Dh5α cells

Stable shRNA-mediated GCN2 knockdown was achieved using pLKO.1 cloning vector (Addgene, Cambridge, MA, USA) with GFP replacing the puromycin insert. Briefly, pLKO stuffer was released upon digestion with EcoRI and AgeI (New England Biolabs, MA, USA). Oligos were annealed and ligated into the pLKO.1, producing a final plasmid expressing the shRNA of interest (shGCN2(1), shGCN2(2), and non-targeting shNTC (Supplementary Table 1)). DH5α cells (New England Biolabs) were transformed with ligation mix and plated on LB agar plates containing ampicillin. Positive clones were identified by sequencing with a pLKO.1 sequencing primer (5′ CAA GGC TGT TAG AGA GAT AAT TGG A 3′). Maxi prep using GeneJet Plasmid Maxiprep Kit (ThermoFisher) was carried out to produce large-scale quantities of the plasmid. Lentiviral particles were generated with HEK-293T (GenHunter) to carry out the viral transfection using X-treme Gene HP kit (Roche, Switzerland). Transfection efficiency was confirmed after 48 h; 72 h post-transfection virus was collected by ultracentrifugation. A549 cells were transduced and GFP-positive cells were sorted by FACS.

All strains, primers, and plasmids used in this study are listed in Table S1. Construction of plasmids was performed by PCR amplification from R. sphaeroides genomic DNA using Herculase II Fusion DNA Polymerase (Agilent, Santa Clara, CA). PCR products were assembled into PCR linearized pIND5 or pk18mobsacB vectors by Gibson Assembly (New England BioLabs, Ipswich, MA) and transformed into DH5α cells (New England BioLabs). Transformants were screened by colony PCR and sequenced to confirm there were no mutations in coding regions. Plasmids were mobilized into R. sphaeroides via conjugal mating with E. coli S17-1 (87 (link)). Colony PCR of Kan^RR. sphaeroides colonies was used to confirm successful mobilization of plasmids. R. sphaeroides genomic insertion on the chromosome was constructed by allelic exchange using the suicide vector pk18mobsacB as described previously (28 (link)). Gene insertions were confirmed by colony PCR of chromosomal loci and sequencing of genomic DNA with gene-specific primers (Table S1).

All strains, primers, and plasmids used in this study are listed in Table S1. Construction of pIND5-cenR or pIND5-cenK was performed by PCR amplification of cenR or cenK from R. sphaeroides genomic DNA using Herculase II Fusion DNA Polymerase (Agilent). PCR products were assembled into PCR linearized pIND5 vector by Gibson Assembly (New England BioLabs) and transformed into DH5α cells (New England BioLabs). Transformants were screened by colony PCR and sequenced to confirm there were no mutations in coding regions. Plasmids were mobilized into R. sphaeroides via conjugal mating with E. coli S17-1 (28 (link)). Colony PCR of Kan^RR. sphaeroides colonies was used to confirm successful mobilization of pIND5-cenR or pIND5-cenK. Translational fusions to fluorescent reporters were constructed by allelic exchange using the suicide vector pk18mobsacB (29 (link)) as described previously (25 (link)). Gene insertions were confirmed by colony PCR of chromosomal loci and sequencing of genomic DNA with gene-specific primers (Table S1).