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

2008 ◽  
Vol 190 (17) ◽  
pp. 5981-5988 ◽  
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.

scholarly journals Antibiotic Sensitivity Profiles Determined with an Escherichia coli Gene Knockout Collection: Generating an Antibiotic Bar Code

2010 ◽  
Vol 54 (4) ◽  
pp. 1393-1403 ◽  
Author(s):  
Anne Liu ◽  
Lillian Tran ◽  
Elinne Becket ◽  
Kim Lee ◽  
Laney Chinn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Single Gene ◽  
Antibiotic Sensitivity ◽  
Multidrug Resistant ◽  
Intrinsic Resistance ◽  
Bar Code ◽  
Entire Collection ◽  
Data Points ◽  
Increased Sensitivity

ABSTRACT We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics.

scholarly journals Determination of hypersensitivity to genotoxic agents among Escherichia coli single gene knockout mutants

DNA Repair ◽  
2010 ◽  
Vol 9 (9) ◽  
pp. 949-957 ◽  
Author(s):  
Elinne Becket ◽  
Frank Chen ◽  
Cindy Tamae ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Single Gene ◽  
Knockout Mutants ◽  
Genotoxic Agents

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

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

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.

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

2019 ◽  
Vol 201 (19) ◽  
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.

scholarly journals Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection

2006 ◽  
Vol 2 (1) ◽  
Author(s):  
Tomoya Baba ◽  
Takeshi Ara ◽  
Miki Hasegawa ◽  
Yuki Takai ◽  
Yoshiko Okumura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Single Gene ◽  
Knockout Mutants ◽  
Keio Collection ◽  
K 12

scholarly journals The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

2012 ◽  
Vol 56 (6) ◽  
pp. 3216-3223 ◽  
Author(s):  
Tina Manzhu Kang ◽  
Jessica Yuan ◽  
Angelyn Nguyen ◽  
Elinne Becket ◽  
Hanjing Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Gene Knockout ◽  
Aminoglycoside Antibiotic ◽  
Double Strand Breaks ◽  
Repair Capacity ◽  
Content Type ◽  
Strand Breaks ◽  
Knockout Mutants ◽  
Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts

2010 ◽  
Vol 87 (2) ◽  
pp. 647-655 ◽  
Author(s):  
Ying Zhou ◽  
Takeshi Minami ◽  
Kohsuke Honda ◽  
Takeshi Omasa ◽  
Hisao Ohtake
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Single Gene ◽  
Systematic Screening ◽  
Whole Cell ◽  
Knockout Mutants

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

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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