Zhongge Zhang

Interdependence of cell growth and gene expression: origins and consequences.

Theory of Growth Control Although quantitative studies of growth in bacterial cultures have been made for over 50 years, the relationship between cell proliferation and gene expression has not been clear. Scott et al. (p. 1099; see the Perspective by Lerman and Palsson) have revealed that mass per cell exponentially increased with linear increases in growth rate and that ribosome abundance increased linearly with growth rate depending on the rate of translation. Hence, the systems properties of the biological processes involved in growth can be derived without any molecular understanding of their basis and can be used to establish fundamental properties for the design of biotechnological procedures. Simple mathematical models describe the relationship between bacterial replication, cellular resources, and protein expression. In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

Growth Rate-Dependent Global Effects on Gene Expression in Bacteria

Bacterial gene expression depends not only on specific regulatory mechanisms, but also on bacterial growth, because important global parameters such as the abundance of RNA polymerases and ribosomes are all growth-rate dependent. Understanding of these global effects is necessary for a quantitative understanding of gene regulation and for the design of synthetic genetic circuits. We find that the observed growth-rate dependence of constitutive gene expression can be explained by a simple model using the measured growth-rate dependence of the relevant cellular parameters. More complex growth dependencies for genetic circuits involving activators, repressors, and feedback control were analyzed and verified experimentally with synthetic circuits. Additional results suggest a feedback mechanism mediated by general growth-dependent effects that does not require explicit gene regulation if the expressed protein affects cell growth. This mechanism can lead to growth bistability and promote the acquisition of important physiological functions such as antibiotic resistance and tolerance (persistence).

Combinatorial transcriptional control of the lactose operon of Escherichia coli

The goal of systems biology is to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents involved in a biological system and their complex web of interactions. The lac promoter of Escherichia coli offers the possibility of confronting “system-level” properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offer insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators Lac repressor (LacR) and cAMP-receptor protein (CRP). A previous in vivo study [Setty Y, Mayo AE, Surette MG, Alon U (2003) Proc Natl Acad Sci USA 100:7702–7707] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors that significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we were able to account for the combinatorial control of the lac promoter quantitatively, in terms of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP.

Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli.

We report here the transcriptome analyses of highly expressed genes that are subject to catabolite repression or activation mediated by the cyclic AMP receptor protein (Crp). The results reveal that many operons encoding enzymes of central carbon metabolic pathways (e.g., Krebs cycle enzymes), as well as transporters and enzymes that initiate carbon metabolism, are subject to direct Crp-mediated catabolite repression. By contrast, few enzyme-encoding genes (direct regulation) but many ribosomal protein- and tRNA-encoding genes (indirect regulation) are subject to Crp-dependent glucose activation. Additionally, Crp mediates strong indirect catabolite repression of many cytoplasmic stress response proteins, including the major chaperone proteins, five ATP-dependent protease complexes, and several cold and heat shock proteins. These results were confirmed by (i) phenotypic analyses, (ii) real-time PCR studies, (iii) reporter gene fusion assays, and (iv) previously published reports about representative genes. The results serve to define and extend our appreciation of the Crp regulon.

Coordination of bacterial proteome with metabolism by cyclic AMP signalling

The cyclic AMP (cAMP)-dependent catabolite repression effect in Escherichia coli is among the most intensely studied regulatory processes in biology. However, the physiological function(s) of cAMP signalling and its molecular triggers remain elusive. Here we use a quantitative physiological approach to show that cAMP signalling tightly coordinates the expression of catabolic proteins with biosynthetic and ribosomal proteins, in accordance with the cellular metabolic needs during exponential growth. The expression of carbon catabolic genes increased linearly with decreasing growth rates upon limitation of carbon influx, but decreased linearly with decreasing growth rate upon limitation of nitrogen or sulphur influx. In contrast, the expression of biosynthetic genes showed the opposite linear growth-rate dependence as the catabolic genes. A coarse-grained mathematical model provides a quantitative framework for understanding and predicting gene expression responses to catabolic and anabolic limitations. A scheme of integral feedback control featuring the inhibition of cAMP signalling by metabolic precursors is proposed and validated. These results reveal a key physiological role of cAMP-dependent catabolite repression: to ensure that proteomic resources are spent on distinct metabolic sectors as needed in different nutrient environments. Our findings underscore the power of quantitative physiology in unravelling the underlying functions of complex molecular signalling networks.

Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in Escherichia coli

In Escherichia coli, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the sodB gene, encoding superoxide dismutase, and the aceBAK operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the sdhA-D, sucA-D, and fumA genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under sigmaS control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.

Overflow metabolism in Escherichia coli results from efficient proteome allocation

Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as the Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast-growing cells, including bacteria, fungi and mammalian cells, but its origin has remained unclear despite decades of research. Here we study metabolic overflow in Escherichia coli, and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviours, and accurately predict responses to new perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry.

