Synthetic Genetic Interactions Reveal a Dense and Cryptic Regulatory Network of Small Noncoding RNAs in Escherichia coli

Abstract

sRNAs have long been purported to be a critical mechanism by which bacteria respond to stress; however, uncovering growth phenotypes for sRNA deletion strains in E. coli and related bacteria has proven particularly challenging. In contrast, the deletion of hfq , a chaperone required for the activity of many sRNAs in E. coli , results in striking growth defects in E. coli under a variety of medium conditions and chemical stressors.

A mobile CRISPRi collection enables genetic interaction studies for the essential genes of Escherichia coli

“…High-throughput conjugation of the mobile arrayed CRISPRi collection was conducted as previously described with some modifications. 20 , 21 , 22 For high-throughput conjugation and screening of the CRISPRi collection into a genetic background of interest such as E. coli Δlpp , the query strain (kanamycin resistant E. coli Δlpp ) was arrayed on LB agar with kanamycin at 1,536-colony density using the Singer Rotor HDA. The mobile arrayed CRISPRi collection was likewise pinned at 1,536 colony density, with each strain of the collection in technical quadruplicate, on LB with spectinomycin and DAP.…”

“… 11 , 12 , 13 Likewise, millions of unique double deletion strains have been generated with the Keio collection and screened using synthetic genetic array technology to uncover genetic contexts where genes become unexpectedly essential. 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 In contrast, there is great difficulty in performing such large-scale gene-gene interaction assays that include essential processes in E. coli . Chemical and antibiotic probes are often used to investigate the effects of perturbing essential processes in different genetic backgrounds, in what are described as “chemical genomics” screens.…”

A mobile CRISPRi collection enables genetic interaction studies for the essential genes ofEscherichia coli

“…High-throughput conjugation of the mobile arrayed CRISPRi collection was conducted as previously described with some modifications [20][21][22] . For high-throughput conjugation and screening of the CRISPRi collection into a genetic background of interest such as E. coli ∆lpp, the query strain (kanamycin resistant E. coli ∆lpp) was arrayed on LB agar with kanamycin at 1,536-colony density using the Singer Rotor HDA.…”

“…The Keio collection has been extensively screened to identify the environmental conditions, for example, different growth media and chemical stressors, where canonically non-essential genes are rendered essential for E. coli growth [11][12][13] . Likewise, millions of unique double deletion strains have been generated with the Keio collection and screened using synthetic genetic array technology to uncover genetic contexts where genes become unexpectedly essential [14][15][16][17][18][19][20][21][22] . In contrast, there is great difficulty in performing such large scale gene-gene interaction assays that include essential processes in E. coli.…”

High-resolution small RNAs landscape provides insights into alkane adaptation in the marine alkane-degraderAlcanivorax dieseloleiB-5

“…Firstly, most sRNAs are poorly conserved except those within phylogenetically close species (Lindgreen et al 2014), resulting in the bad performance of bioinformatic predictions to most cases (Barik and Das, 2018; Boutet et al, 2022). Secondly, the classical mutation-based genetic screening strategy to investigate protein-coding genes is usually inefficient for detecting sRNAs due to lacking of discernible phenotypes for most disrupted sRNAs (Sridhar and Gunasekaran, 2013; Rachwalski et al, 2022). Importantly, more and more evidences support that sRNAs can originate from any locations in the genome.…”