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CRISPRi knockdown of essential genes in Pseudomonas aeruginosa

ABSTRACT: To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes.

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE159327 | GEO | 2020/12/21

REPOSITORIES: GEO

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Project description:Thicklip grey mullet hepatic transcriptome comparission from PASAIA harbour, Basque Country Active biomonitoring using sentinel species caged in differentially polluted aquatic environments is being implemented worldwide. To determine the biological effects of pollutants in a heavily polluted Basque harbour (Pasaia, 431700N 015500W), two cages specially designed to deploy thicklip grey mullets (Chelon labrosus) were placed in the inner- and outer-harbour. Cages were deployed for 5 and 21 days to determine possible differences between sites, linked to specific chemical burdens. Hepatic gene expression profiles were studied using Q-PCR and a toxicology tailored low density mullet microarray (160 genes). Chemical analysis showed high concentrations of metals, PAHs, PCBs, phthalates, and organometallic compounds at both sites, most chemicals showing slightly higher concentrations in the inner-harbour. Q-PCR expression studies confirmed chemical data as metallothionein and CYP1A showed significant expression upregulation at day 21 at both sites, but showed no differences between sites. The microarray showed gene expression profiles that were able to distinguish mullets according to the site they were deployed in and the day they were captured. Regarding sites, 39 genes showed significant differences at p<0.05 on the last sampling day. Two families of genes are to be highlighted. Phase I and II biotransformation genes CYP2, CYP3 and UGT were upregulated in the inner-harbour together with AhR2 and AhRR. Similarly, TBT binding protein and genes involved in lipid metabolism such us PPARa and CYP7 were upregulated. The latter two genes have also been reported to be regulated by TBT. In summary, the low density microarray designed for mullets proved to be useful to unveil even slight differences in pollutant burdens in field conditions. 24 samples:6 hepatic samples from Inner harbour after 3 days of exposure, 6 hepatic samples from outer harbour after 3 days of exposure, 6 hepatic samples from Inner harbour after 15 days of exposure and 6 hepatic samples from outer harbour after 15 days of exposure

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CRISPRi knockdown of essential genes in Pseudomonas aeruginosa

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