Protein-level fluctuation correlation at the microcolony level and its application to the Vibrio harveyi quorum-sensing circuit.
ABSTRACT: Gene expression is stochastic, and noise that arises from the stochastic nature of biochemical reactions propagates through active regulatory links. Thus, correlations in gene-expression noise can provide information about regulatory links. We present what to our knowledge is a new approach to measure and interpret such correlated fluctuations at the level of single microcolonies, which derive from single cells. We demonstrated this approach mathematically using stochastic modeling, and applied it to experimental time-lapse fluorescence microscopy data. Specifically, we investigated the relationships among LuxO, LuxR, and the small regulatory RNA qrr4 in the model quorum-sensing bacterium Vibrio harveyi. Our results show that LuxR positively regulates the qrr4 promoter. Under our conditions, we find that qrr regulation weakly depends on total LuxO levels and that LuxO autorepression is saturated. We also find evidence that the fluctuations in LuxO levels are dominated by intrinsic noise. We furthermore propose LuxO and LuxR interact at all autoinducer levels via an unknown mechanism. Of importance, our new method of evaluating correlations at the microcolony level is unaffected by partition noise at cell division. Moreover, the method is first-order accurate and requires less effort for data analysis than single-cell-based approaches. This new correlation approach can be applied to other systems to aid analysis of gene regulatory circuits.
Project description:Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ? 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.
Project description:Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.
Project description:Vibrio vulnificus is a marine bacterium that causes human infections resulting in high mortality. This pathogen harbors five quorum-regulatory RNAs (Qrr1-5) that affect the expression of pathogenicity genes by modulating the expression of the master regulator SmcR. The qrr genes are activated by phosphorylated LuxO to different degrees; qrr2 is strongly activated; qrr3 and qrr5 are moderately activated, and qrr1 and qrr4 are marginally activated and are the only two that do not respond to cell density-dependent regulation. Qrrs function redundantly to inhibit SmcR at low cell density and fully repress when all five are activated. In this study, we found that iron inhibits qrr expression in three distinct ways. First, the iron-ferric uptake regulator (Fur) complex directly binds to qrr promoter regions, inhibiting LuxO activation by competing with LuxO for cis-acting DNA elements. Second, qrr transcription is repressed by iron independently of Fur. Third, LuxO expression is repressed by iron independently of Fur. We also found that, under iron-limiting conditions, the five Qrrs functioned additively, not redundantly, to repress SmcR, suggesting that cells lacking iron enter a high cell density mode earlier and could thereby modulate expression of virulence factors sooner. This study suggests that iron and quorum sensing, along with their cognate regulatory circuits, are linked together in the coordinated expression of virulence factors.
Project description:Quorum sensing is a mechanism of cell-cell communication that bacteria use to control collective behaviours including bioluminescence, biofilm formation and virulence factor production. In the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits, multiple non-coding small regulatory RNAs called the quorum-regulated small RNAs (Qrr sRNAs) function to establish the global quorum-sensing gene expression pattern by modulating translation of multiple mRNAs encoding quorum-sensing regulatory factors. Here we show that the Qrr sRNAs post-transcriptionally activate production of the low cell density master regulator AphA through base pairing to aphA mRNA, and this is crucial for the accumulation of appropriate levels of AphA protein at low cell density. We find that the Qrr sRNAs use unique pairing regions to discriminate between their different targets. Qrr1 is not as effective as Qrr2-5 in activating aphA because Qrr1 lacks one of two required pairing regions. However, Qrr1 is equally effective as the other Qrr sRNAs at controlling targets like luxR and luxO because it harbours all of the required pairing regions for these targets. Sequence comparisons reveal that Vibrionaceae species possessing only qrr1 do not have the aphA gene under Qrr sRNA control. Our findings suggest co-evolving relationships between particular Qrr sRNAs and particular mRNA targets.
Project description:Microarray data for study "The master regulators AphA and LuxR control the Vibrio harveyi quorum-sensing regulon: analysis of their individual and combined effects". Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors AphA and LuxR coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they co-regulate 77 genes. LuxR strongly controls genes both at low-cell-density and high-cell-density, suggesting it is the major quorum-sensing regulator. By contrast, AphA is absent at high-cell-density, and acts to fine-tune quorum-sensing gene expression at low-cell-density. We examined two loci as case studies of co-regulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group lifestyle. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at mid-cell-density. Thus, asymmetric production of AphA and LuxR coupled with differences in their strength and timing of target gene regulation generates a precise temporal pattern of gene expression. Triplicate biological samples of Vibrio harveyi strains KM810 and JV55 were hybridized to Agilent arrays (Amadid design ID: 021087), and one experiment was a control dye-swap, for a total of four experiments in this array set. KM810 contains a luxO D47E phosphomimic allele and a deletion in luxR; JV55 contains a luxO D47E phosphomimic allele and deletions in luxR and aphA.
Project description:All members of the Vibrionaceae harbour LuxO, a response regulator that integrates outputs from various signalling systems, ultimately controlling specific traits that are crucial to the distinct biology of each species. LuxO is phosphorylated in response to low cell density, activating the transcription of a family of small RNAs called Qrrs, which in turn, control the levels of a global regulatory protein conserved within the Vibrionaceae. Although the function of each Qrr is similar, the number of qrr genes varies among the different species. Using a bioinformatics approach, we have determined the number of qrr genes in fully sequenced Vibrionaceae members. Phylogenetic analysis suggests the most recent common ancestor of all Vibrionaceae shared a single, ancestral qrr gene, which duplicated and diverged into multiple qrr genes in some present-day vibrio lineages. To demonstrate that a single qrr gene is sufficient to mediate repression of LitR, the global regulator in Vibrio fischeri, we have performed a series of genetic and phenotypic analyses of the LuxO pathway and its output. Our studies contribute to a better understanding of the ancestral state of these pathways in vibrios, as well as to the evolution and divergence of other sRNAs within different bacterial lineages.
