Subcellular Localization and Assembly Process of the Nisin Biosynthesis Machinery in Lactococcus lactis

Abstract

Nisin, a class I lantibiotic, is synthesized as a precursor peptide by a putative membrane-associated lanthionine synthetase complex consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT. Here, we characterize the subcellular localization and the assembly process of the nisin biosynthesis machinery in Lactococcus lactis by mutational analyses and fluorescence microscopy. Precursor nisin, NisB, and NisC were found to be mainly localized at the cell poles, with a preference for the old poles. They were found to be colocalized at the same spots in these old pole regions, functioning as a nisin modification complex. In contrast, the transporter NisT was found to be distributed uniformly and circumferentially in the membrane. When nisin secretion was blocked by mutagenesis of NisT, the nisin biosynthesis machinery was also visualized directly at a polar position using fluorescence microscopy. The interactions between NisB and other components of the machinery were further studied in vivo, and therefore, the "order of assembly" of the complex was revealed, indicating that NisB directly or indirectly plays the role of a polar "recruiter" in the initial assembly process. Additionally, a potential domain that is located at the surface of the elimination domain of NisB was identified to be crucial for the polar localization of NisB. Based on these data, we propose a model wherein precursor nisin is first completely modified by the nisin biosynthesis machinery, preventing the premature secretion of partially modified peptides, and subsequently secreted by recruited NisT, preferentially at the old pole regions.IMPORTANCE Nisin is the model peptide for LanBC-modified lantibiotics that are commonly modified and exported by a putative synthetase complex. Although the mechanism of maturation, transport, immunity, and regulation is relatively well understood, and structural information is available for some of the proteins involved (B. Li, J. P. J. Yu, J. S. Brunzelle, G. N. Moll, et al., Science 311:1464-1467, 2006, https://doi.org/10.1126/science.1121422; M. A. Ortega, Y. Hao, Q. Zhang, M. C. Walker, et al., Nature 517:509-512, 2015, https://doi.org/10.1038/nature13888; C. Hacker, N. A. Christ, E. Duchardt-Ferner, S. Korn, et al., J Biol Chem 290:28869-28886, 2015, https://doi.org/10.1074/jbc.M115.679969; Y. Y. Xu, X. Li, R. Q. Li, S. S. Li, et al., Acta Crystallogr D Biol Crystallogr 70:1499-1505, 2014, https://doi.org/10.1107/S1399004714004234), the subcellular localization and assembly process of the biosynthesis complex remain to be elucidated. In this study, we determined the spatial distribution of nisin synthesis-related enzymes and the transporter, revealing that the modification and secretion of the precursor nisin mainly occur at the old cell poles of L. lactis and that the transporter NisT is probably recruited later to this spot after the completion of the modification reactions by NisB and NisC. Fluorescently labeled nisin biosynthesis machinery was visualized directly by fluorescence microscopy. To our knowledge, this is the first study to provide direct evidence of the existence of such a complex in vivo Importantly, the elucidation of the "order of assembly" of the complex will facilitate future endeavors in the investigation of the nisin secretion mechanism and even the isolation and structural characterization of the complete complex.

Keywords: Lactococcus lactis; assembly; biosynthesis machinery; lantibiotics; subcellular localization.

FIG 1

Biosynthesis, regulation, and immunity of nisin in L. lactis. Precursor nisin (NisA) is a ribosomally synthesized peptide with a leader peptide and a core peptide that is then targeted to putative nisin biosynthesis machinery consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT. NisB converts serine and threonine residues into dehydroalanine and dehydrobutyrine, respectively. NisC catalyzes the addition of a thiol group in cysteine to an N-terminally located dehydroamino acid, resulting in the characteristic lanthionine rings. Subsequently, the transporter NisT exports the fully modified precursor nisin outside the cells, where the serine protease NisP extracellularly removes the leader peptide, releasing active nisin. Immunity is conferred by two different systems, the lipoprotein NisI and the ABC transporter NisFEG, protecting the host from the antimicrobial action of nisin. Extracellularly present nisin binds to NisK, a histidine sensor kinase, which starts the autophorylation of a histidine of NisK. Subsequently, phosphate is transferred to NisR, a transcriptional activator, and therefore the promoters indicated by P* are activated by phosphorylated NisR. The other two promoters (P) are constitutive.

FIG 2

Production of nisin in plasmid-based and chromosomally integrated expression systems. (A) Extracellular and intracellular precursor nisin (NisA) detected by Western blotting using anti-leader peptide antibody. (B) Antimicrobial activity assay. The supernatant of the culture was incubated with the purified protease NisP. The indicator strain is Micrococcus flavus. (C) MALDI-TOF MS data. The predicted mass of fully modified precursor nisin is 5,688 Da. (a) Wild-type strain NZ9000, which does not contain nisin biosynthetic genes; (b) NZ9000/pTLR3-nisABTC; (c) NZ9000 pseudo10::nisABTC; pseudo 10, an integration locus in the chromosome of L. lactis NZ9000.

