Constructing Promoter-luxCDABE Reporter Plasmids

The pigA promoter on plasmid pMQ713 [56 (link)] was replaced with the SMDB11_1194, SMDB11_1637, and SMDB11_2817 using yeast homologous recombination, as previously described [57 (link),58 (link)]. Plasmids are listed in Table S1. The pMQ713 plasmid was linearized by restriction enzyme digestion with EcoR1 and Sal1 (New England Biolabs, Ipswich, MA, USA). DNA for the three promoter regions were synthesized as linear double-stranded DNA fragments (Integrated DNA Technologies, Coralville, IA, USA) that include DNA for the promoter region and for site-directed recombination with pMQ713 that places the luxCDABE reporter under transcriptional control of the respective promoter (listed in Table S2). The lengths of the cloned promoters were 338 bp for SMDB11_1194, 354 bp for SMDB11_1637, and 337 bp for SMDB11_2817. To generate the nptII-driven luxCDABE plasmid, the tdtomato gene from pMQ414 was digested with BamH1 and EcoR1 enzymes, and the luxCDABE operon was amplified by PCR from pMQ670 [59 (link)] using primers 3805 and 3806 via PrimeSTAR DNA polymerase (Takara Bio, San Jose, CA, USA). The linearized plasmid and luxCDABE amplicon were combined as above. The plasmids were isolated, and the cloned promoter region was sequenced to validate the constructs.