Pfccgi

Protein expression vector pMAL-c5Xa (NEB) was used as the backbone for constructing the mCherry reporter. The malE gene and the lacI promoter were restricted from the vector using SacI and KasI restriction enzymes. The entCEBA promoter was amplified with primers GK073-F/GK073-R (Table S1) and inserted into the pMAL-c5Xa backbone. The reporter constructs were transformed into respective strains as indicated. An inducible GFP was made by inserting the Yersinia operon 1 promoter sequence into plasmid pFCcGi (Addgene) upon restriction with HindIII and XbaI (NEB). The resulting construct was called pybtP:GFP (30 (link)). The reporter constructs were transformed into respective strains as indicated.

S. Typhimurium 14028s and E. coli B/r were transformed by electroporation with plasmid pFCcGi (Addgene plasmid number 59324), encoding the red fluorescent protein mCherry expressed constitutively (Figueira et al., 2013 (link)). Axenic D. discoideum AX2 cnxA-GFP (∼2 × 10⁷ cells) was co-incubated with each bacteria at 22°C for 24 h in 10 mL of Soerensen buffer, using a MOI of 10 bacteria/amoeba. Images of infected cells were acquired every hour using a Zeiss LSM 710 laser scanning confocal microscope equipped with a 63x 1.4 NA optic setup. Prior to observation, cells were mounted on a thin layer of 1% agarose in PBS buffer deposited on a glass slide. To visualize GFP-associated fluorescence (amoebae), the sample was excited at 488 nm with an argon laser and emission was detected using a filter in the 493–549 nm range. To visualize mCherry-associated fluorescence (bacteria), the sample was excited at 543 nm with a HeNe laser and emission was detected using a filter in the 548–679 nm range. Images were acquired using the ZEN 2012 Black software (Zeiss), and analyzed using Fiji and ImageJ softwares (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)).