High-throughput transcriptomics of 409 bacteria-drug pairs reveals drivers of gut microbiota perturbation. - Related Documents




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469601.0000High-throughput transcriptomics of 409 bacteria-drug pairs reveals drivers of gut microbiota perturbation. Many drugs can perturb the gut microbiome, potentially leading to negative health consequences. However, mechanisms of most microorganism-drug responses have not been elucidated at the genetic level. Using high-throughput bacterial transcriptomics, we systematically characterized the gene expression profiles of prevalent human gut bacteria exposed to the most frequently prescribed orally administered pharmaceuticals. Across >400 drug-microorganism pairs, significant and reproducible transcriptional responses were observed, including pathways involved in multidrug resistance, metabolite transport, tartrate metabolism and riboflavin biosynthesis. Importantly, we discovered that statin-mediated upregulation of the AcrAB-TolC efflux pump in Bacteroidales species enhances microbial sensitivity to vitamin A and secondary bile acids. Moreover, gut bacteria carrying acrAB-tolC genes are depleted in patients taking simvastatin, suggesting that drug-efflux interactions generate collateral toxicity that depletes pump-containing microorganisms from patient microbiomes. This study provides a resource to further understand the drivers of drug-mediated microbiota shifts for better informed clinical interventions.202438233648
77210.9997A Transcriptomic Approach to Identify Novel Drug Efflux Pumps in Bacteria. The core genomes of most bacterial species include a large number of genes encoding putative efflux pumps. The functional roles of most of these pumps are unknown, however, they are often under tight regulatory control and expressed in response to their substrates. Therefore, one way to identify pumps that function in antimicrobial resistance is to examine the transcriptional responses of efflux pump genes to antimicrobial shock. By conducting complete transcriptomic experiments following antimicrobial shock treatments, it may be possible to identify novel drug efflux pumps encoded in bacterial genomes. In this chapter we describe a complete workflow for conducting transcriptomic analyses by RNA sequencing, to determine transcriptional changes in bacteria responding to antimicrobials.201829177833
78720.9996Multidrug-resistance efflux pumps - not just for resistance. It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host-defence molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.200616845433
896630.9996Gene expression profile of Campylobacter jejuni in response to macrolide antibiotics. Campylobacter jejuni is a foodborne pathogen that causes gastroenteritis in humans and has developed resistance to various antibiotics. The primary objective of this research was to examine the network of antibiotic resistance in C. jejuni. The study involved the wild and antibiotic-resistant strains placed in the presence and absence of antibiotics to review their gene expression profiles in response to ciprofloxacin via microarray. Differentially expressed genes (DEGs) analysis and Protein-Protein Interaction (PPI) Network studies were performed for these genes. The results showed that the resistance network of C. jejuni is modular, with different genes involved in bacterial motility, capsule synthesis, efflux, and amino acid and sugar synthesis. Antibiotic treatment resulted in the down-regulation of cluster genes related to translation, flagellum formation, and chemotaxis. In contrast, cluster genes involved in homeostasis, capsule formation, and cation efflux were up-regulated. The study also found that macrolide antibiotics inhibit the progression of C. jejuni infection by inactivating topoisomerase enzymes and increasing the activity of epimerase enzymes, trying to compensate for the effect of DNA twisting. Then, the bacterium limits the movement to conserve energy. Identifying the antibiotic resistance network in C. jejuni can aid in developing drugs to combat these bacteria. Genes involved in cell division, capsule formation, and substance transport may be potential targets for inhibitory drugs. Future research must be directed toward comprehending the underlying mechanisms contributing to the modularity of antibiotic resistance and developing strategies to disrupt and mitigate the growing threat of antibiotic resistance effectively.202438393387
951040.9996The Role of Efflux Pumps in the Transition from Low-Level to Clinical Antibiotic Resistance. Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors.202033266054
960650.9996Rapid identification of key antibiotic resistance genes in E. coli using high-resolution genome-scale CRISPRi screening. Bacteria possess a vast repertoire of genes to adapt to environmental challenges. Understanding the gene fitness landscape under antibiotic stress is crucial for elucidating bacterial resistance mechanisms and antibiotic action. To explore this, we conducted a genome-scale CRISPRi screen using a high-density sgRNA library in Escherichia coli exposed to various antibiotics. This screen identified essential genes under antibiotic-induced stress and offered insights into the molecular mechanisms underlying bacterial responses. We uncovered previously unrecognized genes involved in antibiotic resistance, including essential membrane proteins. The screen also underscored the importance of transcriptional modulation of essential genes in antibiotic tolerance. Our findings emphasize the utility of genome-wide CRISPRi screening in mapping the genetic landscape of antibiotic resistance. This study provides a valuable resource for identifying potential targets for antibiotics or antimicrobial strategies. Moreover, it offers a framework for exploring transcriptional regulatory networks and resistance mechanisms in E. coli and other bacterial pathogens.202540352728
