Bacterial strains and reagents

Reagents were sourced from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. K. pneumoniae strains were cultured overnight at 37°C with shaking in LB broth (Fisher Bioreagents, Ottowa, ON) or at 30°C on LB agar plates. Media were supplemented with 40μg/mL kanamycin to select for transposon mutants and isogenic knockout strains, or with 50μg/mL chloramphenicol to select for strains containing the plasmid pACYC184 and its derivatives. All bacterial strains in this study are detailed in S3 Table, and primers are detailed in S4 Table.

Complementation plasmids were generated as previously described [16]. The complementation vector, pACYC184 was linearized by BamHI and HindIII digestion (New England Biolabs, Ipswitch, MA). The locus for arnD, purD, sspA, pdxA, or pdxA-ksgA-apaG-apaH, along with upstream regions within 500 base pairs of the open reading frame predicted to contain the native promoter (predicted by SoftBerry BPROM; Softberry Inc, Mount Kisco, NY), were amplified from KPPR1 using primers with 5’ homology to linearized pACYC. For each gene, Gibson assembly was performed using the generated PCR products and linearized pACYC184 according to the manufacturer’s protocol with HiFi DNA Assembly Master Mix (New England Biolabs). The pdxA_operon amplicon and pACYC184 was ligated after digestion using T4 DNA ligase according to the manufacturer’s protocol (New England Biolabs). The Gibson or ligated product was transformed into E. coli TOP10 (New England Biolabs), and constructs were confirmed using full length plasmid sequencing (Plasmidsaurus, Eugene, OR). Verified plasmids were mobilized into K. pneumoniae by electroporation.

To generate a pdxA (VK055_2525) isogenic knockout, Lambda Red mutagenesis was performed as described [10,11,16,54]. Briefly, electrocompetent KPPR1 harboring the pKD46 plasmid was generated using an overnight culture grown at 30°C. The culture was diluted into LB broth with 50μg/mL spectinomycin, 50mM L-arabinose, 0.5mM EDTA (Promega, Madison, WI), and 10μM salicylic acid and grown at 30°C until exponential phase. Cells were cooled on ice for 30 minutes and then pelleted at 8,000xg for 15 minutes. Serial washes were performed at 4°C using 50mL 1mM HEPEs pH 7.4 (Gibco, Grand Island, NY), 50mL diH₂O, and 20mL 10% glycerol. To generate site specific targets for pdxA, a kanamycin resistance cassette from the pKD4 plasmid was amplified with primers also containing 65 base pairs of homology at the 5’ end to the chromosome flanking the pdxA open reading frame (S4 Table). This fragment was electroporated into competent KPPR1-pKD46 and transformants were recovered overnight at 30°C, then selected on agar containing kanamycin after a 37° incubation. Knockouts were confirmed with colony PCR using primers flanking and internal to pdxA.