Construction of a mini-Tn5 transposon library.

Transposon mutagenesis was performed based on the mini-Tn5Km element, which was constructed as previously described (61). Cycloclasticus sp. strain P1 was grown in ML medium until the stationary growth phase, and the cells were then centrifuged at 3,200 × g at 4°C. The E. coli donor (λ pir, pUT min-Tn5 Km) and helper (pRK2013) strains were grown overnight at 37°C in LB medium supplemented with kanamycin (25 μg/ml) and then washed with fresh LB and centrifuged at 3,200 × g at 4°C. The pellets obtained after centrifugation (Cycloclasticus sp. strain P1, E. coli donor, and E. coli helper) were mixed in a 4:1:1 ratio (respectively, by volume) and placed on a membrane filter on a plate with LB agar, salts (0.45 g/liter Na₂HPO₄·2H₂O, 2.5 g/liter NaNO₃, 11.5 g/liter NaCl, 0.38 g liter⁻¹ KCl, and 0.7 g/liter CaCl₂·2H₂O), and 2% acetate (wt vol⁻¹) as the carbon and energy source. The plates were incubated for 24 h at 30°C, and cells were then washed with 10 mM MgSO₄. Finally, transconjugants were selected on modified solid ML medium containing 30 g/liter NaCl, 1 g/liter NH₄NO₃, 0.35 g/liter KCl, 1 g/liter KH₂PO₄, 1 g/liter K₂HPO₄, 0.08 g/liter KBr, 0.05 g/liter CaCl₂, 24 mg/liter SrCl₂·6H₂O, 1 × 10⁻⁴ g/liter ZnSO₄·7H₂O, 3.5 g/liter MgSO₄·7H₂O, 0.01 g/liter FeCl₃, 3 g/liter sodium pyruvate, 3 g/liter sodium citrate, 0.5 g/liter yeast extract, 1 g/liter tryptone, 10 g/liter agar, 100 ml of strain P1 (OD₆₀₀ of 1.1) liquid ML medium culture added after the ultrasonic crushing and filtration sterilization, and 900 ml of H₂O (pH 7.5) supplemented with 25 μg/ml kanamycin as required.

A transposon library consisting of approximately 15,000 colonies was obtained based on the mini-Tn5 transposon element. The Cycloclasticus sp. strain P1 genome contained 2,248 genes. Thus, this library represented approximately 7-fold coverage of the genome. However, mutant clones with identical disrupted genes were isolated, indicating that the library was saturated. The quality of the library was verified using Southern blot hybridization, which confirmed the randomness and uniqueness of the transposon insertions.