protocol.

Conductance of fully equilibrated quantum wires.

We study the conductance of a quantum wire in the presence of weak electron-electron scattering. In a sufficiently long wire the scattering leads to full equilibration of the electron distribution function in the frame moving with the electric current. At nonzero temperature this equilibrium distribution differs from the one supplied by the leads. As a result the contact resistance increases, and the quantized conductance of the wire acquires a quadratic in temperature correction. The magnitude of the correction is found by analysis of the conservation laws of the system and does not depend on the details of the interaction mechanism responsible for equilibration.

Resistivity of inhomogeneous quantum wires.

We study the effect of electron-electron interactions on the transport in an inhomogeneous quantum wire. We show that contrary to the well-known Luttinger liquid result, nonuniform interactions contribute substantially to the resistance of the wire. In the regime of weakly interacting electrons and moderately low temperatures we find a linear in T resistivity induced by the interactions. We then use the bosonization technique to generalize this result to the case of arbitrarily strong interactions.