title: Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli.
abstract: Essential 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.
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United Kingdom sgoh@rvc.ac.uk.
authors.forename: Shan
authors.initials: S
authors.lastname: Goh
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United Kingdom.
authors.forename: Angela
authors.initials: A
authors.lastname: Hohmeier
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United Kingdom Central Biotechnology Services and Institute of Translation, Innovation, Methodology and Engagement, School of Medicine, Heath Park, Cardiff University, Cardiff, United Kingdom Institute of Psychological Medicine and Clinical Neurosciences, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Stone
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United Kingdom.
authors.forename: Victoria
authors.initials: V
authors.lastname: Offord
authors.affiliation: Department of Organic Chemistry, Faculty of Sciences, University of Malaga, Malaga, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Sarabia
authors.affiliation: Department of Organic Chemistry, Faculty of Sciences, University of Malaga, Malaga, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Garcia-Ruiz
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United Kingdom.
authors.forename: Liam
authors.initials: L
authors.lastname: Good
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D020043:Genes, Essential; D005822:Genetic Vectors; D009876:Operon; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism
chemical_list: D016372:RNA, Antisense