The Innate Growth Bistability and Fitness Landscapes of Antibiotic-Resistant Bacteria

Introduction Understanding how bacteria harboring antibiotic resistance grow in the presence of antibiotics is critical for predicting the spread and evolution of drug resistance. Because drugs inhibit cell growth and a cell’s growth state globally influences its gene expression, the expression of drug resistance is subject to an innate, growth-mediated feedback, leading to complex behaviors that affect both the characterization and the prevention of antibiotic resistance. We characterized the consequences of this feedback for the growth of antibiotic-resistant bacteria. Fitness landscape and growth bistability. (A) This fitness landscape describes the fitness, or growth rates, of bacterial strains exposed to antibiotics (colored lines indicate the fitness of four example strains). Fitness drops abruptly at high drug concentrations. The shaded area shows a broad region of growth bistability, throughout which we observe that genetically identical cells possessing drug resistance are split into subpopulations of growing and nongrowing cells in response to antibiotics (B, top). Methods We studied the growth of Escherichia coli strains expressing resistance to translation-inhibiting antibiotics, by using both bulk and single-cell techniques. The growth of each strain was quantified in a broad range of drug concentrations by using time-lapse microscopy (to track the responses of individual cells to antibiotics inside a microfluidic chemostat) and by the enrichment of batch cultures for nongrowing cells. We formulated a quantitative phenomenological model to predict the growth rates of drug-resistant strains in the presence of drugs, based on the well-characterized biochemistry of drug and drug-resistance interactions and on bacterial growth laws that dictate relations between cell growth and gene expression. We tested the model predictions for various drugs and resistance mechanisms by constructing strains that constitutively express varying degrees of drug resistance. Results In strains expressing a moderate degree of drug resistance, growth rates dropped abruptly above a critical drug concentration, the minimum inhibitory concentration (MIC), whose value increased linearly with the basal level of resistance expression (see figure below, panel A). Cells exhibited growth bistability over a broad range of drug concentrations below the MIC: Isogenic cells expressing drug resistance coexisted in growing and nongrowing states in a homogeneous environment (panel B). Our model accurately predicted the range of drug concentrations in which growth bistability occurred, as well as the growth rates of the growing subpopulation, without any ad hoc fitting parameters. These findings reveal a plateau-like fitness landscape (panel A), which can be used to study the evolution of drug resistance in environments with varying drug concentrations. Discussion The broad occurrence of growth bistability in drug-resistant bacteria challenges the common notions and measures of drug efficacy and resistance. And because growth bistability can arise without complex regulation when gene expression is coupled to the state of cell growth, similar physiological links may underlie the growth bistability implicated in causing bacterial persistence. The availability of quantitative, predictive models will facilitate the formulation of strategies to limit the efficacy and evolvability of drug resistance. Keeping Quiet Many bacteria overcome antibiotic treatment by expressing proteins that confer antibiotic resistance, for instance, efflux pumps. But when a strain that expresses these antibiotic resistance proteins encounters an environment containing the corresponding drug, the resistance against the drug may paradoxically become silenced in many cells. In this case, a fraction of a population of genetically identical cells will grow in the presence of antibiotics while other subpopulations fail to grow at all. Deris et al. (10.1126/science.1237435) show that this bistable response arises from a built-in global feedback originating in antibiotic-mediated inhibition of growth, which reduces the expression of proteins that protect against growth inhibition. The resulting populations of dormant cells can exceed 50%, among otherwise identical resistance-expressing cells. This is important for antimicrobial treatment strategies because many bacterial cells may remain vulnerable to an antibiotic even when they apparently display strong resistance to it. Identical bacteria can have low-level resistance to low concentrations of drugs that can flip subpopulations into dormancy. To predict the emergence of antibiotic resistance, quantitative relations must be established between the fitness of drug-resistant organisms and the molecular mechanisms conferring resistance. These relations are often unknown and may depend on the state of bacterial growth. To bridge this gap, we have investigated Escherichia coli strains expressing resistance to translation-inhibiting antibiotics. We show that resistance expression and drug inhibition are linked in a positive feedback loop arising from an innate, global effect of drug-inhibited growth on gene expression. A quantitative model of bacterial growth based on this innate feedback accurately predicts the rich phenomena observed: a plateau-shaped fitness landscape, with an abrupt drop in the growth rates of cultures at a threshold drug concentration, and the coexistence of growing and nongrowing populations, that is, growth bistability, below the threshold.

The Ascorbate Transporter of Escherichia coli

The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane alpha-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of L-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent K(m) for L-ascorbate of 9 micro M and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for L-ascorbate utilization and probably encodes a cytoplasmic L-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to L-ascorbate via the phosphoryl transfer pathway: [pathway: see text].

A transporter of Escherichia coli specific for l- and d-methionine is the prototype for a new family within the ABC superfamily

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds.