VMD-PhD Student, University of Pennsylvania
Many bacteria colonize humans or other animals and play a significant role in maintaining health or causing disease. Bacteria can generate the energy needed for cell growth and division by metabolizing numerous different nutrients. How bacterial cells decide which compounds to use for maximal energy production when entering a host is not well understood and depends on sensing what nutrients are available and, in some cases, anticipating what nutrients might soon become available. Our research is aimed at understanding how bacteria that colonize the intestinal tract regulate the metabolism of a particular nutrient when moving from an environment where oxygen is available to one where oxygen is unavailable.
The bacterium Escherichia coli regulates the metabolism of a specific nutrient (trimethylamine oxide, or TMAO) differently when oxygen is present or absent, but we don’t understand how or why E. coli does this. To find out, our lab developed strains of E. coli containing markers that tell when and how strongly genes in the TMAO metabolic pathway are expressed. When we observe these strains with a microscope, we can tell how much each gene is expressed in each individual bacterial cell and build a model of how all the genes work together to regulate the TMAO metabolic pathway. Using this approach, we found that individual cells express the TMAO metabolism genes in random bursts when oxygen is present but uniformly when oxygen is absent. With further investigation, we went on to discover that a single protein controls this behavior by sensing oxygen in the environment and specifically regulating the expression of two other proteins that are required for sensing TMAO and turning on expression of the TMAO metabolism genes. Additionally, we showed that the random bursts of expression when oxygen is present help cells survive a transition to a low-oxygen environment.
TMAO in the bloodstream is an established risk factor for the development of cardiovascular disease, and the accumulation of TMAO in the blood is dependent on the metabolic activity of gut bacteria. We want to understand aspects of this activity that haven’t been explained and that may be important in the development of TMAO-related cardiovascular disease and infectious endocarditis. We hope our work will provide insights into bacterial bet-hedging strategies and signal transduction, which are involved in the development of many bacterial diseases.
Abstract: Escherichia coli senses and responds to trimethylamine-N-oxide (TMAO) in the environment through the TorT-TorS-TorR signal transduction system. The periplasmic protein TorT binds TMAO and stimulates the hybrid kinase TorS to phosphorylate the response regulator TorR through a phosphorelay. Phosphorylated TorR, in turn, activates transcription of the torCAD operon, which encodes the proteins required for anaerobic respiration via reduction of TMAO to trimethylamine. Interestingly, E. coli respires TMAO in both the presence and absence of oxygen, a behavior that is markedly different from the utilization of other alternative electron acceptors by this bacterium. Here we describe an unusual form of regulation by oxygen for this system. While the average level of torCAD transcription is the same for aerobic and anaerobic cultures containing TMAO, the behavior across the population of cells is strikingly different under the two growth conditions. Cellular levels of torCAD transcription in aerobic cultures are highly heterogeneous, in contrast to the relatively homogeneous distribution in anaerobic cultures. Thus, oxygen regulates the variance of the output but not the mean for the Tor system. We further show that this oxygen-dependent variability stems from the phosphorelay.Trimethylamine-N-oxide (TMAO) is utilized by numerous bacteria as an electron acceptor for anaerobic respiration. In E. coli, expression of the proteins required for TMAO respiration is tightly regulated by a signal transduction system that is activated by TMAO. Curiously, although oxygen is the energetically preferred electron acceptor, TMAO is respired even in the presence of oxygen. Here we describe an interesting and unexpected form of regulation for this system in which oxygen produces highly variable expression of the TMAO utilization proteins across a population of cells without affecting the mean expression of these proteins. To our knowledge, this is the first reported example of a stimulus regulating the variance but not the mean output of a signaling system.
Pub.: 01 Apr '15, Pinned: 23 Aug '17
Abstract: In the absence of oxygen, Escherichia coli can use alternative exogenous electron acceptors, including trimethylamine oxide (TMAO), to generate energy. In this study, we showed that in contrast to the other anaerobic respiratory systems, the TMAO reductase (Tor) system was expressed during both aerobiosis and anaerobiosis. By using a torA-lacZ fusion and quantitative reverse transcription polymerase chain reaction, we established that the torCAD operon encoding the Tor system was induced in the presence of TMAO mainly during exponential phase, and that optimal induction required a certain level of DNA supercoiling. We also showed that the presence of oxygen prevented neither the biogenesis of the Tor system nor the reduction of TMAO. The physiological role of TMAO reduction during aerobiosis has not been yet established, but our experiments suggest that alkaline TMA production could enhance the growth conditions by increasing the pH of the culture.
