Abstract
ABSTRACTA membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation.
Highlights
A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis
In our initial attempt to produce precursor nisin (NisA) in B. subtilis, the nisin biosynthetic operon nisABTC controlled by the nisin-inducible promoter (PnisA) was integrated into the chromosome of B. subtilis in which the two-component regulatory system NisRK had been introduced
The production of fully modified NisA was not detected after the induction by nisin Z, which was probably caused by the deficient expression of one or more proteins due to the wild-type ribosomal binding sites (RBSs) used, which were from L. lactis
Summary
A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. While NisT could transport unmodified or dehydrated precursor nisin in the absence of either NisB or NisC, the yield of the secreted peptide was severely decreased [28] It seems that the formation of the lantibiotic synthetase complex is not a prerequisite for the correct functioning of any of the enzymes but is crucial for the efficiency of peptide transportation, and the complex is probably highly unstable and transient in nature [2]. This is likely to be the main reason for the difficulty in visually probing the assembly of the machinery by fluorescence microscopy and direct isolation of the whole complex from a wild-type situation in L. lactis.
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