Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000

Abstract

Background: Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.

Regulation and Function of Pilus Island 1 in Group B Streptococcus

“…To overcome these problems, we utilized an inducible expression system, the nisin-controlled expression system in Lactococcus lactis (1,13). The ape1 gene was amplified and cloned as an ape1-His 6 fusion in the plasmid vector pNZ8048 under the control of a nisin-inducible promoter (12).…”

“…For experiments testing the effects of pH, GBS was cultured at 37°C in complex medium (CM; 10 g/liter proteose peptone, 5 g/liter Trypticase peptone, 5 g/liter yeast extract, 2.5 g/liter KCl, 1 mM urea, 1 mM arginine) (30). E. coli was grown in Luria-Bertani broth, and L. lactis was grown in GM17 broth (M17 broth [Oxoid] supplemented with 0.5% glucose) (1). When appropriate, antibiotics were used at the following concentrations: for E. coli, erythromycin (erm) at 200 g/ml, kanamycin at 25 g/ml, and ampicillin at 100 g/ml; for L. lactis, chloramphenicol at 10 g/ml; and for GBS, erm at 1 g/ml and chloramphenicol at 10 g/ml.…”

“…An overnight culture of L. lactis NZ9000 (pNZ-ape1) was subcultured into fresh GM17 broth containing 10 g/ml chloramphenicol and incubated statically at 30°C. Filter-sterilized culture supernatant of the nisin-secreting strain L. lactis NZ9700 was used as a source of nisin (1,14). Nisin was added when the culture density reached an A 600 of 0.5.…”

“…To express the AP1-1 adhesin protein in Lactococcus lactis NZ9000, the gene encoding the protein, sag0649, was amplified by PCR using GBS 2603 chromosomal DNA as the template and using primers 1461 and 1450. The PCR product was digested with BamHI and XbaI and then ligated to similarly digested pNZ-SEC vector, which harbors the P44 promoter from plasmid pNZ44 and the SEC signal sequence from the L. lactis MG1363 chromosome (1). The recombinant plasmid pNZ-SEC-AP-1 was transformed into E. coli DH5␣ chemically competent cells (Zymo Research).…”

BsaB, a Novel Adherence Factor of Group B Streptococcus

“…GBS strains were 515 (21), 2603 (2603V/R) (23), NEM316 (19), A909, H36B, 18RS21 (24), COH1 (25), CJB111 (Carol Baker, Baylor College of Medicine, Houston, TX), and the derivative mutants 515⌬csrR and 2603⌬csrR (26). Unless otherwise specified, GBS strains were grown in Todd-Hewitt broth (THB; Difco) or on Tryp- (27). When appropriate, antibiotics were used at the following concentrations: for E. coli, erythromycin (ERM) at 200 g/ml and ampicillin at 100 g/ml; for L. lactis, chloramphenicol at 10 g/ml; and for GBS, ERM at 1 g/ml and chloramphenicol at 10 g/ml.…”