Citrulline

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.

Acetonitrile (gradient grade for liquid chromatography) was obtained from Fisher Chemical. Methanol (gradient grade for liquid chromatography), ammonia hydroxide, formic acid, and citrulline were purchased from Sigma-Aldrich. Ammonium acetate was obtained from Aladdin. Distilled water was purchased from Watsons. Glyceric acid, benzoic acid, L-threonine, and creatine were obtained from Tokyo Chemical Industry. The isotope-labeled internal standard mixture for metabolomic analyses was from Biotree Biomedical Technology. The citrulline analogue L-glutamic acid -2,3,3,4,4-d5 was purchased from Sigma-Aldrich as well and used as an internal standard for targeted metabolite analyses.