Explore the vast range of titles available.

Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ 70 and σ 54 ). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ 70 -controlled stress response is regulated via the burst size, while that of the σ 54 -controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ 54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems. Transcription noise in bacteria is often attributed to burstiness, but the mechanisms are unclear. Here, the authors show that the transition from low to high expression can be regulated via burst size or burst frequency, depending on the mode of transcription initiation determined by different sigma factors.

RNA repair is critical for cellular function. The Rtc system maintains RNA integrity within the translational machinery of bacteria. In E. coli , Rtc expression enables cells to rescue growth and survive treatment by conferring transient resistance to ribosome-targeting antibiotics, yet the mechanisms underpinning this resistance remain obscure. Here, we present a computational model of Rtc-regulated repair of translational RNAs. Integrating model predictions with experimental validations, we uncover notable cell-to-cell heterogeneity in rtc expression that impacts on translational capacity, indicating that rtc may induce a form of heteroresistance. We moreover identify Rtc targets that may reduce the translational capacity of cells and so potentiate antibiotic effects. Our findings elucidate a complex response underpinning resistance conferred by Rtc, offering alternate routes for addressing resistance in E. coli and other relevant pathogens. RNA repair helps bacteria survive antibiotic stress. Here, authors show that Rtc-driven repair causes cell-to-cell variation in resistance levels, revealing a potential form of heteroresistance, and identify key Rtc targets to enhance antibiotic effectiveness.

Abstract The bacterial phage shock protein (Psp) response functions to help cells manage the impacts of agents impairing cell membrane function. The system has relevance to biotechnology and to medicine. Originally discovered in Escherichia coli, Psp proteins and homologues are found in Gram-positive and Gram-negative bacteria, in archaea and in plants. Study of the E. coli and Yersinia enterocolitica Psp systems provides insights into how membrane-associated sensory Psp proteins might perceive membrane stress, signal to the transcription apparatus and use an ATP-hydrolysing transcription activator to produce effector proteins to overcome the stress. Progress in understanding the mechanism of signal transduction by the membrane-bound Psp proteins, regulation of the psp gene-specific transcription activator and the cell biology of the system is presented and discussed. Many features of the action of the Psp system appear to be dominated by states of self-association of the master effector, PspA, and the transcription activator, PspF, alongside a signalling pathway that displays strong conditionality in its requirement.

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane. Fluorescent in situ hybridization is used to identify the subcellular location of mRNAs encoding core photosystem subunits in cyanobacteria. They are clustered at thylakoid surfaces, near the central cytoplasm and nucleoid, by mRNA-binding proteins.

Nitrogen is an essential micronutrient for both plant and animal life and naturally exists in both reactive and inert chemical forms. Modern agriculture is heavily reliant on nitrogen that has been “fixed” into a reactive form via the energetically expensive Haber-Bosch process, with significant environmental consequences. Phenotypic heterogeneity in clonal bacterial batch cultures has been shown for a range of bacterial systems; however, the molecular origins of such heterogeneity and its magnitude are not well understood. Under conditions of extreme low-nitrogen stress in the model diazotroph Klebsiella oxytoca , we found remarkably high heterogeneity of nifHDK gene expression, which codes for the structural genes of nitrogenase, one key enzyme of the global nitrogen cycle. This heterogeneity limited the bulk observed nitrogen-fixing capacity of the population. Using dual-probe, single-cell RNA fluorescent in situ hybridization, we correlated nifHDK expression with that of nifLA and glnK - amtB , which code for the main upstream regulatory components. Through stochastic transcription models and mutual information analysis, we revealed likely molecular origins for heterogeneity in nitrogenase expression. In the wild type and regulatory variants, we found that nifHDK transcription was inherently bursty, but we established that noise propagation through signaling was also significant. The regulatory gene glnK had the highest discernible effect on nifHDK variance, while noise from factors outside the regulatory pathway were negligible. Understanding the basis of inherent heterogeneity of nitrogenase expression and its origins can inform biotechnology strategies seeking to enhance biological nitrogen fixation. Finally, we speculate on potential benefits of diazotrophic heterogeneity in natural soil environments. IMPORTANCE Nitrogen is an essential micronutrient for both plant and animal life and naturally exists in both reactive and inert chemical forms. Modern agriculture is heavily reliant on nitrogen that has been “fixed” into a reactive form via the energetically expensive Haber-Bosch process, with significant environmental consequences. Nitrogen-fixing bacteria provide an alternative source of fixed nitrogen for use in both biotechnological and agricultural settings, but this relies on a firm understanding of how the fixation process is regulated within individual bacterial cells. We examined the cell-to-cell variability in the nitrogen-fixing behavior of Klebsiella oxytoca , a free-living bacterium. The significance of our research is in identifying not only the presence of marked variability but also the specific mechanisms that give rise to it. This understanding gives insight into both the evolutionary advantages of variable behavior as well as strategies for biotechnological applications.

