SIG.b/LMGM/Institut Pasteur (Paris)

Description:

In 2019, SIG.b started a collaboration with the IEVA team (LMGM) aimed at identifying putative BAM-interactors. The Beta-barrel Assembly Machinery (BAM) is an essential complex that promotes the biogenesis of outer membrane proteins in Gram negative bacteria. A quantitative mass-spectrometry approach identified putative BAM-interactors.

Service Objectives:

To gain insights into the function of as yet uncharacterized BAM-interacting factors, a CRISPR interference (CRISPRi) screen was set up to define genes conferring a synthetic fitness defect. These experiments have been conducted by SIG.b in the Laboratory of “biologie de synthèse” (Institut Pasteur) which is having the expertise in high throughput CRISPRi silencing.

We validated our genome-scale approach by selectively targeting via CRISPRi a couple of the genes that were in the screen output and demonstrating that shutting down their expression leads to negative growth fitness.

Our experiments allow to highlight several genes of which further genetic and biochemical analysis showed that they are at the crossroad between outer membrane biogenesis and septal peptidoglycan hydrolysis. Such work not only allowed elucidation of the link between E. coli BAM-dependent pathway and major envelope remodelling processes but also the acquisition in LMGM of a CRISPRi-based screening method, nowadays so central in molecular and cellular microbiology.

Funding:

Costs (consumables and mission expenses) were covered by the collaborating team.

Associated publication:

Elife 2021; doi: 10.7554/eLife.67817. Ranava D, Yang Y, Orenday-Tapia L, Rousset F, Turlan C, Morales V, Cui L, Moulin C, Froment C, Munoz G, Rech J, Marcoux J, Caumont-Sarcos A, Albenne C, Bikard D, Ieva R. Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.