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dc.contributor.authorGoh, Shan
dc.contributor.authorHohmeier, Angela
dc.contributor.authorStone, Timothy C
dc.contributor.authorOfford, Victoria
dc.contributor.authorSarabia, Francisco
dc.contributor.authorGarcia-Ruiz, Cristina
dc.contributor.authorGood, Liam
dc.identifier.citationGoh , S , Hohmeier , A , Stone , T C , Offord , V , Sarabia , F , Garcia-Ruiz , C & Good , L 2015 , ' Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli ' , Applied and Environmental Microbiology , vol. 81 , no. 16 , pp. 5650-9 .
dc.identifier.otherPURE: 11775368
dc.identifier.otherPURE UUID: 8e3a1019-9022-431d-9de9-22ea3979dfb3
dc.identifier.otherPubMed: 26070674
dc.identifier.otherPubMedCentral: PMC4510190
dc.identifier.otherScopus: 84937865312
dc.identifier.otherORCID: /0000-0002-9028-0303/work/62751714
dc.descriptionCopyright © 2015, American Society for Microbiology. All Rights Reserved.
dc.description.abstractEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, the Escherichia coli ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL50 values (means and standard deviations) of ribF, ileS, and lspA were 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P = 0.71 by weighted one-way analysis of variance). The gene requirement for ispH could not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.en
dc.relation.ispartofApplied and Environmental Microbiology
dc.subjectEscherichia coli
dc.subjectGene Expression
dc.subjectGene Knockdown Techniques
dc.subjectGene Silencing
dc.subjectGenes, Essential
dc.subjectGenetic Vectors
dc.subjectRNA, Antisense
dc.subjectJournal Article
dc.subjectResearch Support, Non-U.S. Gov't
dc.titleSilencing of Essential Genes within a Highly Coordinated Operon in Escherichia colien
dc.contributor.institutionSchool of Life and Medical Sciences
dc.description.statusPeer reviewed
rioxxterms.typeJournal Article/Review

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