SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus.

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Internal Motion of Chromatin Fibers Is Governed by Dynamics of Uncompressed Linker Strands.

Chromatin compaction and internal motion are fundamental aspects of gene expression regulation. Here, we have investigated chromatin fibers comprising recombinant histone octamers reconstituted with double-stranded bacteriophage T4-DNA. The size of the fibers approaches the typical size of genomic topologically associated domains. Atomic force and fluorescence (correlation) microscopy have been used to assess the structural organization, histone-induced compaction, and internal motion. In particular, the fibers are stretched on arrays of nanochannels, each channel with a diameter of 60 or 125 nm. Major intrafiber segregation and fast internal fluctuations are observed. Full compaction was only achieved by triggering an attractive nucleosome interaction through the addition of magnesium cations. Besides compaction, histone complexation results in a dramatic decrease in the fiber's relaxation time. The relaxation times are similar to those of naked DNA with a comparable stretch, which indicates that internal motion is governed by the dynamics of uncompressed linker strands. Furthermore, the main reorganization process is association-dissociation of individually compacted regions. We surmise that the modulation of chromatin's internal motion by histone complexation might have implications for transcriptional bursting.

Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response.

Molecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Escherichia coli Hfq is reported, involved in controlling the flow of a DNA solution. A combination of experimental methodologies has been used to probe the interaction of Hfq with DNA and to measure the rheological properties of the complex. A physical gel with a temperature reversible elasticity modulus is formed due to the formation of noncovalent cross-links. The mechanical response of the complexes shows that they are inhomogeneous soft solids. Our experiments indicate that the Hfq C-terminal region could contribute to the genome's mechanical response. The reported viscoelasticity of the DNA-protein complex might have implications for cellular processes involving molecular transport of DNA or segments thereof.

Single-molecule compaction of megabase-long chromatin molecules by multivalent cations.

To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo. Variation of HO loading revealed a number of unique features related to the efficiency of chromatin compaction by multivalent cations, the mechanism of compaction, and the character of partly compact chromatin structures. The observations may be relevant for how DNA accessibility in chromatin is maintained. Compaction of saturated chromatin, in turn, is accompanied by an intra-chain segregation at the level of single chromatin molecules, suggesting an intriguing scenario of selective activation/deactivation of DNA as a result of chromatin fiber heterogeneity due to the nucleosome positioning. We suggest that this chromatin, reconstituted on megabase-long DNA because of its large size, is a useful model of eukaryotic chromatin.

Compaction and condensation of DNA mediated by the C-terminal domain of Hfq.

Hfq is a bacterial protein that is involved in several aspects of nucleic acids metabolism. It has been described as one of the nucleoid associated proteins shaping the bacterial chromosome, although it is better known to influence translation and turnover of cellular RNAs. Here, we explore the role of Escherichia coli Hfq's C-terminal domain in the compaction of double stranded DNA. Various experimental methodologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy, isothermal titration microcalorimetry and electrophoretic mobility assays have been used to follow the assembly of the C-terminal and N-terminal regions of Hfq on DNA. Results highlight the role of Hfq's C-terminal arms in DNA binding, change in mechanical properties of the double helix and compaction of DNA into a condensed form. The propensity for bridging and compaction of DNA by the C-terminal domain might be related to aggregation of bound protein and may have implications for protein binding related gene regulation.

Effect of HU protein on the conformation and compaction of DNA in a nanochannel.

The effect of the heat unstable nucleoid structuring protein HU on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. Pre-incubated DNA molecules contract in the longitudinal direction of the channel with increasing concentration of HU. This contraction is mainly due to HU-mediated bridging of distal DNA segments and is controlled by channel diameter as well as ionic composition and strength of the buffer. For over-threshold concentrations of HU, the DNA molecules compact into an condensed form. Divalent magnesium ions facilitate, but are not required for bridging nor condensation. The conformational response following exposure to HU was investigated with a nanofluidic device that allows an in situ change in environmental solution conditions. The stretch of the nucleoprotein complex first increases, reaches an apex in ∼20 min, and subsequently decreases to an equilibrium value pertaining to pre-incubated DNA molecules after ∼2 h. This observation is rationalised in terms of a time-dependent bending rigidity by structural rearrangement of bound HU protein followed by compaction through bridging interaction. Results are discussed in regard to previous results obtained for nucleoid associated proteins H-NS and Hfq, with important implications for protein binding related gene regulation.

Effects of Hfq on the conformation and compaction of DNA.

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq's interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to previous results obtained for H-NS, with important implications for protein binding related gene regulation.

Structure of the H-NS-DNA nucleoprotein complex.

Nucleoid associated proteins (NAPs) play a key role in the compaction and expression of the prokaryotic genome. Here we report the organisation of a major NAP, the protein H-NS on a double stranded DNA fragment. For this purpose we have carried out a small angle neutron scattering study in conjunction with contrast variation to obtain the contributions to the scattering (structure factors) from DNA and H-NS. The H-NS structure factor agrees with a heterogeneous, two-state binding model with sections of the DNA duplex surrounded by protein and other sections having protein bound to the major groove. In the presence of magnesium chloride, we observed a structural rearrangement through a decrease in cross-sectional diameter of the nucleoprotein complex and an increase in fraction of major groove bound H-NS. The two observed binding modes and their modulation by magnesium ions provide a structural basis for H-NS-mediated genome organisation and expression regulation.

Revisiting the Anomalous Bending Elasticity of Sharply Bent DNA.

Several recent experiments suggest that sharply bent DNA has a surprisingly high bending flexibility, but the cause of this flexibility is poorly understood. Although excitation of flexible defects can explain these results, whether such excitation can occur with the level of DNA bending in these experiments remains unclear. Intriguingly, the DNA contained preexisting nicks in most of these experiments but whether nicks might play a role in flexibility has never been considered in the interpretation of experimental results. Here, using full-atom molecular dynamics simulations, we show that nicks promote DNA basepair disruption at the nicked sites, which drastically reduces DNA bending energy. In addition, lower temperatures suppress the nick-dependent basepair disruption. In the absence of nicks, basepair disruption can also occur but requires a higher level of DNA bending. Therefore, basepair disruption inside B-form DNA can be suppressed if the DNA contains preexisting nicks. Overall, our results suggest that the reported mechanical anomaly of sharply bent DNA is likely dependent on preexisting nicks, therefore the intrinsic mechanisms of sharply bent nick-free DNA remain an open question.

Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein.

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA-HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ∼2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

Intrachain dynamics of large dsDNA confined to slitlike channels.

Exploration of intrachain hydrodynamics of dsDNA within channels has been limited to indirect analysis of global coil dynamics. In this Letter, we isolate hydrodynamic interactions within single molecules of dsDNA confined to slitlike channels by making use of density covariance measurements. We show that the strength of hydrodynamic interactions in DNA is dependent on the intrachain correlation length and that screening by symmetry in slitlike confinement results in a screening length that is proportional channel height. Moreover, we directly show the partial draining nature of the blobs formed by dsDNA in slits and predict under what conditions a dsDNA blob should obey nondraining Zimm behavior.

Revisiting blob theory for DNA diffusivity in slitlike confinement.

Blob theory has been widely applied to describe polymer conformations and dynamics in nanoconfinement. In slit confinement, blob theory predicts a scaling exponent of 2/3 for polymer diffusivity as a function of slit height, yet a large body of experimental studies using DNA produce a scaling exponent significantly less than 2/3. In this work, we develop a theory that predicts that this discrepancy occurs because the segment correlation function for a semiflexible chain such as DNA does not follow the Flory exponent for length scales smaller than the persistence length. We show that these short length scale effects contribute significantly to the scaling for the DNA diffusivity, but do not appreciably affect the scalings for static properties. Our theory is fully supported by Monte Carlo simulations, quantitative agreement with DNA experiments, and the results reconcile this outstanding problem for confined polymers.

Effect of H-NS on the elongation and compaction of single DNA molecules in a nanospace.

The effect of the bacterial heat-stable nucleoid-structuring protein (H-NS) on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. With increasing concentration of H-NS, the DNA molecules either elongate or contract. The conformational response is related to filamentation of H-NS on DNA through oligomerization and H-NS mediated bridging of distal DNA segments and is controlled by the concentration and ionic composition of the buffer. Confinement in a nanochannel also facilitates compaction of DNA into a condensed form for over-threshold concentrations of H-NS. Divalent ions such as magnesium facilitate but are not required for bridging nor condensation. The time scale of the collapse after exposure to H-NS was determined to be on the order of minutes, which is much shorter than the measured time required for filamentation of around one hour. We found that the effect of H-NS is not only related to its binding properties but also the confinement is of paramount importance. The interplay between confinement, H-NS-mediated attraction, and filamentation controls the conformation and compaction of DNA. This finding might have implications for gene silencing and chromosome organisation, because the cross-sectional dimensions of the channels are comparable to those of the bacterial nucleoid.

Form factor of helical structures and twisted fibres.

A general formalism is presented for the isotropically averaged single-chain scattering function (form factor) of single, double, triple and higher-order helices, as well as twisted fibres consisting of concentric layers of strands. Form factors for double and triple helices with differently sized grooves have also been derived. The formulas include the longitudinal and transverse interference over the pitch and radius of the helices, respectively. The results may be useful for the analysis of small-angle scattering data of (bio)macromolecules or molecular assemblies exhibiting a helical arrangement.

Bipedal nanowalker by pure physical mechanisms.

Artificial nanowalkers are inspired by biomolecular counterparts from living cells, but remain far from comparable to the latter in design principles. The walkers reported to date mostly rely on chemical mechanisms to gain a direction; they all produce chemical wastes. Here we report a light-powered DNA bipedal walker based on a design principle derived from cellular walkers. The walker has two identical feet and the track has equal binding sites; yet the walker gains a direction by pure physical mechanisms that autonomously amplify an intrasite asymmetry into a ratchet effect. The nanowalker is free of any chemical waste. It has a distinct thermodynamic feature that it possesses the same equilibrium before and after operation, but generates a truly nonequilibrium distribution during operation. The demonstrated design principle exploits mechanical effects and is adaptable for use in other nanomachines.

Macromolecular crowding induced elongation and compaction of single DNA molecules confined in a nanochannel.

The effect of dextran nanoparticles on the conformation and compaction of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. It was observed that the DNA molecules elongate and eventually condense into a compact form with increasing volume fraction of the crowding agent. Under crowded conditions, the channel diameter is effectively reduced, which is interpreted in terms of depletion in DNA segment density in the interfacial region next to the channel wall. Confinement in a nanochannel also facilitates compaction with a neutral crowding agent at low ionic strength. The threshold volume fraction for condensation is proportional to the size of the nanoparticle, due to depletion induced attraction between DNA segments. We found that the effect of crowding is not only related to the colligative properties of the agent and that confinement is also important. It is the interplay between anisotropic confinement and osmotic pressure which gives the elongated conformation and the possibility for condensation at low ionic strength.

Effect of nanochannel geometry on DNA structure in the presence of macromolecular crowding agent.

We experimentally and numerically study the effects of macromolecular crowding agents on DNA structure when confined to a nanochannel. Curiously, DNA response to crowding is significantly different between bulk phase, nanoslit confinement, and nanotube confinement. Coarse grained Brownian dynamics simulations reproduce trends seen in the experiments and allow us to develop a deeper understanding of the key physics at play in these systems. It is proposed that the occupancy of free volume next to the channel wall by crowders causes an effective reduction in confining dimensions of the channel that initially swells DNA in nanoconfinement.

Probing Amyloid-DNA Interaction with Nanofluidics.

Nanofluidics is an emerging methodology to investigate single biomacromolecules without functionalization and/or attachment of the molecules to a substrate. In conjunction with fluorescence microscopy, it can be used to investigate structural and dynamical aspects of amyloid-DNA interaction. Here, we summarize the methodology for fabricating lab-on-chip devices in relatively cheap polymer resins and featuring quasi one-dimensional nanochannels with a cross-sectional diameter of tens to a few hundred nanometers. Site-specific staining of amyloid-forming protein Hfq with a fluorescence dye is also described. The methodology is illustrated with two application studies. The first study involves assembling bacterial amyloid proteins such as Hfq on double-stranded DNA and monitoring the folding and compaction of DNA in a condensed state. The second study is about the concerted motion of Hfq on DNA and how this is related to DNA's internal motion. Explicit details of procedures and workflows are given throughout.

Mobility of Bacterial Protein Hfq on dsDNA: Role of C-Terminus-Mediated Transient Binding.

The mobility of protein is fundamental in the machinery of life. Here, we have investigated the effect of DNA binding in conjunction with DNA segmental fluctuation (internal motion) of the bacterial Hfq master regulator devoid of its amyloid C-terminus domain. Hfq is one of the most abundant nucleoid associated proteins that shape the bacterial chromosome and is involved in several aspects of nucleic acid metabolism. Fluorescence microscopy has been used to track a C-terminus domain lacking mutant form of Hfq on double-stranded DNA, which is stretched by confinement to a rectangular nanofluidic channel. The mobility of the mutant is strongly accelerated with respect to the wild-type variant. Furthermore, it shows a reverse dependence on the internal motion of DNA, in that slower motion results in slower protein diffusion. The results demonstrate the subtle role of DNA internal motion in controlling the mobility of a nucleoid associated protein, and, in particular, the importance of transient binding and moving DNA strands out of the way.