Isopropyl β d 1 thiogalactopyranoside (iptg)

The UPEC strains used in this study are all derivatives of the human cystitis isolate UTI89 and are listed in Table S1 (17 (link), 91 (link)). In general, bacteria were grown and propagated in Luria-Bertani (LB) broth and plated for isolation on LB agar (BD). Where necessary, the medium was supplemented with 50 μg/ml kanamycin, 50 μg/ml spectinomycin, 100 μg/ml ampicillin, and/or 1 mM isopropyl β-d-1-thiogalactopyranoside (Gold-Bio). Human urine was collected from at least 2 healthy volunteers and filter sterilized through a 0.22-μm filter (Millipore). Biological replicates of growth curves were conducted with different batches of urine collected on different days. Where indicated, urine was supplemented with 10 mM ornithine, citrulline, or arginine (Sigma-Aldrich).
All deletion mutants were generated in the prototypical cystitis isolate UTI89 using the λRed recombinase system (92 (link), 93 (link)). The allelic replacement of argI was performed using a previously described, λRed recombinase-based negative-selection system (65 (link)). To facilitate in vivo competition assays, kanamycin or chloramphenicol resistance markers were inserted into the HK site using the standard λRed recombinase protocol (49 (link), 94 (link)). All deletion and allelic replacement mutants were confirmed by Sanger sequencing.

L. pneumophila strains, plasmids, and primers used in this study are listed in Tables A, B, and C in S1 Text, respectively. L. pneumophila strains were cultured on supplemented charcoal N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (CYE) and grown at 37°C, as described [96 (link)]. Liquid cultures were grown overnight with shaking at 37°C in supplemented ACES-buffered yeast extract (AYE) medium, as described [20 (link),97 (link)]. CYE was supplemented with 10 μg mL^-1 chloramphenicol for plasmid maintenance and legC4 expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; GoldBio) where indicated. Escherichia coli strains used for cloning (Top10; Invitrogen) and protein expression [BL21 (DE3); New England Biolabs] were grown at 37°C in Luria-Bertani (LB) medium supplemented with antibiotics as appropriate for plasmid selection [50 μg mL^-1 kanamycin (GoldBio), 25 μg mL^-1 chloramphenicol (GoldBio), or 100 μg mL^-1 ampicillin (GoldBio)]. Protein expression in BL21 (DE3) was induced with 1 mM IPTG. Unless otherwise indicated, all chemicals were obtained from Millipore Sigma (St. Louis, MO).

B. desmolans ATCC 43058 and Bifidobacterium scardovii ATCC BAA-773 were purchased from ATCC. C. scindens ATCC 35704 was maintained as −80°C glycerol stocks in our laboratory. Propionimicrobium lymphophilum ACS-093-V-SCH5 was obtained from the Culture Collection, University of Götesborg, Sweden. Escherichia coli DH5α (turbo) competent cells were from New England Biolabs (Ipswich, MA) and E. coli BL21-CodonPlus(DE3) RIPL was purchased from Stratagene (La Jolla, CA). The pET-51b(+) vector was obtained from Novagen (San Diego, CA). Restriction enzymes were purchased from New England Biolabs; QIAprep Spin Miniprep kit was obtained from Qiagen (Valencia, CA). Isopropyl β-D-1-thiogalactopyranoside was purchased from Gold Biotechnology (St. Louis, MO). Strep-Tactin resin was purchased from IBA GmbH (Gottingen, Germany). Steroids were purchased from Steraloids (Newport, RI). Amicon Ultra-15 centrifugal filter units with 30 and 50 kDa molecular-mass cutoffs were obtained from Millipore (Billerica, MA). All other reagents were of the highest possible purity and were purchased from Fisher Scientific (Pittsburgh, PA).