Development of a versatile method for targeted gene deletion and insertion by using the pyrF gene in the psychrotrophic bacterium, Shewanella livingstonensis Ac10

Ito, Tomokazu; Gong, Chunjie; Kawamoto, Jun; Kurihara, Tatsuo

doi:10.1016/j.jbiosc.2016.06.004

Cited by 13 publications

(15 citation statements)

References 21 publications

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“…The PLs prepared as described above were hydrolyzed with Phospholipase A2 (PLA2, P6534, Sigma, St. Louis, MO). The resulting sn-2 fatty acyl groups were extracted by the Dole's method [34,43,44] and analyzed by gas chromatography-mass spectrometry [GC-MS, Clarus 680 gas chromatograph interfaced with Clarus SQ 8C mass spectrometer (Perkin Elmer, Wellesley, MA) equipped with an Agilent J&W GC column DB-1 (Agilent Technologies Inc., Santa Clara, CA, USA)], as described previously [45]. Lysophospholipids (LPLs) were analyzed using the total lipid extracts from an aliquot of the PLA2 reaction product by ESI-MS as described above.…”

Section: Analysis Of the Sn-1 And Sn-2 Fatty Acyl Groups Of Plsmentioning

confidence: 99%

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation

et al. 2020

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Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into the sn-2 position of membrane phospholipids (PLs). Various bacteria produce multiple LPAATs, whereas it is believed that Escherichia coli produces only one essential LPAAT homolog, PlsC—the deletion of which is lethal. However, we found that E. coli possesses another LPAAT homolog named YihG. Here, we show that overexpression of YihG in E. coli carrying a temperature-sensitive mutation in plsC allowed its growth at non-permissive temperatures. Analysis of the fatty acyl composition of PLs from the yihG-deletion mutant (∆yihG) revealed that endogenous YihG introduces the cis-vaccenoyl group into the sn-2 position of PLs. Loss of YihG did not affect cell growth or morphology, but ∆yihG cells swam well in liquid medium in contrast to wild-type cells. Immunoblot analysis showed that FliC was highly expressed in ∆yihG cells, and this phenotype was suppressed by expression of recombinant YihG in ∆yihG cells. Transmission electron microscopy confirmed that the flagellar structure was observed only in ∆yihG cells. These results suggest that YihG has specific functions related to flagellar formation through modulation of the fatty acyl composition of membrane PLs.

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Section: Analysis Of the Sn-1 And Sn-2 Fatty Acyl Groups Of Plsmentioning

confidence: 99%

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation

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“…The EPA strain, which was derived from the pyrF strain of S. livingstonensis Ac10 by deleting the orf5 gene (Ito et al, 2016), and its derivatives were cultivated at 4 • C in Luria-Bertani (LB) medium that was, when indicated, supplemented with 50 µg/mL kanamycin (Km), 50 µg/mL chloramphenicol (Cm), 50 µg/mL rifampicin (Rf), and/or 40 µg/mL uracil (Ura). EPA and DHA were dissolved in ethanol and added to LB medium at a final concentration of 32 µM (or 128 µM when indicated).…”

Section: Bacterial Strain and Cultivationmentioning

confidence: 99%

Bioconversion From Docosahexaenoic Acid to Eicosapentaenoic Acid in the Marine Bacterium Shewanella livingstonensis Ac10

et al. 2020

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Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which belong to the same class of long chain ω-3 polyunsaturated fatty acids (PUFAs), are present in marine γ-proteobacteria. In contrast to their de novo biosynthesis that has been intensively studied, their metabolic fates remain largely unknown. Detailed information regarding bacterial ω-3 PUFA metabolism would be beneficial for understanding the physiological roles of EPA/DHA as well as the industrial production of EPA, DHA, and other PUFAs. Our previous studies revealed that the EPA-producing marine bacterium Shewanella livingstonensis Ac10 produces EPA from exogenous DHA independently of de novo EPA biosynthesis, indicating the presence of an unidentified metabolic pathway that converts DHA into EPA. In this study, we attempted to reveal the molecular basis for the bioconversion through both in vivo and in vitro analyses. Mutagenesis experiments showed that the gene disruption of fadH, which encodes an auxiliary β-oxidation enzyme 2,4-dienoyl-CoA reductase, impaired EPA production under DHA-supplemented conditions, and the estimated conversion rate decreased by 86% compared to that of the parent strain. We also found that the recombinant FadH had reductase activity toward the 2,4-dienoyl-CoA derivative of DHA, whereas the intermediate did not undergo β-oxidation in the absence of the FadH protein. These results indicate that a typical β-oxidation pathway is responsible for the conversion. Furthermore, we assessed whether DHA can act as a substitute for EPA by using an EPA-less and conversion-deficient mutant. The cold-sensitive phenotype of the mutant, which is caused by the lack of EPA, was suppressed by supplementation with EPA, whereas the DHA-supplementation suppressed it to a lesser extent. Therefore, DHA can partly substitute for, but is not biologically equivalent to, EPA in S. livingstonensis Ac10.

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“…Auxotrophic makers, such as amino acid auxotrophy, NADPH auxotrophy, thymidine auxotrophy and uracil auxotrophy, are useful tools in gene editing and vaccine products [21][22][23][24][25]. The uracil auxotrophy has been successfully constructed in multiple strains, and some uracil related genes, such as pyrF and ura3, are also widely used [23,[26][27][28]. Uracil nucleotides are indispensable in the process of bacterial life, and orotate is a key intermediate in the synthesis of uracil nucleotides.…”

Section: Baumanniimentioning

confidence: 99%

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.05.370742 doi: bioRxiv preprint bacteria could only use exogenous uracil nucleotides, and could not grow in medium lacking uracil nucleotides. 5-fluoroorotic acid , an analogue of orotic acid, can be converted to a highly toxic compound (5-fluoro-UMP, 5-F-UMP) by the product of pyrF [28]. Therefore, the pyrF gene can be used as the selection and counter-selection marker in the pyrF-deleted mutants.…”

Section: Baumanniimentioning

confidence: 99%

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Developing a broadly applicablepyrF-based genetic manipulation system inAcinetobacter baumannii

Gao

et al. 2020

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Acinetobacter baumannii is an emergency pathogenic bacterium for its multidrug-resistance and high mortality rates after infection. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. Existing methods for genetic manipulation of A. baumannii mainly rely on the use of antibiotic as the selectable marker, and the sacB/sucrose as the counter-selectable marker, which is inconvenient and inappropriate for all research of A. baumannii. Based on the highly conserved pyrF gene and its conserved 500bp-flanking sequence, we quickly and easily generated the pyrF-deleted mutants as the uracil auxotrophic host strain in three model strains and 11 clinical strains. The pyrF-carried vectors constructed for gene editing with pyrF/5-FOA as the counter-selection were conveniently and time-saving in these pyrF-deleted mutants. Utilizing the pyrF-based genetic manipulation system, we easily and efficiently modified the cas gene and CRISPR sequence of I-F CRISPR-Cas system in A. baumannii AYE, and detected the CRISPR interference and adaptation in these mutants. In summary, the pyrF-based genetic manipulation system could be broadly applicable used for efficiently maker-less gene editing in most A. baumannii strains.

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Development of a versatile method for targeted gene deletion and insertion by using the pyrF gene in the psychrotrophic bacterium, Shewanella livingstonensis Ac10

Cited by 13 publications

References 21 publications

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation

Bioconversion From Docosahexaenoic Acid to Eicosapentaenoic Acid in the Marine Bacterium Shewanella livingstonensis Ac10

Developing a broadly applicablepyrF-based genetic manipulation system inAcinetobacter baumannii

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