FIG 3

Determination of the subcellular localization of the nisin biosynthesis machinery-associated components using fluorescent protein labeling. (A) Western blot analysis of fusion proteins in the lysate (L), cytosol (C), and membrane (M) fractions. NisA-sfGFP, NisB-sfGFP, NisT-sfGFP, NisC-sfGFP, and sfGFP were determined in different fractions of the strains NZ9000/pTLR3-nisA_sfgfp-nisBTC, NZ9000/pTLR3-nisA-nisB_sfgfp-nisTC, NZ9000/pTLR3-nisAB-nisT_sfgfp-nisC, NZ9000/pTLR3-nisABT-nisC_sfgfp, and NZ9000/pTLR3-sfgfp, respectively. The monoclonal anti-GFP antibody was used. (B) Antimicrobial activity assay. (1) NZ9000/pTLR3, used as a negative control; (2) NZ9000/pTLR3-nisABTC; (3) NZ9000/pTLR3-nisA_sfgfp-nisBTC; (4) NZ9000/pTLR3-nisA-nisB_sfgfp-nisTC; (5) NZ9000/pTLR3-nisAB-nisT_sfgfp-nisC; (6) NZ9000/pTLR3-nisABT-nisC_sfgfp. All the samples were treated with the purified protease NisP. The indicator strain is Micrococcus flavus. (C) Determination of the extent of modification of the NisA portion of the fusion protein NisA-sfGFP. (Left) Purification of NisA-sfGFP_His and removal of sfGFP. Ni-NTA, NisA-sfGFP_His primarily purified using Ni-NTA agarose; SEC, NisA-sfGFP_His further purified by size exclusion chromatography; SEC+Factor Xa protease, sfGFP_His removed by incubation with the protease factor Xa. The factor Xa site IEGR was located between NisA and sfGFP_His. (Middle) MALDI-TOF MS data. The predicted mass of modified NisA-IEGR with 7 dehydrations is 6,161.3 Da. The observed mass is 6,162.2 Da. (Right) Antimicrobial activity assay of NisA-IEGR. The indicator strain is Micrococcus flavus. (D to G) Subcellular localization of sfGFP-labeled proteins and quantification of the proportion of cells with polar fluorescent foci in the strains NZ9000/pTLR3-nisA_sfgfp-nisBTC (D), NZ9000/pTLR3-nisA-nisB_sfgfp-nisTC (E), NZ9000/pTLR3-nisABT-nisC_sfgfp (F), and NZ9000/pTLR3-nisAB-nisT_sfgfp-nisC (G). N is the number of counted cells from 3 independent experiments. All the above-described analyses were conducted in the plasmid-based expression system.

FIG 4

Determination of the localization of precursor nisin using FlAsH labeling. (A) Design of precursor nisin (NisA) tagged by the FlAsH tag. The FlAsH tag was added to the N terminus or the C terminus of precursor nisin, generating MCCPGCC-NisA (peptide b) and NisA-CCPGCC (peptide c). Between precursor nisin and the FlAsH tag, a factor Xa site and a flexible linker were inserted into peptide c. (B) Antimicrobial activity assay. The supernatant of the culture was incubated with purified NisP. The indicator strain is Micrococcus flavus. (C to E) Subcellular localization of precursor nisin with the FlAsH tag and quantification of the proportion of cells with polar fluorescent foci in strains a, b, and c. (a) NZ9000/pTLR3-nisABTC, producing precursor nisin without labeling; (b) NZ9000/pTLR3-_FlAsHnisA-nisBTC, producing precursor nisin with the FlAsH tag at the N terminus; (c) NZ9000/pTLR3-nisA_FlAsH-nisBTC, producing precursor nisin with the FlAsH tag at the C terminus. In panels D and E, percentages were normalized to the total number of bacteria showing a fluorescent signal. N is the number of counted cells from 3 independent experiments. All the above-described analyses were performed using the plasmid-based expression system.

FIG 5

Colocalization of NisA, NisB, and NisC. (A) The fusion proteins NisA-sfGFP and NisB-mCherry were colocalized at the cell poles in the strain NZ9000/pTLR3-nisA_sfgfp-nisB_mCherry-nisTC in the presence of NisT and NisC. (B) The fusion protein NisA-sfGFP was colocalized with mCherry-NisC to the cell poles in the strain NZ9000/pTLR3-nisA_sfgfp-nisBT-_mCherrynisC when NisB and NisT were coexpressed. (C) The fusion proteins NisB-sfGFP and mCherry-NisC were colocalized at the same spots of the cell poles of the strain NZ9000/pTLR3-nisA-nisB_sfgfp-nisT-_mCherrynisC with the coexpression of NisA and NisT. In panels A to C, quantitative analysis of the respective localizations of proteins tagged by sfGFP or mCherry was also performed. Same pole, green foci and red foci colocalized to the same cell poles; Different pole, green foci and red foci localized to different cell poles; Others, no fluorescent focus or weak signal. N is the number of counted cells from 3 independent experiments.

