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

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 Nis-FEG, 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 hexahistidinetagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.In recent years peptide antibiotics have gained increasing attention as therapeutics (1, 2) and food preservatives (3). Nisin represents the most prominent member of lantibiotics, peptide antibiotics with intramolecular lanthionine bridges (4 -9). The nisin producer Lactococcus lactis 6F3 contains a gene cluster encoding proteins for the biosynthesis and transport (10 -14), immunity (15), and regulation (16 -18) of nisin. Subtilin (19,20) and ericin S (21) produced by Bacillus subtilis ATCC 6633 and A1/3, respectively, are closely related lantibiotics. Lantibiotics form voltage-dependent pores in the bacterial cytoplasmic membrane that are lethal for the target cells but also for the producer. For nisin, the mode of action was investigated in several model systems such as black lipid bilayers and membrane vesicles (22)(23)(24)(25). Recent findings demonstrated that specific binding of nisin to the cell wall precursor lipid II coincides with pore formation (26, 27). Specific selfprotection (immunity) mechanisms are necessary to protect the lantibiotic-producing organisms from the action of their own lantibiotics. For nisin and subtilin (28) Although numerous genes involved in lantibiotic immunity are known, the mechanism by which the encoded proteins confer immunity remain unclear. For full nisin or subtilin immunity, both are required, i.e. the lipoprotein as well as the immunity transporter. The lack of each component diminished the tolerance to nisin (35) or subtilin (28) significantly. Here we report for the first time on the establishment of nisin immunity in the heterologous host B. subtilis. Functional analyses of its different components provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules fro...

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

“…It is conceivable that the putative lipoprotein encoded by pspQ also functions to mediate resistance in the producing strains by sequestering the lantibiotic, preventing interaction with lipid II. A similar function has been proposed for other lipoproteins encoded in lantibiotic biosynthetic gene clusters (37,38).…”

Cloning and Analysis of the Planosporicin Lantibiotic Biosynthetic Gene Cluster of Planomonospora alba

“…PspQ contains a predicted signal peptide sequence containing the lipobox motif LTAC found in lipoproteins (52). The biosynthetic gene clusters for nisin and subtilin also encode lipoproteins (NisI and SpaI, respectively) that provide immunity by sequestering the corresponding lantibiotic, preventing its interaction with lipid II (53,54). Thus, PspQ could function in concert with PspEF as a self-resistance mechanism against planosporicin.…”

Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7

“…For their characterization and applications, bacteriocins of LAB have been purified employing standard methods of purification, which involves more than 3-steps to achieve purification to homogeneity (Daba et al 1994;Elegado et al 1997). However, purification through these routine methods resulted in low levels of recovery and the possibility of the immunity protein remaining contaminated due to their similar molecular weight (MW) and possible in vitro interactions (Stein et al 2003). The main difficulty in exploiting bacteriocins commercially is the low production rates in their native conditions.…”