951160.9996Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.200919207745
78670.9996The Varied Role of Efflux Pumps of the MFS Family in the Interplay of Bacteria with Animal and Plant Cells. Efflux pumps represent an important and large group of transporter proteins found in all organisms. The importance of efflux pumps resides in their ability to extrude a wide range of antibiotics, resulting in the emergence of multidrug resistance in many bacteria. Besides antibiotics, multidrug efflux pumps can also extrude a large variety of compounds: Bacterial metabolites, plant-produced compounds, quorum-sensing molecules, and virulence factors. This versatility makes efflux pumps relevant players in interactions not only with other bacteria, but also with plant or animal cells. The multidrug efflux pumps belonging to the major facilitator superfamily (MFS) are widely distributed in microbial genomes and exhibit a large spectrum of substrate specificities. Multidrug MFS efflux pumps are present either as single-component transporters or as tripartite complexes. In this review, we will summarize how the multidrug MFS efflux pumps contribute to the interplay between bacteria and targeted host cells, with emphasis on their role in bacterial virulence, in the colonization of plant and animal host cells and in biofilm formation. We will also address the complexity of these interactions in the light of the underlying regulatory networks required for the effective activation of efflux pump genes.201931443538
961380.9996Using Selection by Nonantibiotic Stressors to Sensitize Bacteria to Antibiotics. Evolutionary adaptation of bacteria to nonantibiotic selective forces, such as osmotic stress, has been previously associated with increased antibiotic resistance, but much less is known about potentially sensitizing effects of nonantibiotic stressors. In this study, we use laboratory evolution to investigate adaptation of Enterococcus faecalis, an opportunistic bacterial pathogen, to a broad collection of environmental agents, ranging from antibiotics and biocides to extreme pH and osmotic stress. We find that nonantibiotic selection frequently leads to increased sensitivity to other conditions, including multiple antibiotics. Using population sequencing and whole-genome sequencing of single isolates from the evolved populations, we identify multiple mutations in genes previously linked with resistance to the selecting conditions, including genes corresponding to known drug targets or multidrug efflux systems previously tied to collateral sensitivity. Finally, we hypothesized based on the measured sensitivity profiles that sequential rounds of antibiotic and nonantibiotic selection may lead to hypersensitive populations by harnessing the orthogonal collateral effects of particular pairs of selective forces. To test this hypothesis, we show experimentally that populations evolved to a sequence of linezolid (an oxazolidinone antibiotic) and sodium benzoate (a common preservative) exhibit increased sensitivity to more stressors than adaptation to either condition alone. The results demonstrate how sequential adaptation to drug and nondrug environments can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints governing adaptation to diverse environmental challenges.202031851309
897090.9996Transcriptomic Analyses to Unravel Cronobacter sakazakii Resistance Pathways. The proliferation of antibiotic usage has precipitated the emergence of drug-resistant variants of bacteria, thereby augmenting their capacity to withstand pharmaceutical interventions. Among these variants, Cronobacter sakazakii (C. sakazakii), prevalent in powdered infant formula (PIF), poses a grave threat to the well-being of infants. Presently, global contamination by C. sakazakii is being observed. Consequently, research endeavors have been initiated to explore the strain's drug resistance capabilities, alterations in virulence levels, and resistance mechanisms. The primary objective of this study is to investigate the resistance mechanisms and virulence levels of C. sakazakii induced by five distinct antibiotics, while concurrently conducting transcriptomic analyses. Compared to the susceptible strains prior to induction, the drug-resistant strains exhibited differential gene expression, resulting in modifications in the activity of relevant enzymes and biofilm secretion. Transcriptomic studies have shown that the expression of glutathione S-transferase and other genes were significantly upregulated after induction, leading to a notable enhancement in biofilm formation ability, alongside the existence of antibiotic resistance mechanisms associated with efflux pumps, cationic antimicrobial peptides, and biofilm formation pathways. These alterations significantly influence the strain's resistance profile.202439272551
790100.9996The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.201525788514
8923110.9996The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli. Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. IMPORTANCE: With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.201627879333
