Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>

ABSTRACT 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. Here, we used advanced fluorescence microscopy to visualize the...

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Autores principales:	Jingqi Chen, Auke J. van Heel, Oscar P. Kuipers
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	Bacillus subtilis nisin lantibiotic biosynthesis machinery subcellular localization assembly dynamics fluorescence microscopy Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ce8c268bde174d4e971f8e6fd8cc0089
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spelling	oai:doaj.org-article:ce8c268bde174d4e971f8e6fd8cc00892021-11-10T18:37:51ZVisualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>10.1128/mBio.01219-212150-7511https://doaj.org/article/ce8c268bde174d4e971f8e6fd8cc00892021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01219-21https://doaj.org/toc/2150-7511ABSTRACT 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. 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. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.Jingqi ChenAuke J. van HeelOscar P. KuipersAmerican Society for MicrobiologyarticleBacillus subtilisnisinlantibiotic biosynthesis machinerysubcellular localizationassembly dynamicsfluorescence microscopyMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Bacillus subtilis nisin lantibiotic biosynthesis machinery subcellular localization assembly dynamics fluorescence microscopy Microbiology QR1-502
spellingShingle	Bacillus subtilis nisin lantibiotic biosynthesis machinery subcellular localization assembly dynamics fluorescence microscopy Microbiology QR1-502 Jingqi Chen Auke J. van Heel Oscar P. Kuipers Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
description	ABSTRACT 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. 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. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.
format	article
author	Jingqi Chen Auke J. van Heel Oscar P. Kuipers
author_facet	Jingqi Chen Auke J. van Heel Oscar P. Kuipers
author_sort	Jingqi Chen
title	Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
title_short	Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
title_full	Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
title_fullStr	Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
title_full_unstemmed	Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>
title_sort	visualization and analysis of the dynamic assembly of a heterologous lantibiotic biosynthesis complex in <named-content content-type="genus-species">bacillus subtilis</named-content>
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/ce8c268bde174d4e971f8e6fd8cc0089
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Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in <named-content content-type="genus-species">Bacillus subtilis</named-content>

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