scholarly journals High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

2015 ◽  
Vol 81 (8) ◽  
pp. 2827-2840 ◽  
Author(s):  
Yannick D. N. Tremblay ◽  
Philippe Vogeleer ◽  
Mario Jacques ◽  
Josée Harel
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Biofilm Formation ◽  
Cell Biology ◽  
Shear Force ◽  
Host Pathogen Interactions ◽  
Content Type ◽  
E Coli ◽  
Rpmi Medium ◽  
Host Pathogen

ABSTRACTBiofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemblein vivoconditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenicEscherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenicE. coliin RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherentE. colimutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved inE. colibiofilm formation and host-pathogen interactions under shear force.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Kelvin G. K. Goh ◽  
Danilo G. Moriel ◽  
Steven J. Hancock ◽  
Minh-Duy Phan ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Secretion System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Type V Secretion ◽  
K 12 ◽  
Type V

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

scholarly journals Flagellum-Mediated Mechanosensing and RflP Control Motility State of Pathogenic Escherichia coli

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Leanid Laganenka ◽  
María Esteban López ◽  
Remy Colin ◽  
Victor Sourjik
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Porous Medium ◽  
Biofilm Formation ◽  
Liquid Culture ◽  
Content Type ◽  
E Coli ◽  
Surface Contact ◽  
Conditional Inhibition ◽  
Flagellar Genes

ABSTRACT Bacterial flagellar motility plays an important role in many processes that occur at surfaces or in hydrogels, including adhesion, biofilm formation, and bacterium-host interactions. Consequently, expression of flagellar genes, as well as genes involved in biofilm formation and virulence, can be regulated by the surface contact. In a few bacterial species, flagella themselves are known to serve as mechanosensors, where an increased load on flagella experienced during surface contact or swimming in viscous media controls gene expression. In this study, we show that gene regulation by motility-dependent mechanosensing is common among pathogenic Escherichia coli strains. This regulatory mechanism requires flagellar rotation, and it enables pathogenic E. coli to repress flagellar genes at low loads in liquid culture, while activating motility in porous medium (soft agar) or upon surface contact. It also controls several other cellular functions, including metabolism and signaling. The mechanosensing response in pathogenic E. coli depends on the negative regulator of motility, RflP (YdiV), which inhibits basal expression of flagellar genes in liquid. While no conditional inhibition of flagellar gene expression in liquid and therefore no upregulation in porous medium was observed in the wild-type commensal or laboratory strains of E. coli, mechanosensitive regulation could be recovered by overexpression of RflP in the laboratory strain. We hypothesize that this conditional activation of flagellar genes in pathogenic E. coli reflects adaptation to the dual role played by flagella and motility during infection. IMPORTANCE Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.

scholarly journals The Catabolite Repressor Protein-Cyclic AMP Complex RegulatescsgDand Biofilm Formation in Uropathogenic Escherichia coli

2016 ◽  
Vol 198 (24) ◽  
pp. 3329-3334 ◽  
Author(s):  
David A. Hufnagel ◽  
Margery L. Evans ◽  
Sarah E. Greene ◽  
Jerome S. Pinkner ◽  
Scott J. Hultgren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Extracellular Matrix ◽  
Cyclic Amp ◽  
Intergenic Region ◽  
Biofilm Formation ◽  
Cellulose Production ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
K1 Capsule

ABSTRACTThe extracellular matrix protectsEscherichia colifrom immune cells, oxidative stress, predation, and other environmental stresses. Production of theE. coliextracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenicE. coli(UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms throughcsgD. The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion ofcyaAresulted in reduced extracellular matrix production and biofilm formation. Thecataboliterepressorprotein (CRP) positively regulatedcsgDtranscription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaAand Δcrpdid not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within thecsgD-csgBintergenic region, and purified CRP could gel shift thecsgD-csgBintergenic region. Additionally, we found that CRP binded upstream ofkpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influenceE. colibiofilms through transcriptional regulation ofcsgD.IMPORTANCEThecataboliterepressorprotein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on theEscherichia colichromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874–5893, 2004,https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibitsE. colibiofilm formation, and ΔcyaAand Δcrpmutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406–3410, 2002,https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms throughcsgD. Additionally, we propose that cAMP may work as a signaling compound for uropathogenicE. coli(UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

Uncovering complex molecular networks in host–pathogen interactions using systems biology

2019 ◽  
Vol 3 (4) ◽  
pp. 371-378
Author(s):  
Joshua M. Peters ◽  
Sydney L. Solomon ◽  
Christopher Y. Itoh ◽  
Bryan D. Bryson
Keyword(s):  
Systems Biology ◽  
High Throughput ◽  
Cell Biology ◽  
Molecular Networks ◽  
Host Pathogen Interactions ◽  
Exciting Field ◽  
Specific Host ◽  
Biomolecular Network ◽  
Host Pathogen ◽  
Analytical Frameworks

