A 3' external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise.

During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. However, we report that the 3'ETS of the glyW-cysT-leuZ polycistronic tRNA precursor is highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs are shown to base pair with the same region in the 3'ETS of leuZ (3'ETS(leuZ)). Disrupting the pairing by mutating 3'ETS(leuZ) strongly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3'ETS(leuZ) prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and decreases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3'ETS(leuZ) functions as a sponge to absorb transcriptional noise from repressed sRNAs. Additional data showing RybB and MicF sRNAs are co-purified with ITS(metZ-metW) and ITS(metW-metV) strongly suggest a wide distribution of this phenomenon.

Synchronized switching of multiple toxin-antitoxin modules by (p)ppGpp fluctuation.

Toxin-antitoxin (TA) loci are widespread in bacteria including important pathogenic species. Recent studies suggest that TA systems play a key role in persister formation. However, the persistence phenotype shows only weak dependence on the number of TA systems, i.e. they are functionally redundant. We use a mathematical model to investigate the interaction of multiple TA systems in the switching between growth and persistence. We explore two scenarios: (i) TA systems are bistable and each TA system experiences its own noise and (ii) the noise in the level of common stress signal (e.g. (p)ppGpp) coordinates all TA systems simultaneously. We find that in the first scenario the exit from the persister state strongly depends on the number of TA systems. However in the second case, we could reproduce the weak dependence. The duration of the high (p)ppGpp state was found to be the key parameter for persistence. The (p)ppGpp-driven synchronized transition of all TA systems results in the redundancy.

Quantification of Loading and Laser-Assisted Release of RNA from Single Gold Nanoparticles.

Novel RNA-based technologies provide an avenue of possibilities to control the regulation of gene expression in cells. To realize the full potential of small interfering RNA (siRNA)-based therapy, efficient delivery vehicles and novel strategies for triggering release from carrier vehicles have to be developed. Gold nanoparticles (AuNPs) with sizes of ∼50-150 nm have the ability to accumulate in tumor tissue and can be transported across the membrane by endocytosis. Therefore, a laser-controlled oligonucleotide release from such particles is of particular interest. Here, we quantify the loading of specifically attached microRNA oligonucleotides (miRNA) onto single gold nanoparticles with diameters of 80, 100, 150, and 200 nm. We show that AuNPs have a curvature-dependent density of miRNA loading: the higher the curvature, the higher the loading density. Moreover, we demonstrate how one sensing strand of an RNA duplex can be dehybridized and hence released from the AuNP by heating the AuNP by irradiation with a near-infrared (NIR) laser. Laser-induced release is also demonstrated inside living cells. Together, these findings show that plasmonic nanoparticles with high curvatures are ideal carriers of oligonucleotides into cells, and their cargo can be released in a controlled manner by a thermoplasmonic mechanism. Importantly, this remotely controlled release strategy can be applied to any cargo attached to a plasmonic nanocarrier, on either the single particle or ensemble level.

Vesicle Fusion Triggered by Optically Heated Gold Nanoparticles.

Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We locally heated the membranes of two adjacent vesicles by laser irradiating gold nanoparticles, thus causing vesicle fusion with associated membrane and cargo mixing. The mixing time scales were consistent with diffusive mixing of the membrane dyes and the aqueous content. This method is useful for nanoscale reactions as demonstrated here by I-BAR protein-mediated membrane tubulation triggered by fusion.

The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli.

The lactose operon of Escherichia coli is a paradigm system for quantitative understanding of gene regulation in prokaryotes. Yet, none of the many mathematical models built so far to study the dynamics of this system considered the fact that the Lac repressor regulates its own transcription by forming a transcriptional roadblock at the O3 operator site. Here we study the effect of autoregulation on intracellular LacI levels and also show that cAMP-CRP binding does not affect the efficiency of autoregulation. We built a mathematical model to study the role of LacI autoregulation in the lactose utilization system. Previously, it has been argued that negative autoregulation can significantly reduce noise as well as increase the speed of response. We show that the particular molecular mechanism, a transcriptional roadblock, used to achieve self-repression in the lac system does neither. Instead, LacI autoregulation balances two opposing states, one that allows quicker response to smaller pulses of external lactose, and the other that minimizes production costs in the absence of lactose.

Commitment to lysogeny is preceded by a prolonged period of sensitivity to the late lytic regulator Q in bacteriophage λ.

