Proteus mirabilis HI4320 Transposon Mutant Library

Proteus mirabilis HI4320 was isolated in a prior study from the urine of a catheterized patient in a chronic care facility in Baltimore, Maryland [2 (link), 59 (link)]. The P. mirabilis HI4320 transposon mutant library was previously constructed, validated, and successfully utilized in a mouse model of CAUTI [28 (link)]. Bacteria were routinely cultured at 37°C with aeration in 5 ml LB broth (10 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl) or on LB solidified with 1.5% agar. Proteus mirabilis minimal salts medium (PMSM) (10.5 g/L K₂HPO₄, 4.5 g/L KH₂PO₄, 0.47 g/L sodium citrate, 1 g/L (NH₄)₂SO₄, supplemented with 0.001% nicotinic acid, 1mM MgSO₄, and 0.2% glycerol) [60 (link)] or RPMI with 2 mM L-glutamine (Sigma). Transposon mutants were cultured in LB containing 25 μg/ml kanamycin (Sigma). Additional P. mirabilis mutants for validation of candidate fitness factors were constructed by insertion of a kanamycin resistance cassette as previously described using the TargeTron system (Sigma), and are listed in S8 Table [61 (link)]. Resulting mutants were screened by kanamycin selection and PCR. All primers for generation and verification of mutants are provided in S9 Table.