Bacterial enhancer-dependent transcription systems support major adaptive responses and offer a singular paradigm in gene control analogous to complex eukaryotic systems. Here we report new mechanistic insights into the control of one-membrane stress-responsive bacterial enhancer-dependent system. Using millisecond single-molecule fluorescence microscopy of live cells we determine the localizations, two-dimensional diffusion dynamics and stoichiometries of complexes of the bacterial enhancer-binding ATPase PspF during its action at promoters as regulated by inner membrane interacting negative controller PspA. We establish that a stable repressive PspF–PspA complex is located in the nucleoid, transiently communicating with the inner membrane via PspA. The PspF as a hexamer stably binds only one of the two psp promoters at a time, suggesting that psp promoters will fire asynchronously and cooperative interactions of PspF with the basal transcription complex influence dynamics of the PspF hexamer–DNA complex and regulation of the psp promoters. Cellular adaptive responses require temporal and spatial control of key regulatory protein complexes. Mehta et al. describe the dynamic interaction of a transcriptional activator mediating membrane stress response in E. coli with its negative regulator, the cell membrane and the transcription machinery.

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H⁺-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response.

The phage shock protein (Psp) response is found in many Gram-negative enterobacteria, where it helps to maintain the proton motive force (PMF) when the integrity of the inner membrane (IM) is impaired and promotes virulence of pathogens such as Yersinia or Salmonella. In Escherichia coli, Psp comprises seven genes (pspF pspABCDE and pspG) which are organised in a regulon under the control of two Sigma54-dependent promoters. Despite considerable advances, neither the mechanism of Psp induction nor the functioning of the Psp response is fully understood. Recent findings comparing the roles of ArcB in Yersinia enterocolitica and E. coli caused a dispute over the requirement of the twocomponent system ArcAB in Psp signal-transduction. The present study now establishes that ArcAB involvement is conditional and appears to be mediated via protein-protein interactions between ArcB and PspB. The study further suggests that the cellular ubiquinone pool, which acts upstream of ArcAB, may also play a role in Psp signalling whereas dissipation of proton motive force (PMF), generally inferred to be the inducing signal, is not sufficient to mount a Psp response. To gain further insight into its functioning, PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp) were visualised in vivo using fusions to Green fluorescent protein (GFP). To maintain PMF, PspA was proposed to uniformly cover the cytoplasmic face of the IM. However, the present study demonstrates that PspA (and PspG) is highly organised into distinct complexes at the cell pole and the lateral cell membrane. Real-time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain PMF under stress conditions.

RNA is susceptible to damage from various internal and external factors. Given the integral role of RNA, its repair is essential for maintaining proper cell function. A highly conserved RNA repair system, the Rtc system, maintains core RNA components of the translational apparatus. Rtc expression is induced upon various stresses, including exposure to ribosome-targeting antibiotics. Its expression enables cells to rescue growth and survive treatment by conferring transient resistance to these antibiotics. The mechanisms by which Rtc-induced resistance arises are largely unknown. Here, we develop and analyse a computational model of Rtc-regulated maintenance of long-lived RNAs that form part of the translational apparatus. We investigate the mechanistic action of Rtc leading to transient resistance and find that its regulatory structure promotes heterogeneity across bacterial populations, where a resistant sub-population can co-exist with non-expressing, susceptible cells. The finding suggests a complex response underlying adaptive resistance conferred by Rtc with individual cell fates determining antibiotic efficacy. We further analyse the molecular determinants that lead to growth rescue vs suppression and identify components within the Rtc system that may be targeted to potentiate antibiotic effects. Our results provide novel tools for the systems analysis of Rtc-regulated RNA repair and pinpoint testable hypotheses to advance knowledge of RNA repair and its physiological implications.Competing Interest StatementThe authors have declared no competing interest.Footnotes* This revised manuscript contains a correction of a mis-assigned affiliation in the author section.

RNA is susceptible to damage from various internal and external factors. Given the integral role of RNA, its repair is essential for maintaining proper cell function. A highly conserved RNA repair system, the Rtc system, maintains core RNA components of the translational apparatus. In E. coli, Rtc expression is induced upon various stresses, including exposure to ribosome-targeting antibiotics. Its expression enables cells to rescue growth and survive treatment by conferring transient resistance to these antibiotics. The mechanisms by which Rtc-induced resistance arises are largely unknown. Here, we develop and analyse a computational model of Rtc-regulated maintenance of long-lived RNAs that form part of the translational apparatus. Our model analysis and experimental validation provide evidence of cell-to-cell heterogeneity in rtc expression and effects on the translational capacity of cells, indicating that individual cell fates determine antibiotic efficacy and thus rtc may induce a form of heteroresistance. Through further analysis and quantification of single-cell ribosome levels, we then identify targets within the Rtc system that can reduce translational capacity of cells, rendering cells more susceptible and so potentiating antibiotic effects. Our results provide novel tools for the systems analysis of Rtc-regulated RNA repair, they reveal a complex response underpinning resistance conferred by the innate repair system, and they suggest novel avenues to address resistance in E. coli and related clinical pathogens.