A key event in development is the irreversible commitment to a particular cell fate, which may be concurrent with or delayed with respect to the initial cell fate decision. In this work, we use the paradigmatic bacteriophage λ lysis-lysogeny decision circuit to study the timing of commitment. The lysis-lysogeny decision is made based on the expression trajectory of CII. The chosen developmental strategy is manifested by repression of the pR and pL promoters by CI (lysogeny) or by antitermination of late gene expression by Q (lysis). We found that expression of Q in trans from a plasmid at the time of infection resulted in a uniform lytic decision. Furthermore, expression of Q up to 50 min after infection results in lysis of the majority of cells which initially chose lysogenic development. In contrast, expression of Q in cells containing a single chromosomal prophage had no effect on cell growth, indicating commitment to lysogeny. Notably, if the prophage was present in 10 plasmid-borne copies, Q expression resulted in lytic development, suggesting that the cellular phage chromosome number is the critical determinant of the timing of lysogenic commitment. Based on our results, we conclude that (i) the lysogenic decision made by the CI-Cro switch soon after infection can be overruled by ectopic Q expression at least for a time equivalent to one phage life cycle, (ii) the presence of multiple λ chromosomes is a prerequisite for a successful Q-mediated switch from lysogenic to lytic development, and (iii) phage chromosomes within the same cell can reach different decisions.

Specific contacts of the -35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression.

The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of 'extended -10' promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor -35 element. To investigate whether specific recognition of the -35 element would affect the regulation of P1 by GalR, we mutagenized the -35 element of P1, isolated variants of the -35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the -10 and -35 elements concomitantly. Our results suggest that promoters that lack specific -35 element recognition allow decoupling of local chromosome structure from transcription initiation.

Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.

Direct and indirect effects in the regulation of overlapping promoters.

Optimal response to environmental stimuli often requires activation of certain genes and repression of others. Dual function regulatory proteins play a key role in the differential regulation of gene expression. While repression can be achieved by any DNA binding protein through steric occlusion of RNA polymerase in the promoter region, activation often requires a surface on the regulatory protein to contact RNAP and thus facilitate transcription initiation. RNAP itself is also a DNA binding protein, therefore it can function as a transcriptional repressor. Searching the Escherichia coli promoter database we found that ∼14% of the identified 'forward' promoters overlap with a promoter oriented in the opposite direction. In this article we combine a mathematical model with experimental analysis of synthetic regulatory regions to investigate interference of overlapping promoters. We find that promoter interference depends on the characteristics of overlapping promoters. The model predicts that promoter strength and interference can be regulated separately, which provides unique opportunities for regulation. Our experimental data suggest that in principle any DNA binding protein can be used for both activation and repression of promoter transcription, depending on the context. These findings can be exploited in the construction of synthetic networks.

Genetic flexibility of regulatory networks.

Gene regulatory networks are based on simple building blocks such as promoters, transcription factors (TFs) and their binding sites on DNA. But how diverse are the functions that can be obtained by different arrangements of promoters and TF binding sites? In this work we constructed synthetic regulatory regions using promoter elements and binding sites of two noninteracting TFs, each sensing a single environmental input signal. We show that simply by combining these three kinds of elements, we can obtain 11 of the 16 Boolean logic gates that integrate two environmental signals in vivo. Further, we demonstrate how combination of logic gates can result in new logic functions. Our results suggest that simple elements of transcription regulation form a highly flexible toolbox that can generate diverse functions under natural selection.

Dynamic features of gene expression control by small regulatory RNAs.

Small regulatory RNAs (sRNAs) in eukaryotes and bacteria play an important role in the regulation of gene expression either by binding to regulatory proteins or directly to target mRNAs. Two of the best-characterized bacterial sRNAs, Spot42 and RyhB, form a complementary pair with the ribosome binding region of their target mRNAs, thereby inhibiting translation or promoting mRNA degradation. To investigate the steady-state and dynamic potential of such sRNAs, we examine the 2 key parameters characterizing sRNA regulation: the capacity to overexpress the sRNA relative to its target mRNA and the speed at which the target mRNA is irreversibly inactivated. We demonstrate different methods to determine these 2 key parameters, for Spot42 and RyhB, which combine biochemical and genetic experiments with computational analysis. We have developed a mathematical model that describes the functional properties of sRNAs with various characteristic parameters. We observed that Spot42 and RyhB function in distinctive parameter regimes, which result in divergent mechanisms.

Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in "foci" in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

Filopodia rotate and coil by actively generating twist in their actin shaft.

Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

Timing of gene transcription in the galactose utilization system of Escherichia coli.

