Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis

Torsten Stein,Stefan Heinzmann,Irina Solovieva,Karl-Dieter Entian

doi:10.1074/jbc.m207237200

Torsten Stein, Stefan Heinzmann + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m207237200

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Abstract

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.

Highlights

Lantibiotic-producing organisms from the action of their own lantibiotics
Fusion of nisI to nisFEG and Transfer to the Chromosome of B. subtilis—In contrast to subtilin immunity (Fig. 1A) [46], the L. lactis nisin immunity genes nisI and nisFEG reside on different transcriptional units (Fig. 1B) [18, 47]
These data clearly showed that B. subtilis MO1099 represents a surrogate host for the functional expression of nisin immunity genes and that the action of two immunity systems, the lipoprotein NisI and the ABC transporter-homologous protein NisFEG, is needed to obtain full nisin tolerance

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Growth Conditions—B. subtilis MO1099 [36] and ATCC 6633 were grown at 37 °C on Difco sporulation medium or M9 medium [37] supplemented with 50 ␮g/ml phenylalanine, 20 ␮g/ml tryptophan, and 0.1% casamino acids. For subtilin production B. subtilis ATCC 6633 was grown at 37 °C in TY medium (0.8% tryptone, 0.5% yeast extract, 0.5% NaCl). Recombinant plasmids were amplified in Escherichia coli DH5␣, TP611, or M15. Antibiotics were used in the following concentrations: 80 ␮g/ml ampicillin for E. coli, and 5 ␮g/ml chloramphenicol, 1 ␮g/ml erythromycin, and 25 ␮g/ml lincomycin for B. subtilis. The alkaline extraction procedure [39] was followed to isolate plasmids of E. coli. Nisin immunity genes were amplified from L. lactis chromosomal DNA or the nisIFEGcontaining plasmid pSI22 [41]. A copy of nisI (EcoRV/XbaI) was cloned into pUC19 [42], resulting in pHZ39, and fused to nisF

Nisin Immunity in Bacillus subtilis

RESULTS

DISCUSSION