Systematic Dissection and Trajectory-Scanning Mutagenesis of the Molecular Interface That Ensures Specificity of Two-Component Signaling Pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk

Temporal Controls of the Asymmetric Cell Division Cycle in Caulobacter crescentus

The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella)

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

Cross-Talk and Information Transfer in Mammalian and Bacterial Signaling

In mammalian and bacterial cells simple phosphorylation circuits play an important role in signaling. Bacteria have hundreds of two-component signaling systems that involve phosphotransfer between a receptor and a response regulator. In mammalian cells a similar pathway is the TGF-beta pathway, where extracellular TGF-beta ligands activate cell surface receptors that phosphorylate Smad proteins, which in turn activate many genes. In TGF-beta signaling the multiplicity of ligands begs the question as to whether cells can distinguish signals coming from different ligands, but transduced through a small set of Smads. Here we use information theory with stochastic simulations of networks to address this question. We find that when signals are transduced through only one Smad, the cell cannot distinguish between different levels of the external ligands. Increasing the number of Smads from one to two significantly improves information transmission as well as the ability to discriminate between ligands. Surprisingly, both total information transmitted and the capacity to discriminate between ligands are quite insensitive to high levels of cross-talk between the two Smads. Robustness against cross-talk requires that the average amplitude of the signals are large. We find that smaller systems, as exemplified by some two-component systems in bacteria, are significantly much less robust against cross-talk. For such system sizes phosphotransfer is also less robust against cross-talk than phosphorylation. This suggests that mammalian signal transduction can tolerate a high amount of cross-talk without degrading information content. This may have played a role in the evolution of new functionalities from small mutations in signaling pathways, allowed for the development of cross-regulation and led to increased overall robustness due to redundancy in signaling pathways. On the other hand the lack of cross-regulation observed in many bacterial two-component systems may partly be due to the loss of information content due to cross-talk

Model-Based Deconvolution of Cell Cycle Time-Series Data Reveals Gene Expression Details at High Resolution

In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure “just-in-time” assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract “single-cell”-like information from population-level time-series expression data. This method removes the effects of 1) variance in growth rate and 2) variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell

Regulatory Response to Carbon Starvation in Caulobacter crescentus

Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells

The genetic organisation of prokaryotic two-component system signalling pathways

<p>Abstract</p> <p>Background</p> <p>Two-component systems (TCSs) are modular and diverse signalling pathways, involving a stimulus-responsive transfer of phosphoryl groups from transmitter to partner receiver domains. TCS gene and domain organisation are both potentially informative regarding biological function, interaction partnerships and molecular mechanisms. However, there is currently little understanding of the relationships between domain architecture, gene organisation and TCS pathway structure.</p> <p>Results</p> <p>Here we classify the gene and domain organisation of TCS gene loci from 1405 prokaryotic replicons (>40,000 TCS proteins). We find that 200 bp is the most appropriate distance cut-off for defining whether two TCS genes are functionally linked. More than 90% of all TCS gene loci encode just one or two transmitter and/or receiver domains, however numerous other geometries exist, often with large numbers of encoded TCS domains. Such information provides insights into the distribution of TCS domains between genes, and within genes. As expected, the organisation of TCS genes and domains is affected by phylogeny, and plasmid-encoded TCS exhibit differences in organisation from their chromosomally-encoded counterparts.</p> <p>Conclusions</p> <p>We provide here an overview of the genomic and genetic organisation of TCS domains, as a resource for further research. We also propose novel metrics that build upon TCS gene/domain organisation data and allow comparisons between genomic complements of TCSs. In particular, '<it>percentage orphaned TCS genes</it>' (or 'Dissemination') and '<it>percentage of complex loci</it>' (or 'Sophistication') appear to be useful discriminators, and to reflect mechanistic aspects of TCS organisation not captured by existing metrics.</p

Trade-Offs and Constraints in Allosteric Sensing

Sensing extracellular changes initiates signal transduction and is the first stage of cellular decision-making. Yet relatively little is known about why one form of sensing biochemistry has been selected over another. To gain insight into this question, we studied the sensing characteristics of one of the biochemically simplest of sensors: the allosteric transcription factor. Such proteins, common in microbes, directly transduce the detection of a sensed molecule to changes in gene regulation. Using the Monod-Wyman-Changeux model, we determined six sensing characteristics – the dynamic range, the Hill number, the intrinsic noise, the information transfer capacity, the static gain, and the mean response time – as a function of the biochemical parameters of individual sensors and of the number of sensors. We found that specifying one characteristic strongly constrains others. For example, a high dynamic range implies a high Hill number and a high capacity, and vice versa. Perhaps surprisingly, these constraints are so strong that most of the space of characteristics is inaccessible given biophysically plausible ranges of parameter values. Within our approximations, we can calculate the probability distribution of the numbers of input molecules that maximizes information transfer and show that a population of one hundred allosteric transcription factors can in principle distinguish between more than four bands of input concentrations. Our results imply that allosteric sensors are unlikely to have been selected for high performance in one sensing characteristic but for a compromise in the performance of many

Adaptable Functionality of Transcriptional Feedback in Bacterial Two-Component Systems

A widespread mechanism of bacterial signaling occurs through two-component systems, comprised of a sensor histidine kinase (SHK) and a transcriptional response regulator (RR). The SHK activates RR by phosphorylation. The most common two-component system structure involves expression from a single operon, the transcription of which is activated by its own phosphorylated RR. The role of this feedback is poorly understood, but it has been associated with an overshooting kinetic response and with fast recovery of previous interrupted signaling events in different systems. Mathematical models show that overshoot is only attainable with negative feedback that also improves response time. Our models also predict that fast recovery of previous interrupted signaling depends on high accumulation of SHK and RR, which is more likely in a positive feedback regime. We use Monte Carlo sampling of the parameter space to explore the range of attainable model behaviors. The model predicts that the effective feedback sign can change from negative to positive depending on the signal level. Variations in two-component system architectures and parameters may therefore have evolved to optimize responses in different bacterial lifestyles. We propose a conceptual model where low signal conditions result in a responsive system with effectively negative feedback while high signal conditions with positive feedback favor persistence of system output