E coli dh10b

The subcloning and construction of recombinant plasmids was carried out in E. coli DH10B (Invitrogen). The P. pastoris GS115 strain (Invitrogen) was used as a host for the expression of heterogenous proteins. The vectors pPIC3.5K and pPICZαA were purchased from Invitrogen. The vector pTSC (Yan et al., 2008 (link)) was stocked in this lab. pUC57-MCS7 (the original plasmid used for construction of recombinant expression vectors) was from GeneCreate Biological Engineering Co., Ltd, Wuhan, China. E. coli DH10B was cultured in Luria-Bertani medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, pH 7.0] at 37°C. The P. pastoris yeast strain was cultured in YPD medium [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose]. YPDZ plates containing Zeocin (100 μg/mL) were used for the selection of positive P. pastoris transformants. Buffered minimal glycerol-complex medium (BMGY) was prepared with 2% (w/v) peptone, 1% (w/v) yeast extract, 1% (w/v) glycerol, 1.34% (w/v) yeast nitrogen base with ammonium sulfate but without amino acids, and 4 × 10⁻⁵% (w/v) biotin in 100 mM potassium phosphate buffer. The phosphate buffer was adjusted to pH 6.0. Buffered minimal methanol-complex medium (BMMY) was the same as BMGY, except that 0.5% (w/v) methanol was replaced with glycerol.

The pathogenic M. agalactiae type strain PG2 [28 (link), 29 (link)] and the pdhB transposon mutant were grown at 37°C in SP4 medium supplemented with penicillin, pyruvate, and phenol red as an indicator, as described before [21 (link)]. Gentamicin sulphate (50 μg/ml) was added to both broth and agar plates for the propagation of the pdhB transposon mutant. E. coli DH10B (Invitrogen GmbH, Lofer, Austria) transformants containing the transposon vector pISM2062 or the M. agalactiae oriC vector pMM21–7 were grown in LB broth (10 g tryptone, 5 g yeast extract, and 5 g of NaCl per litre) supplemented with ampicillin (100 μg/ml), gentamicin sulphate (7 μg/ml) or tetracycline (100 μg/ml). The M. agalactiae pdhB mutant containing the complementation plasmid pPlacZ was grown in SP4 broth containing both tetracycline (2 μg/ml) and gentamicin (50 μg/ml). To monitor lacZ expression on the basis of blue-white colonies, M. agalactiae clones were grown on SP4 agar supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at a concentration of 160 μg/ml [30 (link)].

All strains used in this study are listed in Table 1. The serotype 1/2a strain L. monocytogenes EGD-e (Bécavin et al., 2014 (link)) was used to generate the sensor strain for detection of membrane damage. E. coli DH10B (Invitrogen, Darmstadt, Germany) was used as a cloning host. L. lactis strain B1627 (strain collection Dzung B. Diep, NMBU, Norway) was used to produce nisin-containing culture supernatants and L. lactis MG1363 (Gasson, 1983 (link)) served as nisin-negative control. Bacteria were grown under standard conditions in LB (E. coli), BHI (L. monocytogenes) or GM17 (L. lactis) broth aerobically at 37°C with agitation (E. coli, L. monocytogenes) or 30°C without aeration (L. lactis; Kuipers et al., 1995 (link)). Where indicated chloramphenicol was added to the media at a final concentration of 20 μg/ml (E. coli) or 15 μg/ml (L. monocytogenes).
For preparation of supernatants, overnight (o/N) cultures of L. lactis strains were diluted in 20 ml of fresh GM17 broth in an Erlenmeyer flask to an optical density at 600 nm (OD₆₀₀) of 0.1 and grown under standard conditions. Growth was monitored by measuring OD₆₀₀. At the indicated timepoints, aliquots were collected and supernatants were prepared by centrifugation at 3200 × g for 3 min and filtration (pore size: 0.2 μm).