Oligonucleotides, bacterial strains, and plasmids.

All bacterial strains and plasmids used in this study are listed in Table 1. The oligonucleotides used in this work were synthesized by STAB Vida and are listed in Table 2. All Salmonella strains used are isogenic derivatives of the wild-type S. Typhimurium strain SL1344. Restriction enzymes, T4 DNA ligase, and Phusion DNA polymerase were purchased from Thermo Fisher Scientific and used according to the supplier's instructions.

Strains and plasmids used in this work

Oligonucleotides used in this work

The λ-Red-mediated mutagenesis method (38) was used to obtain the bolA deletion strain. Briefly, a PCR fragment was obtained by amplification of a pKD3 plasmid using the primer pair RNMS003/RNMS004 (Table 2). The resulting fragment, carrying the chloramphenicol (Cat) cassette flanked by 70-nucleotide (nt) homologous extension regions adjacent to the bolA gene, was transformed in S. Typhimurium SL1344 electrocompetent cells containing pKD46 to allow recombination with the bacterial chromosome. P22 HT105/1 int-201 transduction was used to obtain the SL1344 ΔbolA strain (CMA820) in a fresh background (39).

The pRMA04 plasmid was constructed by inserting a PCR-amplified DNA fragment carrying the bolA coding sequence and promoter region (using primers RMNS011/RMNS012) (Table 2) into XbaI and KpnI sites of a pWSK29 vector. The resulting pRMA04 plasmid was transformed in CMA820 competent cells to obtain the complemented strain CMA821. All constructs were confirmed by DNA sequencing at STAB Vida.