Pdonr221

Genes encoding Cbx-KRAB domain fusions were synthesized as gene fragments (Integrated DNA Technologies) and cloned with the Gateway BP Clonase II system (Thermo Fisher, 11789020) into the Gateway Entry vector pDONR221 (Thermo Fisher) according to the manufacturer’s protocols. Cbx-KRAB domain fusions cloned into pDONR221 were then transferred, via Gateway LR Clonase II system (Thermo Fisher, 11791020), into the pLX303-dCas9 vector^{38 (link)} (a kind gift of Dr. Taipale, University of Toronto). The pLX303-dCas9 vector encoded a Streptococcus pyogenes dCas9 with two C-terminal and one N-terminal SV40 nuclear localization signals. See Supplementary Dataset 5 for sequences of all dCas9-repressors used in this study.

To generate Bag101 (SPBC16G5.11c) constructs, FL (full length), BAG domain (amino acids 78–190) and UBL domain (1–77) cDNAs were amplified from S. pombe genomic DNA and inserted into pDONR221 (Invitrogen) and pGEX-KG (GE Healthcare). Full length bag102⁺ (SPBC530.03c), ΔTM (31–206), BAG (122–206) and UBL (31–121) cDNAs were also inserted into the pDONR221 and pGEX-KG vectors. For expression in S. pombe, the inserts from the pDONR221 vectors were transferred to the pDUAL vector system [54] (link) using Gateway cloning technology (Invitrogen). Both ubp3 variants were cloned via XhoI/NotI into the pJR2-3XL vector with the nmt1⁺ promoter [55] (link). The HA-tagged Spc7-23 construct was obtained by QuikChange site directed mutagenesis (Stratagene) on pJR-XU41 plasmid encoding Spc7-3HA. The expression construct for 6His-ubiquitin has been described before [56] (link). The expression construct for San1 was kindly provided by Dr Makoto Kawamukai [57] (link).

pDONR221 (Invitrogen) and pABB-CRS2 [20 (link)] were used as cloning and positive suicide vectors, respectively. The construction of a deletion mutant using pABB-CRS2 has been described previously [10 (link)]. A 7.1-kbp DNA fragment containing the cyaA gene and its flanking region was amplified by PCR with the primers B1-cyaA and B2-cyaA using B. bronchiseptica S798 genomic DNA as a template. The resulting PCR product was cloned into pDONR221 using the adaptor PCR method (Gateway cloning system, Invitrogen) to obtain pDONR-cyaA. For the deletion of cyaA in pDONR-cyaA, inverse PCR was then carried out with the primers R1-cyaA and R2-cyaA using circular pDONR-cyaA as a template. The resulting PCR products were digested with HindIII and self-ligated to obtain pDONR-ΔcyaA, which contained a HindIII site in addition to a 4959-bp deletion including the start codon of cyaA. The cyaA fragment with internal deletion was transferred to pABB-CRS2 to obtain pABB-CRS2-ΔcyaA using the Gateway cloning system. pABB-CRS2-ΔcyaA was then introduced into E. coli SM10λpir and was transconjugated into B. bronchiseptica S798 ΔbteA, which is unable to induce necrosis to mammalian cells as described previously [10 (link)]. The resulting mutant strain was designated S798 ΔbteA ΔcyaA.

Complete deletion of the oxyR gene was carried out as previously described [57] (link). All primer sequences are described in Table S2. Briefly, a first round of three PCR reactions was performed in which the 5′ and 3′ flanking regions of oxyR, as well as a Gm resistance cassette were amplified from plasmid pPS856 [58] (link) using four gene-specific primers (Oxy-UpF-GWL, Oxy-UpR-Gm, Oxy-DnF-Gm and Oxy-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generated three fragments with partial overlaps either to each other or the attB1 and attB2 recombination sites. The purified fragments were then assembled in vitro by overlap extension during the second round PCR using the common primers GW-attB1 and GW-attB2. This resulted in an oxyR-deletion-mutant PCR fragment which was subsequently cloned into pDONR221 (Invitrogen) via the BP clonase reaction to create pDONR221-oxyR::Gm. This construct served as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-oxyR::Gm was then transferred to P. aeruginosa and the plasmid-borne oxyR-deletion mutation was exchanged with the chromosome via homologous recombination to generate the chromosomal deletion mutant.