Project description:Gene regulatory interactions are context dependent, active in some cellular states but not in others. Stochastic fluctuations, or 'noise', in gene expression propagate through active, but not inactive, regulatory links. Thus, correlations in gene expression noise could provide a noninvasive means to probe the activity states of regulatory links. However, global, 'extrinsic', noise sources generate correlations even without direct regulatory links. Here we show that single-cell time-lapse microscopy, by revealing time lags due to regulation, can discriminate between active regulatory connections and extrinsic noise. We demonstrate this principle mathematically, using stochastic modeling, and experimentally, using simple synthetic gene circuits. We then use this approach to analyze dynamic noise correlations in the galactose metabolism genes of Escherichia coli. We find that the CRPGalS-GalE feed-forward loop is inactive in standard conditions but can become active in a GalR mutant. These results show how noise can help analyze the context dependence of regulatory interactions in endogenous gene circuits.
Project description:BACKGROUND: A wide range of bacteria species are known to communicate through the so called quorum sensing (QS) mechanism by means of which they produce a small molecule that can freely diffuse in the environment and in the cells. Upon reaching a threshold concentration, the signalling molecule activates the QS-controlled genes that promote phenotypic changes. This mechanism, for its simplicity, has become the model system for studying the emergence of a global response in prokaryotic cells. Yet, how cells precisely measure the signal concentration and act coordinately, despite the presence of fluctuations that unavoidably affects cell regulation and signalling, remains unclear. RESULTS: We propose a model for the QS signalling mechanism in Vibrio fischeri based on the synthetic strains lux01 and lux02. Our approach takes into account the key regulatory interactions between LuxR and LuxI, the autoinducer transport, the cellular growth and the division dynamics. By using both deterministic and stochastic models, we analyze the response and dynamics at the single-cell level and compare them to the global response at the population level. Our results show how fluctuations interfere with the synchronization of the cell activation and lead to a bimodal phenotypic distribution. In this context, we introduce the concept of precision in order to characterize the reliability of the QS communication process in the colony. We show that increasing the noise in the expression of LuxR helps cells to get activated at lower autoinducer concentrations but, at the same time, slows down the global response. The precision of the QS switch under non-stationary conditions decreases with noise, while at steady-state it is independent of the noise value. CONCLUSIONS: Our in silico experiments show that the response of the LuxR/LuxI system depends on the interplay between non-stationary and stochastic effects and that the burst size of the transcription/translation noise at the level of LuxR controls the phenotypic variability of the population. These results, together with recent experimental evidences on LuxR regulation in wild-type species, suggest that bacteria have evolved mechanisms to regulate the intensity of those fluctuations.
Project description:Type VI secretion is critical for Vibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V.?cholerae type VI secretion system genes are encoded in one large and two small clusters. In V.?cholerae, type VI secretion is controlled by quorum sensing, the cell-cell communication process that enables bacteria to orchestrate group behaviours. The quorum-sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum-sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs (Qrr sRNAs) that function between LuxO and HapR in the quorum-sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA-dependent regulation of the type VI secretion system is conserved in pandemic and non-pandemic V.?cholerae strains.
Project description:Quorum sensing (QS) is a process enabling a bacterial population to communicate via small molecules called autoinducers (AIs). This intercellular communication process allows single cells to synchronize their behavior within a population. The marine bacterium Vibrio harveyi ATCC BAA-1116 channels the information of three AI signals into one QS cascade. Three receptors perceive these AIs, the hybrid histidine kinases LuxN, Lux(P)Q and CqsS, to transduce the information to the histidine phosphotransfer (HPt) protein LuxU via phosphorelay, and finally to the response regulator LuxO. Hence, the level of phosphorylated LuxO depends on the AI concentrations. The phosphorylated LuxO (P-LuxO) controls the expression of small regulatory RNAs (sRNAs), which together with the RNA chaperon Hfq, destabilize the transcript of the master regulator luxR. LuxR is responsible for the induction and repression of several genes (e.g., for bioluminescence, exoprotease and siderophore production). In vivo studies with various mutants have demonstrated that the ratio between kinase and phosphatase activities of the individual QS receptors and therefore the P-LuxO/LuxO ratio is crucial not only for the output strength but also for the degree of noise. This study was undertaken to better understand the inherent design principles of this complex signaling cascade, which allows sensing and integration of different signals, but also the differentiated output in individual cells. Therefore, we quantitatively analyzed not only the enzymatic activities, but also the abundance and localization of the three QS receptors. We found that LuxN presents the highest capacity to phosphorylate LuxU, while the phosphatase activity was comparable to LuxQ and CqsS in vitro. In whole cells the copy number of LuxN was higher than that of LuxQ and CqsS, and further increased in the late exponential growth phase. Microscopy experiments indicate that LuxN and LuxQ form independent clusters. Altogether, these results suggest, that the three QS receptors act in parallel, and V. harveyi has developed with LuxN the most dynamic sensing range for HAI-1, the species-specific AI.