Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00259-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 2

Author(s):

Darren J. Parker ◽

Pınar Demetci ◽

Gene-Wei Li

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Liquid Medium ◽

Regulatory Networks ◽

Gene Knockout ◽

Single Gene ◽

Point Mutations ◽

Content Type ◽

E Coli ◽

Keio Collection

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.

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Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01824-18 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 3

Author(s):

Jun Li ◽

Haihong Hao ◽

Menghong Dai ◽

Heying Zhang ◽

Jianan Ning ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pumps ◽

Genetic Alterations ◽

Site Directed Mutagenesis ◽

Single Mutation ◽

Genetic Characteristics ◽

Content Type ◽

E Coli ◽

Low Level ◽

High Level

ABSTRACT This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 μg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 μg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

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mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Applied and Environmental Microbiology ◽

10.1128/aem.02110-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6362-6368 ◽

Cited By ~ 12

Author(s):

Ying Xu ◽

Bing Chen ◽

Hongjun Chao ◽

Ning-Yi Zhou

Keyword(s):

Escherichia Coli ◽

Wild Type Strain ◽

Fluorescent Protein ◽

Gene Knockout ◽

Transport Activity ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Green Fluorescent ◽

K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

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Escherichia coli Genomic Diversity within Extraintestinal Acute Infections Argues for Adaptive Evolution at Play

mSphere ◽

10.1128/msphere.01176-20 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Antoine Bridier-Nahmias ◽

Adrien Launay ◽

Alexandre Bleibtreu ◽

Mélanie Magnan ◽

Violaine Walewski ◽

...

Keyword(s):

Escherichia Coli ◽

Mouse Model ◽

Bacterial Infections ◽

Single Gene ◽

Genomic Diversity ◽

Mismatch Repair Gene ◽

Content Type ◽

E Coli ◽

Synonymous Mutations ◽

Acute Infections

ABSTRACT Adaptive processes in chronic bacterial infections are well described, but much less is known about the processes at play during acute infections. Here, by sequencing seven randomly selected isolates per patient, we analyzed Escherichia coli populations from three acute extraintestinal infections in adults (meningitis, pyelonephritis, and peritonitis), in which a high-mutation-rate isolate or mutator isolate was found. The isolates of single patients displayed between a few dozen and more than 200 independent mutations, with up to half being specific to the mutator isolate. Multiple signs of positive selection were evidenced: a high ratio of nonsynonymous to synonymous mutations (Ka/Ks ratio) and strong mutational convergence within and between patients, some of them at loci well known for their adaptive potential, such as rpoS, rbsR, fimH, and fliC. For all patients, the mutator isolate was likely due to a large deletion of a methyl-directed mismatch repair gene, and in two instances, the deletion extended to genes involved in some genetic convergence, suggesting potential coselection. Intrinsic extraintestinal virulence assessed in a mouse model of sepsis showed variable patterns of virulence ranging from non-mouse killer to mouse killer for the isolates from single patients. However, genomic signature and gene inactivation experiments did not establish a link between a single gene and the capacity to kill mice, highlighting the complex and multifactorial nature of the virulence. Altogether, these data indicate that E. coli isolates are adapting under strong selective pressure when colonizing an extraintestinal site. IMPORTANCE Little is known about the dynamics of adaptation in acute bacterial infections. By sequencing multiple isolates from monoclonal extraintestinal Escherichia coli infections in several patients, we were able to uncover traces of selection taking place at short time scales compared to chronic infection. High genomic diversity was observed in the patient isolates, with an excess of nonsynonymous mutations, and the comparison within and between different infections showed patterns of convergence at the gene level, both constituting strong signs of adaptation. The genes targeted were coding mostly for proteins involved in global regulation, metabolism, and adhesion/motility. Moreover, virulence assessed in a mouse model of sepsis was variable among the isolates of single patients, but this difference was left unexplained at the molecular level. This work gives us clues about the E. coli lifestyle transition between commensalism and pathogenicity.