Abstract Interactions between pathogens and their hosts can induce complex changes in both host and pathogen states to privilege pathogen survival or host clearance of the pathogen. To determine the consequences of specific host–pathogen interactions, a variety of techniques in microbiology, cell biology, and immunology are available to researchers. Systems biology that enables unbiased measurements of transcriptomes, proteomes, and other biomolecules has become increasingly common in the study of host–pathogen interactions. These approaches can be used to generate novel hypotheses or to characterize the effects of particular perturbations across an entire biomolecular network. With proper experimental design and complementary data analysis tools, high-throughput omics techniques can provide novel insights into the mechanisms that underlie processes from phagocytosis to pathogen immune evasion. Here, we provide an overview of the suite of biochemical approaches for high-throughput analyses of host–pathogen interactions, analytical frameworks for understanding the resulting datasets, and a vision for the future of this exciting field.

scholarly journals Analysis of the Resistome of a Multidrug-Resistant NDM-1-Producing Escherichia coli Strain by High-Throughput Genome Sequencing

2011 ◽  
Vol 55 (9) ◽  
pp. 4224-4229 ◽  
Author(s):  
Laurent Poirel ◽  
Rémy A. Bonnin ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Genome Sequencing ◽  
High Throughput ◽  
Multidrug Resistant ◽  
Replicase Gene ◽  
Content Type ◽  
E Coli ◽  
Resistance Determinants ◽  
16S Rna ◽  
Carbapenemase Gene

ABSTRACTThe resistome of the multidrug-resistantEscherichia colistrain 271 carrying the plasmid-mediatedblaNDM-1carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying theblaNDM-1gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of theblaNDM-1gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression ofblaNDM-1was associated with the insertion sequence ISAba125likely originating fromAcinetobacter baumannii. E. coli271 accumulated multiple resistance determinants, including five β-lactamase genes (comprising the extended-spectrum β-lactamase CTX-M-15), two 16S RNA methylase ArmA- and RmtB-encoding genes, and theqepAgene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsicampCgene ofE. colithat was weakly expressed. The multidrug resistance pattern observed forE. coli271 was therefore the result of combined chromosome- and plasmid-encoded mechanisms.

scholarly journals Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

2016 ◽  
Vol 82 (17) ◽  
pp. 5320-5331 ◽  
Author(s):  
Cathy L. Abberton ◽  
Ludmila Bereschenko ◽  
Paul W. J. J. van der Wielen ◽  
Cindy J. Smith
Keyword(s):  
Escherichia Coli ◽  
Shear Stress ◽  
Drinking Water ◽  
Biofilm Formation ◽  
Pipe Wall ◽  
Health Concern ◽  
Wall Material ◽  
Content Type ◽  
E Coli ◽  
Contamination Events

ABSTRACTEscherichia coliis the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is thatE. colibacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate ofE. coliin drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of threeE. colistrains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, withT90(time taken for a reduction in cell number of 1 log10unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA,tuf,uidA, androdAgenes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm−2; BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P> 0.05 byttest). Finally,E. coliregrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods forE. coli. The results of this work highlight that whenE. colienters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely.IMPORTANCEThe provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coliis used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate ofE. colion entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, ifE. colibacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains ofE. coliin drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely.

scholarly journals Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats

2011 ◽  
Vol 79 (12) ◽  
pp. 4819-4827 ◽  
Author(s):  
Jin-Hyung Lee ◽  
Sushil Chandra Regmi ◽  
Jung-Ae Kim ◽  
Moo Hwan Cho ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Trinitrobenzene Sulfonic Acid ◽  
Content Type ◽  
E Coli ◽  
Colon Inflammation ◽  
K 12

ABSTRACTPathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagicEscherichia coliO157:H7. The antioxidant phloretin, which is abundant in apples, markedly reducedE. coliO157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensalE. coliK-12 biofilms. Also, phloretin reducedE. coliO157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyEandstx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgAandcsgB), and dozens of prophage genes inE. coliO157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production inE. coliO157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory responsein vitrousing human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor ofE. coliO157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensalE. colibiofilms.

scholarly journals Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12

2018 ◽  
Vol 200 (7) ◽  
Author(s):  
Soonkyu Hwang ◽  
Donghui Choe ◽  
Minseob Yoo ◽  
Sanghyuk Cho ◽  
Sun Chang Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Metabolic Pathways ◽  
Genetic Information ◽  
Peptide Transporter ◽  
Pyruvate Metabolism ◽  
Content Type ◽  
E Coli ◽  
Uptake Activity ◽  
K 12

ABSTRACT Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli . Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 10 5 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli , which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli . IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli .

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