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 (MTL 50 ). 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 MTL 50 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.

Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design inEscherichia coli

“…Because of the general existence of operon structure and their importance at transcription regulation in prokaryotic genomes, further investigation is needed to more comprehensively understand the potential interactions when applying CRISPRi technology to operon structures at molecular level. We recommend that CRISPRi-based arrayed or pooled screen can be used to study the gene expression regulation in operons, as a complementary method to gene knockout (DNA sequence) and antisense RNA technology (currently known transcription vs. mRNA stability and protein translation)(58) functioning at a different level, the combination of which should give us novel insight into the mechanism of expression regulation in operons, such as the unidentified promoter activities within the operon suggested by our screen result (Figure 3, nirBDC_cysG and aroF_tyrA operons).…”

Development of Design Rules for Reliable Antisense RNA Behavior in E. coli

“…Antisense RNA (asRNA) is a well-studied category of RNA regulators that has been used extensively in engineered systems . Synthetic asRNA regulation has been used in many metabolic engineering studies to optimize expression levels of genes within a target pathway or to down-regulate competing pathways. − asRNA has also been used as an antagonistic regulator that sequesters a small guide RNA (sgRNA), as a component of a counter-selection method, and as a tool to study essential gene knockdown . Furthermore, work is underway to use asRNA in various pharmaceutical applications. , Despite its wide range of applications, there remains no consensus regarding the design rules governing asRNA behavior.…”

“…7−10 asRNA has also been used as an antagonistic regulator that sequesters a small guide RNA (sgRNA), 11 as a component of a counter-selection method, 12 and as a tool to study essential gene knockdown. 13 Furthermore, work is underway to use asRNA in various pharmaceutical applications. 14,15 Despite its wide range of applications, there remains no consensus regarding the design rules governing asRNA behavior.…”

CRISPR–dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria

“…Building on the foundation of robust gene repression achievable with the CRISPR–dCas13a system, we embarked on the challenge of achieving specific gene repression within a polycistronic gene operon, a feat not easily accomplished using the CRISPR–dCas9 system, which tends to repress all genes within operons. To illustrate this capability, we selected the ribF – ileS – lspA – fkpB – ispH operon in E. coli as a representative example ( 45 ). This operon encompasses distinct functions: ribF is associated with riboflavin kinase and FMN adenylyltransferase activities, ileS encodes an isoleucyl-tRNA synthetase, lspA is involved in prolipoprotein signal peptidase activity associated with cell wall processes, fkpB encodes a peptidylprolyl cis - trans isomerase affecting protein function, and ispH plays a vital role in isoprenoid biosynthesis.…”