Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

Shan Goh, Angela Hohmeier, Timothy C Stone, Victoria Offord, Francisco Sarabia, Cristina Garcia-Ruiz, Liam Good

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)5650-9
Number of pages10
JournalApplied and Environmental Microbiology
Issue number16
Publication statusPublished - 15 Aug 2015


  • Escherichia coli
  • Gene Expression
  • Gene Knockdown Techniques
  • Gene Silencing
  • Genes, Essential
  • Genetic Vectors
  • Operon
  • Plasmids
  • RNA, Antisense
  • Journal Article
  • Research Support, Non-U.S. Gov't


Dive into the research topics of 'Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli'. Together they form a unique fingerprint.

Cite this