Beneficial and detrimental genes in the cellular response to replication arrest.

DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

Phenotypic diversity and temporal variability in a bacterial signaling network revealed by single-cell FRET.

We present in vivo single-cell FRET measurements in the Escherichia coli chemotaxis system that reveal pervasive signaling variability, both across cells in isogenic populations and within individual cells over time. We quantify cell-to-cell variability of adaptation, ligand response, as well as steady-state output level, and analyze the role of network design in shaping this diversity from gene expression noise. In the absence of changes in gene expression, we find that single cells demonstrate strong temporal fluctuations. We provide evidence that such signaling noise can arise from at least two sources: (i) stochastic activities of adaptation enzymes, and (ii) receptor-kinase dynamics in the absence of adaptation. We demonstrate that under certain conditions, (ii) can generate giant fluctuations that drive signaling activity of the entire cell into a stochastic two-state switching regime. Our findings underscore the importance of molecular noise, arising not only in gene expression but also in protein networks.

Dose-Response Analysis of Chemotactic Signaling Response in Salmonella typhimurium LT2 upon Exposure to Cysteine/Cystine Redox Pair.

The chemotaxis system enables motile bacteria to search for an optimum level of environmental factors. Salmonella typhimurium senses the amino acid cysteine as an attractant and its oxidized dimeric form, cystine, as a repellent. We investigated the dose-response dependence of changes in chemotactic signaling activity upon exposure to cysteine and cystine of S. typhimurium LT2 using in vivo fluorescence resonance energy transfer (FRET) measurements. The dose-response curve of the attractant response to cysteine had a sigmoidal shape, typical for receptor-ligand interactions. However, in a knockout strain of the chemoreceptor genes tsr and tar, we detected a repellent response to cysteine solutions, scaling linearly with the logarithm of the cysteine concentration. Interestingly, the magnitude of the repellent response to cystine also showed linear dependence to the logarithm of the cystine concentration. This linear dependence was observed over more than four orders of magnitude, where detection started at nanomolar concentrations. Notably, low concentrations of another oxidized compound, benzoquinone, triggered similar responses. In contrast to S. typhimurium 14028, where no response to cystine was observed in a knockout strain of chemoreceptor genes mcpB and mcpC, here we showed that McpB/McpC-independent responses to cystine existed in the strain S. typhimurium LT2 even at nanomolar concentrations. Additionally, knocking out mcpB and mcpC did not affect the linear dose-response dependence, whereas enhanced responses were only observed to solutions that where not pH neutral (>100 µM cystine) in the case of McpC overexpression. We discuss that the linear dependence of the response on the logarithm of cystine concentrations could be a result of a McpB/C-independent redox-sensing pathway that exists in S. typhimurium LT2. We supported this hypothesis with experiments with defined cysteine/cystine mixed solutions, where a transition from repellent to attractant response occurred depending on the estimated redox potential.

Salmonella chemoreceptors McpB and McpC mediate a repellent response to L-cystine: a potential mechanism to avoid oxidative conditions.

Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility-plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L-cysteine and its oxidized form, L-cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild-type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for Escherichia coli. The repellent response to cystine was mediated by McpB/C. Adaptive recovery upon cystine exposure required the methyl-transferase/-esterase pair, CheR/CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant-like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.

Response rescaling in bacterial chemotaxis.

Sensory systems rescale their response sensitivity upon adaptation according to simple strategies that recur in processes as diverse as single-cell signaling, neural network responses, and whole-organism perception. Here, we study response rescaling in Escherichia coli chemotaxis, where adaptation dynamically tunes the cells' motile response during searches for nutrients. Using in vivo fluorescence resonance energy transfer (FRET) measurements on immobilized cells, we demonstrate that the design of this prokaryotic signaling network follows the fold-change detection (FCD) strategy, responding faithfully to the shape of the input profile irrespective of its absolute intensity. Using a microfluidics-based assay for free swimming cells, we confirm intensity-independent gradient responses at the behavioral level. By theoretical analysis, we identify a set of sufficient conditions for FCD in E. coli chemotaxis, which leads to the prediction that the adaptation timescale is invariant with respect to the background input level. Additional FRET experiments confirm that the adaptation timescale is invariant over an ∼10,000-fold range of background concentrations. These observations in a highly optimized bacterial system support the concept that FCD represents a robust sensing strategy for spatial searches. To our knowledge, these experiments provide a unique demonstration of FCD in any biological sensory system.

Mobility of G proteins is heterogeneous and polarized during chemotaxis.

The interaction of G-protein-coupled receptors with G proteins is a key event in transmembrane signal transduction that leads to vital decision-making by the cell. Here, we applied single-molecule epifluorescence microscopy to study the mobility of both the Gbetagamma and the Galpha2 subunits of the G protein heterotrimer in comparison with the cAMP receptor responsible for chemotactic signaling in Dictyostelium discoideum. Our experimental results suggest that approximately 30% of the G protein heterotrimers exist in receptor-precoupled complexes. Upon stimulation in a chemotactic gradient, this complex dissociates, subsequently leading to a linear diffusion and collision amplification of the external signal. We further found that Gbetagamma was partially immobilized and confined in an agonist-, F-actin- and Galpha2-dependent fashion. This led to the hypothesis that functional nanometric domains exist in the plasma membrane, which locally restrict the activation signal, and in turn, lead to faithful and efficient chemotactic signaling.