FIG 6

The nisin modification complex represented by NisB-sfGFP was primarily localized to the old pole. Cells from the culture of the strain NZ9000/pTLR3-nisA-nisB_sfgfp-nisTC were transferred to a microscope slide with an agarose patch containing growth medium with 5 ng/ml nisin Z as an inducer. Images were captured at 15-min intervals using time-lapse microscopy. The red arrows indicate the appearance of old fluorescent poles. The blue arrows show the appearance of new fluorescent poles.

FIG 7

Visualization of the mutant nisin biosynthesis machinery NisBT^H551AC using fluorescence microscopy. (A) Blocking the secretion of nisin by introducing a mutation, H551A, into NisT. (1) NZ9000/pTLR3-nisABTC; (2) NZ9000/pTLR3-nisAB-nisT_sfgfp-nisC; (3) NZ9000/pTLR3-nisAB-nisT^H551A-nisC; (4) NZ9000/pTLR3-nisAB-nisT^H551A_sfgfp-nisC. The supernatant of the culture was incubated with purified NisP. The indicator strain is Micrococcus flavus. (B) Subcellular localization of NisT-sfGFP in the strain NZ9000/pTLR3-nisAB-nisT_sfgfp-nisC. (C) Subcellular localization of NisT^H551A-sfGFP in the strain NZ9000/pTLR3-nisAB-nisT^H551A_sfgfp-nisC. NisT^H551A, mutation H551A introduced into the NBD of the ABC transporter NisT. N is the number of counted cells from 3 independent experiments. (D) Localization of NisT^H551A-sfGFP in the strain NZ9000/pTLR3-nisT^H551A_sfgfp. (E) Colocalization of NisB-mCherry and NisT^H551A-sfGFP in the strain NZ9000/pTLR3-nisA-nisB_mCherry-nisT^H551A_sfgfp-nisC in the presence of NisA and NisC. Same pole, green foci and red foci colocalized to the same cell poles; Different pole, green foci and red foci localized to different cell poles; Others, no fluorescent focus or weak signal. N is the number of counted cells from 3 independent experiments.

FIG 8

NisC and NisT were targeted to NisB when they were coexpressed with NisB. (A) Effect of coexpression with NisA, NisC, or NisT on the localization of NisB-sfGFP. (B) Effect of coexpression with NisA, NisB, or NisT on the localization of NisC-sfGFP. (C) Effect of coexpression with NisA, NisB, and NisC on the localization of NisT-sfGFP. Same pole, green foci and red foci colocalized to the same cell poles; Different pole, green foci and red foci localized to different cell poles; Others, no fluorescent focus or weak signal. N is the number of counted cells from 3 independent experiments.

FIG 9

Identification of the domain required for the polar localization of NisB. (A) Native NisB labeled by sfGFP at the C terminus. (B) NisB with deletions based on the predicted transmembrane domain (NisB_838–851) labeled by sfGFP. (C) NisB with deletions based on the predicted degradation site (residue 760) labeled by sfGFP. (D) NisB with deletion of amino acid residues 750 to 769 labeled by sfGFP. N, N-terminal domain of NisB; D (760), predicted degradation site (residue 760), which is located in the N-terminal domain of NisB; T, predicted transmembrane domain NisB_838–851; C, C-terminal domain of NisB. (E) Expression of truncated NisB labeled by sfGFP. The monoclonal anti-GFP antibody was used. When the proportion of cells containing polar foci was quantified, the number of cells was higher than 100, and the cells were from 3 independent experiments.

FIG 10

Location of the domain NisB_750–769 within the crystal structure of NisB (PDB accession no. 4WD9) (15). (A) Overall structure of the NisB homodimer showing the disposition of the domain NisB_750–769. The NisB homodimer is shown with one monomer in blue and the other monomer in green. The NisB_750–769 domains are shown in pink and red. The elimination domain is shown in the black square. (B) The majority of residues 750 to 769 are located on the surface of the elimination domain of NisB.

FIG 11

Proposed model of the assembly process and subcellular localization of nisin biosynthesis machinery. (A) NisB plays the role of a “recruiter” in the assembly of the complex NisABTC. NisB is localized to the old cell poles at an early stage. Precursor nisin, NisC, and NisT travel to the poles by binding to NisB via the “diffusion-and-capture” mechanism, generating the nisin biosynthesis machinery. (B) Recruited NisT transports fully modified precursor nisin released from the complex NisBC once the (methyl)lanthionine rings are formed.