Pub.: 14 Sep '07, Pinned: 27 Aug '17
Abstract: Multistability, the capacity to achieve multiple internal states in response to a single set of external inputs, is the defining characteristic of a switch. Biological switches are essential for the determination of cell fate in multicellular organisms, the regulation of cell-cycle oscillations during mitosis and the maintenance of epigenetic traits in microbes. The multistability of several natural and synthetic systems has been attributed to positive feedback loops in their regulatory networks. However, feedback alone does not guarantee multistability. The phase diagram of a multistable system, a concise description of internal states as key parameters are varied, reveals the conditions required to produce a functional switch. Here we present the phase diagram of the bistable lactose utilization network of Escherichia coli. We use this phase diagram, coupled with a mathematical model of the network, to quantitatively investigate processes such as sugar uptake and transcriptional regulation in vivo. We then show how the hysteretic response of the wild-type system can be converted to an ultrasensitive graded response. The phase diagram thus serves as a sensitive probe of molecular interactions and as a powerful tool for rational network design.
Pub.: 20 Feb '04, Pinned: 27 Aug '17
Abstract: Stochastic mechanisms are ubiquitous in biological systems. Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations. This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-lambda, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals. We sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations within single cells (biochemical noise) could explain the variation of gene expression levels between cells in a genetically identical population (phenotypic noise). We have isolated the biochemical contribution to phenotypic noise from that of other noise sources by carrying out a series of differential measurements. We varied independently the rates of transcription and translation of a single fluorescent reporter gene in the chromosome of Bacillus subtilis, and we quantitatively measured the resulting changes in the phenotypic noise characteristics. We report that of these two parameters, increased translational efficiency is the predominant source of increased phenotypic noise. This effect is consistent with a stochastic model of gene expression in which proteins are produced in random and sharp bursts. Our results thus provide the first direct experimental evidence of the biochemical origin of phenotypic noise, demonstrating that the level of phenotypic variation in an isogenic population can be regulated by genetic parameters.
Pub.: 23 Apr '02, Pinned: 27 Aug '17
Abstract: Organisms in fluctuating environments must constantly adapt their behavior to survive. In clonal populations, this may be achieved through sensing followed by response or through the generation of diversity by stochastic phenotype switching. Here we show that stochastic switching can be favored over sensing when the environment changes infrequently. The optimal switching rates then mimic the statistics of environmental changes. We derive a relation between the long-term growth rate of the organism and the information available about its fluctuating environment.
Pub.: 27 Aug '05, Pinned: 27 Aug '17
Abstract: In response to impending anoxic conditions, denitrifying bacteria sustain respiratory metabolism by producing enzymes for reducing nitrogen oxyanions/-oxides (NOx) to N2 (denitrification). Since denitrifying bacteria are non-fermentative, the initial production of denitrification proteome depends on energy from aerobic respiration. Thus, if a cell fails to synthesise a minimum of denitrification proteome before O2 is completely exhausted, it will be unable to produce it later due to energy-limitation. Such entrapment in anoxia is recently claimed to be a major phenomenon in batch cultures of the model organism Paracoccus denitrificans on the basis of measured e(-)-flow rates to O2 and NOx. Here we constructed a dynamic model and explicitly simulated actual kinetics of recruitment of the cells to denitrification to directly and more accurately estimate the recruited fraction (Fden). Transcription of nirS is pivotal for denitrification, for it triggers a cascade of events leading to the synthesis of a full-fledged denitrification proteome. The model is based on the hypothesis that nirS has a low probability (rden, h(-1)) of initial transcription, but once initiated, the transcription is greatly enhanced through positive feedback by NO, resulting in the recruitment of the transcribing cell to denitrification. We assume that the recruitment is initiated as [O2] falls below a critical threshold and terminates (assuming energy-limitation) as [O2] exhausts. With rden = 0.005 h(-1), the model robustly simulates observed denitrification kinetics for a range of culture conditions. The resulting Fden (fraction of the cells recruited to denitrification) falls within 0.038-0.161. In contrast, if the recruitment of the entire population is assumed, the simulated denitrification kinetics deviate grossly from those observed. The phenomenon can be understood as a 'bet-hedging strategy': switching to denitrification is a gain if anoxic spell lasts long but is a waste of energy if anoxia turns out to be a 'false alarm'.
