Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition

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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An integrated view on the metabolic regulation in response to culture environments and the specific gene knockout in Escherichia coli

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.08.334 ◽

2009 ◽

Vol 108 ◽

pp. S114

Author(s):

Kazuyuki Shimizu

Keyword(s):

Escherichia Coli ◽

Metabolic Regulation ◽

Gene Knockout ◽

Specific Gene

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Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

Malaysian Journal of Microbiology ◽

10.21161/mjm.220605 ◽

2006 ◽

Author(s):

Aini, A. R.* ◽

Shirai, Y. ◽

Hassan, M. A. ◽

Shimizu, K.

Keyword(s):

Escherichia Coli ◽

Enzyme Activities ◽

Metabolic Regulation ◽

Gene Knockout ◽

Intracellular Metabolite ◽

Metabolite Concentrations

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The effect of pfl gene knockout on the metabolism for optically pure d-lactate production by Escherichia coli

Applied Microbiology and Biotechnology ◽

10.1007/s00253-003-1499-9 ◽

2004 ◽

Vol 64 (3) ◽

pp. 367-375 ◽

Cited By ~ 96

Author(s):

J. Zhu ◽

K. Shimizu

Keyword(s):

Escherichia Coli ◽

Gene Knockout ◽

Lactate Production ◽

Optically Pure

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00906-09 ◽

2010 ◽

Vol 54 (4) ◽

pp. 1393-1403 ◽

Cited By ~ 180

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.

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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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