In the natural environment, bacterial cells have to adjust their metabolism to alterations in the availability of food sources. The order and timing of gene expression are crucial in these situations to produce an appropriate response. We used the galactose regulation in Escherichia coli as a model system for understanding how cells integrate information about food availability and cAMP levels to adjust the timing and intensity of gene expression. We simulated the feast-famine cycle of bacterial growth by diluting stationary phase cells in fresh medium containing galactose as the sole carbon source. We followed the activities of six promoters of the galactose system as cells grew on and ran out of galactose. We found that the cell responds to a decreasing external galactose level by increasing the internal galactose level, which is achieved by limiting galactose metabolism and increasing the expression of transporters. We show that the cell alters gene expression based primarily on the current state of the cell and not on monitoring the level of extracellular galactose in real time. Some decisions have longer term effects; therefore, the current state does subtly encode the history of food availability. In summary, our measurements of timing of gene expression in the galactose system suggest that the system has evolved to respond to environments where future galactose levels are unpredictable rather than regular feast and famine cycles.

Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus: expression dynamics for regulatory and effector genes.

Antlers of deer display the fastest and most robust bone development in the animal kingdom. Deposition of the minerals in the cartilage preceding ossification is a specific feature of the developing antler. We have cloned 28 genes which are upregulated in the cartilaginous section (called mineralized cartilage) of the developing ("velvet") antler of red deer stags, compared to their levels in the fetal cartilage. Fifteen of these genes were further characterized by their expression pattern along the tissue zones (i.e., antler mesenchyme, precartilage, cartilage, bone), and by in situ hybridization of the gene activities at the cellular level. Expression dynamics of genes col1A1, col1A2, col3A1, ibsp, mgp, sparc, runx2, and osteocalcin were monitored and compared in the ossified part of the velvet antler and in the skeleton (in ribs and vertebrae). Expression levels of these genes in the ossified part of the velvet antler exceeded the skeletal levels 10-30-fold or more. Gene expression and comparative sequence analyses of cDNAs and the cognate 5' cis-regulatory regions in deer, cattle, and human suggested that the genes runx2 and osx have a master regulatory role. GC-MS metabolite analyses of glucose, phosphate, ethanolamine-phosphate, and hydroxyproline utilizations confirmed the high activity of mineralization genes in governing the flow of the minerals from the skeleton to the antler bone. Gene expression patterns and quantitative metabolite data for the robust bone development in the antler are discussed in an integrated manner. We also discuss the potential implication of our findings on the deer genes in human osteoporosis research.

Why do phage play dice?

Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into the lytic state, where the phage particles rapidly replicate and eventually lyse the cell, or into a lysogenic state, where the phage goes dormant and replicates along with the cell. Experimental observations by P. Kourilsky are consistent with a single phage infection deterministically choosing lysis and double infection resulting in a stochastic choice. We argue that the phage are playing a "game" of minimizing the chance of extinction and that the shift from determinism to stochasticity is due to a shift from a single-player to a multiplayer game. Crucial to the argument is the clonal identity of the phage.

The antiparallel loops in gal DNA.

Interactions between proteins bound to distant sites along a DNA molecule require bending and twisting deformations in the intervening DNA. In certain systems, the sterically allowed protein-DNA and protein-protein interactions are hypothesized to produce loops with distinct geometries that may also be thermodynamically and biologically distinct. For example, theoretical models of Gal repressor/HU-mediated DNA-looping suggest that the antiparallel DNA loops, A1 and A2, are thermodynamically quite different. They are also biologically different, since in experiments using DNA molecules engineered to form only one of the two loops, the A2 loop failed to repress in vitro transcription. Surprisingly, single molecule measurements show that both loop trajectories form and that they appear to be quite similar energetically and kinetically.

Combinatorics of feedback in cellular uptake and metabolism of small molecules.

We analyze the connection between structure and function for regulatory motifs associated with cellular uptake and usage of small molecules. Based on the boolean logic of the feedback we suggest four classes: the socialist, consumer, fashion, and collector motifs. We find that the socialist motif is good for homeostasis of a useful but potentially poisonous molecule, whereas the consumer motif is optimal for nutrition molecules. Accordingly, examples of these motifs are found in, respectively, the iron homeostasis system in various organisms and in the uptake of sugar molecules in bacteria. The remaining two motifs have no obvious analogs in small molecule regulation, but we illustrate their behavior using analogies to fashion and obesity. These extreme motifs could inspire construction of synthetic systems that exhibit bistable, history-dependent states, and homeostasis of flux (rather than concentration).

A gamut of loops: meandering DNA.

Nucleoprotein complexes comprising short DNA loops (150 base pairs or less) are involved in a wide variety of DNA transactions (e.g. transcription regulation, replication and recombination) in both prokaryotes and eukaryotes, and also can be useful in designing nanostructures. In these higher-order nucleoprotein complexes, proteins bound to spatially separated sites on a DNA interact with each other by looping out the relatively stiff intervening DNA. Recent technological developments have enabled determination of DNA trajectories in a few DNA-loop-containing regulatory complexes. Results show that, in a given system, a specific DNA trajectory is preferred over others.