Pub.: 07 Nov '14, Pinned: 27 Aug '17
Abstract: We present an analytical framework describing the steady-state distribution of protein concentration in live cells, considering that protein production occurs in random bursts with an exponentially distributed number of molecules. We extend this framework for cases of transcription autoregulation and noise propagation in a simple genetic network. This model allows for the extraction of kinetic parameters of gene expression from steady-state distributions of protein concentration in a cell population, which are available from single cell data obtained by flow cytometry or fluorescence microscopy.
Pub.: 13 Dec '06, Pinned: 27 Aug '17
Abstract: Clonal populations of cells exhibit substantial phenotypic variation. Such heterogeneity can be essential for many biological processes and is conjectured to arise from stochasticity, or noise, in gene expression. We constructed strains of Escherichia coli that enable detection of noise and discrimination between the two mechanisms by which it is generated. Both stochasticity inherent in the biochemical process of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) contribute substantially to overall variation. Transcription rate, regulatory dynamics, and genetic factors control the amplitude of noise. These results establish a quantitative foundation for modeling noise in genetic networks and reveal how low intracellular copy numbers of molecules can fundamentally limit the precision of gene regulation.
Pub.: 17 Aug '02, Pinned: 27 Aug '17
Abstract: The genetic circuits that regulate cellular functions are subject to stochastic fluctuations, or 'noise', in the levels of their components. Noise, far from just a nuisance, has begun to be appreciated for its essential role in key cellular activities. Noise functions in both microbial and eukaryotic cells, in multicellular development, and in evolution. It enables coordination of gene expression across large regulons, as well as probabilistic differentiation strategies that function across cell populations. At the longest timescales, noise may facilitate evolutionary transitions. Here we review examples and emerging principles that connect noise, the architecture of the gene circuits in which it is present, and the biological functions it enables. We further indicate some of the important challenges and opportunities going forward.
Pub.: 11 Sep '10, Pinned: 27 Aug '17
Abstract: Transient resistance can allow microorganisms to temporarily survive lethal concentrations of antibiotics. This can be accomplished through stochastic mechanisms, where individual cells within a population display diverse phenotypes to hedge against the appearance of an antibiotic. To date, research on transient stochastic resistance has focused primarily on mechanisms where a subpopulation of cells enters a dormant, drug-tolerant state. However, a fundamental question is whether stochastic gene expression can also generate variable resistance levels among growing cells in a population. We hypothesized that stochastic expression of antibiotic-inducible resistance mechanisms might play such a role. To investigate this, we focused on a prototypical example of such a system: the multiple antibiotic resistance activator MarA. Previous studies have shown that induction of MarA can lead to a multidrug resistant phenotype at the population level. We asked whether MarA expression also has a stochastic component, even when uninduced. Time lapse microscopy showed that isogenic cells express heterogeneous, dynamic levels of MarA, which were correlated with transient antibiotic survival. This finding has important clinical implications, as stochastic expression of resistance genes may be widespread, allowing populations to hedge against the sudden appearance of an antibiotic.