8965120.9996Resistance characterization and transcriptomic analysis of imipenem-induced drug resistance in Escherichia coli. BACKGROUND: Bacteria can develop resistance to various antibiotics under selective pressure, leading to multifaceted changes in resistance mechanisms. Transcriptomic sequencing allows for the observation of transcriptional level alterations in cells under antibiotic stress. Understanding the bacterial response to such stress is essential for deciphering their strategy against drug-resistant antibiotics and identifying potential targets for antibiotic development. METHODS: This study using wild-type (WT) Escherichia coli (E. coli) discovered that continuous in vitro induction screening for imipenem-resistant strains resulted in bacteria with enhanced biofilm-forming ability and mutations in antibiotic target sites. Transcriptomic sequencing of the resistant bacteria revealed significant changes in carbon and amino acid metabolism, nutrient assimilation, substance transport, nucleotide metabolism, protein biosynthesis, and cell wall biosynthesis. The up-regulated drug efflux genes were disrupted using gene knockout technology. Drug sensitivity tests indicated that drug efflux has a minimal effect on imipenem resistance. RESULTS: This suggests a strategy for E. coli drug resistance involving the reduction of unnecessary substance synthesis and metabolism, coupled with an increase in activities that aid in resisting foreign threats.202439624129
9599130.9996Antibiotic export: transporters involved in the final step of natural product production. In the fight against antimicrobial resistance (AMR), antibiotic biosynthetic gene clusters are constantly being discovered. These clusters often include genes for membrane transporters that are involved in the export of the produced natural product during biosynthesis and/or subsequent resistance through active efflux. Despite transporter genes being integral parts of these clusters, study of the function of antibiotic export in natural producers such as Streptomyces spp. remains underexplored, in many cases lagging far behind our understanding of the biosynthetic enzymes. More efficient release of antibiotics by producing cells has potential benefits to industrial biotechnology and understanding the relationships between exporters in natural producers and resistance-associated efflux pumps in pathogens can inform our efforts to understand how AMR spreads. Herein we compile and critically assess the literature on the identification and characterization of antibiotic exporters and their contribution to production in natural antibiotic producers. We evaluate examples of how this knowledge could be used in biotechnology to increase yields of the final product or modulate its chemical nature. Finally, we consider the evidence that natural exporters form a reservoir of protein functions that could be hijacked by pathogens as efflux pumps and emphasize the need for much greater understanding of these exporters to fully exploit their potential for applications around human health.201930964430
8922140.9996Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches. Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.202336856434
788150.9996Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.200616614254
9607160.9996Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution. Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress response processes known as adaptive resistance. Adaptive resistance fosters transient tolerance increases and the emergence of mutations conferring heritable drug resistance. In order to extend the applicable lifetime of new antibiotics, we must seek to hinder the occurrence of bacterial adaptive resistance; however, the regulation of adaptation is difficult to identify due to immense heterogeneity emerging during evolution. This study specifically seeks to generate heterogeneity by adapting bacteria to different stresses and then examines gene expression trends across the disparate populations in order to pinpoint key genes and pathways associated with adaptive resistance. The targets identified here may eventually inform strategies for impeding adaptive resistance and prolonging the effectiveness of antibiotic treatment.201728217741
9115170.9996RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria. The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.202438927168
784180.9996Regulation of the AcrAB-TolC efflux pump in Enterobacteriaceae. Bacterial multidrug efflux systems are a major mechanism of antimicrobial resistance and are fundamental to the physiology of Gram-negative bacteria. The resistance-nodulation-division (RND) family of efflux pumps is the most clinically significant, as it is associated with multidrug resistance. Expression of efflux systems is subject to multiple levels of regulation, involving local and global transcriptional regulation as well as post-transcriptional and post-translational regulation. The best-characterised RND system is AcrAB-TolC, which is present in Enterobacteriaceae. This review describes the current knowledge and new data about the regulation of the acrAB and tolC genes in Escherichia coli and Salmonella enterica.201829128373
8968190.9996Antibiotic stress, genetic response and altered permeability of E. coli. BACKGROUND: Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure.200717426813