Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.

RNA polymerase redistribution supports growth in E. coli strains with a minimal number of rRNA operons.

Bacterial transcription by RNA polymerase (RNAP) is spatially organized. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich medium, with several studies linking RNAP clustering and transcription of rRNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons (Δrrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting five or six of the seven chromosomal rrn operons. Spatial analysis in Δrrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at cell endcaps, where the remaining rrn operons reside. These results support a model where RNAPs in Δrrn strains relocate to copies of the remaining rrn operons. In rich medium, Δrrn strains redistribute RNAP to minimize growth defects due to rrn deletions, with very high RNAP densities on rrn genes leading to genomic instability. Our study links RNAP clusters and rrn transcription, and offers insight into how bacteria maintain growth in the presence of only 1-2 rrn operons.

Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction.

Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool.

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Lipid nanobilayers to host biological nanopores for DNA translocations.

We characterize a recently introduced novel nanobilayer technique [Gornall, J. L., Mahendran, K. R., Pambos, O. J., Steinbock, L. J., Otto, O., Chimerel, C., Winterhalter, M., and Keyser, U. F. Simple reconstitution of protein pores in nano lipid bilayers. Nano Lett. 2011, 11 (8), 3334-3340] and its practical aspects for incorporating the biological nanopore α-hemolysin from Staphylococcus aureus and subsequent studies on the translocation of biomolecules under various conditions. This technique provides advantages over classical bilayer methods, especially the quick formation and extended stability of a bilayer. We have also developed a methodology to prepare a uniform quality of giant unilamellar vesicles (GUVs) in a reproducible way for producing nanobilayers. The process and the characteristics of the reconstitution of α-hemolysin in nanobilayers were examined by exploiting various important parameters, including pH, applied voltage, salt concentration, and number of nanopores. Protonation of α-hemolysin residues in the low pH region affects the translocation durations, which, in turn, changes the statistics of event types as a result of electrostatics and potentially the structural changes in DNA. When the pH and applied voltage were varied, it was possible to investigate and partly control the capture rates and type of translocation events through α-hemolysin nanopores. This study could be helpful to use the nanobilayer technique for further explorations, particularly owing to its advantages and technical ease compared to existing bilayer methods.

Simple reconstitution of protein pores in nano lipid bilayers.

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.