Summary
With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has ...re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.
Oufti is an interactive, open source software package built for high throughput quantification of microscopy images. Oufti performs cell detection, localization of diffraction‐limited spots as well as boundary determination of image objects beyond the diffraction limit. Due to parallel processing, it is able to perform these tasks on large (>1Gb) datasets, which can be further expanded by high performance computing centers. Oufti also provides post‐processing tools for the statistical characterization of data following image analysis.
Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a ...built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.
Display omitted
•Multiple RNA polymerases (RNAPs) translocate faster than a single RNAP•Cooperation between RNAPs is not additive and occurs over long distances•Promoter repression leads to antagonistic dynamics and premature dissociation of RNAPs•Transcription-coupled DNA supercoiling causes these long-distance RNAP dynamics
Supercoiling introduced into DNA strands during the process of transcription influences other transcribing RNA polymerases over long distances, providing a physical basis for cooperative and antagonistic polymerase group behaviors.
Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) ...and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.
Display omitted
•Whole-cell 4Pi single-molecule switching nanoscopy allows 10- to 20-nm 3D resolution•Refined hardware and new data analysis allow imaging of cells as thick as ∼10 μm•Using structure-averaging, the 3D shape of a bacteriophage can be resolved•Wide applicability across diverse research fields is demonstrated
A new super-resolution microscope opens a window deep into cells to image organelles and subcellular structures spanning large volumes.
Post-transcriptional regulatory mechanisms are widespread in bacteria. Interestingly, current published data hint that some of these mechanisms may be non-random with respect to their phylogenetic ...distribution. Although small, trans-acting regulatory RNAs commonly occur in bacterial genomes, they have been better characterized in Gram-negative bacteria, leaving the impression that they may be less important for Firmicutes. It has been presumed that Gram-positive bacteria, in particular the Firmicutes, are likely to utilize cis-acting regulatory RNAs located within the 5' mRNA leader region more often than trans-acting regulatory RNAs. In this analysis we catalog, by a deep sequencing-based approach, both classes of regulatory RNA candidates for Bacillus subtilis, the model microorganism for Firmicutes. We successfully recover most of the known small RNA regulators while also identifying a greater number of new candidate RNAs. We anticipate these data to be a broadly useful resource for analysis of post-transcriptional regulatory strategies in B. subtilis and other Firmicutes.
Organisms maintain the correct balance of intracellular metals primarily through metal-sensing proteins that control transport and storage of the target ion(s). Here, we reveal the basis of metal ...sensing and genetic control by a metalloregulatory RNA. Our data demonstrate that a previously uncharacterized orphan riboswitch, renamed the “M-box,” is a divalent metal-sensing RNA involved in Mg
2+ homeostasis. A combination of genetic, biochemical, and biophysical techniques demonstrate that Mg
2+ induces a compacted tertiary architecture for M-box RNAs that regulates the accessibility of nucleotides involved in genetic control. Molecular details are provided by crystallographic structure determination of a Mg
2+-bound M-box RNA. Given the distribution of this RNA element, it may constitute a common mode for bacterial metal ion regulation, and its discovery suggests the possibility of additional RNA-based metal sensors in modern and primordial organisms.
To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation ...between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.
The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis ...was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.
Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several ...chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.
Formation of a Bacillus subtilis biofilm community requires an abundant matrix protein, TasA and an exopolysaccharide. The transcriptional regulatory pathways that control synthesis of these ...structural features are complex and responsive to multiple physiological and population signals. We report herein that an additional layer of co-transcriptional regulation is required for exopolysaccharide (eps) expression. This mechanism is mediated by a novel cis-acting RNA element, coined 'EAR', located between the second and third gene of the eps operon. The presence of the EAR element within the eps operon is required for readthrough of distally located termination signals. We also find that the EAR element promotes readthrough of heterologous termination sites. Based upon these observations, we hypothesize that the EAR element associates with RNA polymerase to promote processive antitermination, a process wherein the transcription elongation complex is altered by accessory factors to become resistant to pausing and termination signals. It is likely that this mechanism is required for eps expression to ensure full synthesis of the unusually long transcript (16 kb). We also identify the EAR element in other species within the order Bacillales, suggesting that a similar mechanism is required for synthesis of biofilm and capsular polysaccharide operons in other microorganisms.
PDF
