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.

Deep learning and single-cell phenotyping for rapid antimicrobial susceptibility detection in Escherichia coli.

The rise of antimicrobial resistance (AMR) is one of the greatest public health challenges, already causing up to 1.2 million deaths annually and rising. Current culture-based turnaround times for bacterial identification in clinical samples and antimicrobial susceptibility testing (AST) are typically 18-24 h. We present a novel proof-of-concept methodological advance in susceptibility testing based on the deep-learning of single-cell specific morphological phenotypes directly associated with antimicrobial susceptibility in Escherichia coli. Our models can reliably (80% single-cell accuracy) classify untreated and treated susceptible cells for a lab-reference fully susceptible E. coli strain, across four antibiotics (ciprofloxacin, gentamicin, rifampicin and co-amoxiclav). For ciprofloxacin, we demonstrate our models reveal significant (p < 0.001) differences between bacterial cell populations affected and unaffected by antibiotic treatment, and show that given treatment with a fixed concentration of 10 mg/L over 30 min these phenotypic effects correlate with clinical susceptibility defined by established clinical breakpoints. Deploying our approach on cell populations from six E. coli strains obtained from human bloodstream infections with varying degrees of ciprofloxacin resistance and treated with a range of ciprofloxacin concentrations, we show single-cell phenotyping has the potential to provide equivalent information to growth-based AST assays, but in as little as 30 min.

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.

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome.

In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation.

Transcription factor dynamics in plants: Insights and technologies for in vivo imaging.

Biochemical and genetic approaches have been extensively used to study transcription factor (TF) functions, but their dynamic behaviors and the complex ways in which they regulate transcription in plant cells remain unexplored, particularly behaviors such as translocation and binding to DNA. Recent developments in labeling and imaging techniques provide the necessary sensitivity and resolution to study these behaviors in living cells. In this review, we present an up-to-date portrait of the dynamics and regulation of TFs under physiologically relevant conditions and then summarize recent advances in fluorescent labeling strategies and imaging techniques. We then discuss future prospects and challenges associated with the application of these techniques to examine TFs' intricate dance in living plants.

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.

Mapping E. coli Topoisomerase IV Binding and Activity Sites.

This methods article described a protocol aiming at mapping E. coli Topoisomerase IV (Topo IV) binding and cleavage activity sites on the genome. The approach is readily applicable to any Type II topoisomerase on a broad variety of gram-positive and gram-negative bacterial species. Conventional ChIP-seq of flag tagged Topo IV subunits and a novel method aimed at trapping only DNA bound to active Topo IV (called NorfliP) are described. NorfliP relies on the ability of norfloxacin, a quinolone drug, to cross-link the 5' ends of the DNA breaks with the catalytic tyrosine of bacterial Type II topoisomerases. These methods give complementary results and their combination brought important insights on both the function and regulation of Topo IV.

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

Fungal lysis by a soil bacterium fermenting cellulose.

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.