Pub.: 14 Jan '16, Pinned: 27 Aug '17
Abstract: Trimethylamine oxide, which is found in relatively high concentrations in the tissues of marine animals, serves as an electron acceptor in the anaerobic metabolism of a number of bacteria associated primarily with three environments: the marine environment (e.g. Alteromonas and Vibrio), the brackish pond (nonsulfur photosynthetic bacteria), and animal intestines (Enterobacteriaceae). Its reduction to trimethylamine by such bacteria can constitute a major spoilage reaction during the storage of marine fish. In the Enterobacteriaceae, anaerobic respiration with TMAO has been shown to support oxidative phosphorylation. Electron transport to TMAO in these bacteria involves flavin nucleotides, menaquinones, both b- and c-type cytochromes, and a molybdoenzyme reductase. Formate, hydrogen, lactate, and glycerol all serve as electron donors for TMAO respiration. Electrophoretically distinct constitutive and TMAO-induced reductases are synthesized by both E. coli and S. typhimurium. Electron transport to TMAO is repressed both by air and by nitrate. A number of genes involved in TMAO respiration have been mapped, but the structural gene for the inducible TMAO reductase has not yet been firmly established. Oxidative phosphorylation is also supported by TMAO reduction in Alteromonas. In this organism, which is nonfermentative, TMAO respiration resembles aerobic respiration in that intermediates of the TCA cycle are excellent electron donors. Alteromonas exhibits a requirement for NaCl for growth on TMAO and certain electron donors. As in the Enterobacteriaceae, air and nitrate both interfere with TMAO reduction. The role of TMAO reduction in the anaerobic metabolism of nonsulfur purple bacteria has not yet been resolved; it is not clear if TMAO serves simply as an accessory oxidant for fermentation or if TMAO reduction is associated with energy-yielding membrane-bound electron transport. Some of the confusion regarding this bacterial group stems from the fact that much of the work to date has involved parallel studies of TMAO and dimethyl sulfoxide reduction, and it is not yet known whether the two compounds are reduced by the same enzyme. Although our understanding of bacterial TMAO reduction lags far behind our knowledge of bacterial nitrate reduction, it is unlikely that this will always be the case.(ABSTRACT TRUNCATED AT 400 WORDS)
Pub.: 01 Jan '85, Pinned: 27 Aug '17
Abstract: Noise in gene expression is generated at multiple levels, such as transcription and translation, chromatin remodeling and pathway-specific regulation. Studies of individual promoters have suggested different dominating noise sources, raising the question of whether a general trend exists across a large number of genes and conditions. We examined the variation in the expression levels of 43 Saccharomyces cerevisiae proteins, in cells grown under 11 experimental conditions. For all classes of genes and under all conditions, the expression variance was approximately proportional to the mean; the same scaling was observed at steady state and during the transient responses to the perturbations. Theoretical analysis suggests that this scaling behavior reflects variability in mRNA copy number, resulting from random 'birth and death' of mRNA molecules or from promoter fluctuations. Deviation of coexpressed genes from this general trend, including high noise in stress-related genes and low noise in proteasomal genes, may indicate fluctuations in pathway-specific regulators or a differential activation pattern of the underlying gene promoters.
Pub.: 23 May '06, Pinned: 27 Aug '17
Abstract: Induction of the torCAD operon, encoding the trimethylamine N-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443-->Asp723-->His850-->Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)-->His850-->Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of tor operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators.
Pub.: 29 Mar '01, Pinned: 27 Aug '17
Abstract: The gut microbiota is a complex and densely populated community in a dynamic environment determined by host physiology. We investigated how intestinal oxygen levels affect the composition of the fecal and mucosally adherent microbiota.We used the phosphorescence quenching method and a specially designed intraluminal oxygen probe to dynamically quantify gut luminal oxygen levels in mice. 16S ribosomal RNA gene sequencing was used to characterize the microbiota in intestines of mice exposed to hyperbaric oxygen, human rectal biopsy and mucosal swab samples, and paired human stool samples.Average Po2 values in the lumen of the cecum were extremely low (<1 mm Hg). In altering oxygenation of mouse intestines, we observed that oxygen diffused from intestinal tissue and established a radial gradient that extended from the tissue interface into the lumen. Increasing tissue oxygenation with hyperbaric oxygen altered the composition of the gut microbiota in mice. In human beings, 16S ribosomal RNA gene analyses showed an increased proportion of oxygen-tolerant organisms of the Proteobacteria and Actinobacteria phyla associated with rectal mucosa, compared with feces. A consortium of asaccharolytic bacteria of the Firmicute and Bacteroidetes phyla, which primarily metabolize peptones and amino acids, was associated primarily with mucus. This could be owing to the presence of proteinaceous substrates provided by mucus and the shedding of the intestinal epithelium.In an analysis of intestinal microbiota of mice and human beings, we observed a radial gradient of microbes linked to the distribution of oxygen and nutrients provided by host tissue.
Pub.: 22 Jul '14, Pinned: 27 Aug '17
Abstract: Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.
Pub.: 10 Jun '08, Pinned: 23 Aug '17
Abstract: Gene regulation by two-component systems has traditionally been studied using assays that involve averages over large numbers of cells. Single-cell measurements of transcription offer a complementary approach that provides the distribution of gene expression among the population. This chapter focuses on methods for using fluorescence microscopy and fluorescent proteins to study gene expression in single cells.
Pub.: 05 Jul '07, Pinned: 23 Aug '17
Abstract: In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 mum. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.
Pub.: 17 Oct '06, Pinned: 23 Aug '17
Abstract: Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell's ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational "memory" caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.
Pub.: 08 Mar '08, Pinned: 23 Aug '17
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