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Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01982-07 ◽

2008 ◽

Vol 190 (17) ◽

pp. 5981-5988 ◽

Cited By ~ 143

Author(s):

Cindy Tamae ◽

Anne Liu ◽

Katherine Kim ◽

Daniel Sitz ◽

Jeeyoon Hong ◽

...

Keyword(s):

Escherichia Coli ◽

High Throughput Screening ◽

Gene Knockout ◽

Single Gene ◽

E Coli ◽

Knockout Mutants ◽

Data Points ◽

Increased Sensitivity ◽

Gene Knockouts

ABSTRACT We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of “codrugs” that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's “intrinsic resistome,” which provides innate resistance to antibiotics.

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Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

Journal of Bacteriology ◽

10.1128/jb.00969-15 ◽

2016 ◽

Vol 198 (11) ◽

pp. 1683-1693 ◽

Cited By ~ 8

Author(s):

Elyse J. Roach ◽

Charles Wroblewski ◽

Laura Seidel ◽

Alison M. Berezuk ◽

Dyanne Brewer ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Bacterial Cell ◽

Site Directed Mutagenesis ◽

Bacterial Cell Division ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Z Ring

ABSTRACTBacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associatedproteins (Zap) for stability throughout the process of division. InEscherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure ofEscherichia coliZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Usingin vitroFtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments.IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins inE. colithat associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure ofE. coliZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

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A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profiling bacterial membrane transporters

Microbiology ◽

10.1099/mic.0.001016 ◽

2021 ◽

Author(s):

Jesus Enrique Salcedo-Sora ◽

Srijan Jindal ◽

Steve O'Hagan ◽

Douglas B. Kell

Keyword(s):

Escherichia Coli ◽

Fluorescent Dyes ◽

Efflux Pump ◽

Gene Knockout ◽

Single Gene ◽

Wild Type ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Effects Of Ph ◽

Small Collection

Our previous work demonstrated that two commonly used fluorescent dyes that were accumulated by wild-type Escherichia coli MG1655 were differentially transported in single-gene knockout strains, and also that they might be used as surrogates in flow cytometric transporter assays. We summarize the desirable properties of such stains, and here survey 143 candidate dyes. We eventually triage them (on the basis of signal, accumulation levels and cost) to a palette of 39 commercially available and affordable fluorophores that are accumulated significantly by wild-type cells of the ‘Keio’ strain BW25113, as measured flow cytometrically. Cheminformatic analyses indicate both their similarities and their (much more considerable) structural differences. We describe the effects of pH and of the efflux pump inhibitor chlorpromazine on the accumulation of the dyes. Even the ‘wild-type’ MG1655 and BW25113 strains can differ significantly in their ability to take up such dyes. We illustrate the highly differential uptake of our dyes into strains with particular lesions in, or overexpressed levels of, three particular transporters or transporter components (yhjV, yihN and tolC). The relatively small collection of dyes described offers a rapid, inexpensive, convenient and informative approach to the assessment of microbial physiology and phenotyping of membrane transporter function.

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Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

Frontiers in Microbiology ◽

10.3389/fmicb.2021.706525 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yoshihiro Ojima ◽

Tomomi Sawabe ◽

Mao Nakagawa ◽

Yuhei O. Tahara ◽

Makoto Miyata ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Outer Membrane ◽

Fluorescent Protein ◽

Gene Knockout ◽

Single Gene ◽

Membrane Vesicles ◽

Coli Strain ◽

Outer Membrane Vesicles ◽

E Coli

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2147 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2147-2153 ◽

Cited By ~ 69

Author(s):

M Pizza ◽

M R Fontana ◽

M M Giuliani ◽

M Domenighini ◽

C Magagnoli ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Neutralizing Antibodies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

E Coli ◽

Heat Labile ◽

A Subunit ◽

Adp Ribosyltransferase ◽

Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

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