Impact of the nisin modification machinery on the transport kinetics of NisT

Abstract

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied examples is nisin produced by Lactococcus lactis. Nisin is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT that secretes nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the nisin maturation machinery, consisting of the dehydratase NisB and the cyclase NisC. Our determined apparent secretion rates of NisT show how NisB and NisC modulate the transport kinetics of NisA. Additional in vitro studies of the detergent-solubilized NisT revealed how these enzymes and the substrates again influence the activity of transporter. In summary, this study highlights the pivotal role of NisB for NisT in the secretion process.

Figure 1

Scheme of nisin modification and secretion system. (a) The lanthipeptide nisin (NisA, grey) operon encodes for the modification and secretion enzymes. (b) The enzyme NisB (blue) catalyzes the dehydration reaction of unmodified NisA (uNisA), whereas NisC (red) catalyzes the thioether ring formation resulting in modified NisA (mNisA). The ABC transporter NisT (green) translocates mNisA across the membrane to the exterior. Finally, the mature peptide is processed by the serine protease NisP (turquoise) and active nisin is released. The two-component system (TCS) consisting of NisR and NisK (orange) is controlling the expression of these proteins. Please note, that the operon is partial represented and shows only proteins responsible for nisin maturation and secretion.

Figure 2

In vivo secretion assay of different L. lactis NZ9000 strains. (a) The supernatants of NisA secreting L. lactis NZ9000 strains was analyzed by RP-HPLC and the amount of NisA were determined. Amounts of secreted peptides (nmol) are plotted against time (min) and the resulting curves were fitted by an allosteric sigmoidal fit. Modified NisA (mNisA, red) was secreted by strain NZ9000BTC (red rhomb) and can be precluded by nisT deletion (strain NZ9000BC, clear dot) or an ATPase deficient mutant (NZ9000BT_H551AC, red square). Dehydrated NisA (dNisA, blue) was secreted by strains NZ9000BTC_H331A (blue square) and NZ9000BT (blue dots), whereas unmodified NisA (uNisA, grey) was secreted by the strains NZ9000T (grey dots) and NZ9000TC (grey square). Dashed square shows a zoom-in on strains with lower secretion level. (b) The kinetic parameter of V_max (nmol) of secreted peptides was plotted as bars against the various secretion systems. (c) The secretion rate of NisA molecules per NisT molecule was plotted against time (min) and fitted by linear regression. The slope represented the secretion rate of NisA·NisT⁻¹·min⁻¹ for the strains NZ9000BTC and NZ9000T. All data represent secretion experiments from at least five different transformants and are represented as means ± s.d. (n = 5).  + + : WT secretion; o: low secretion; −: no secretion.

Figure 3

Purification and ATPase activity assay of NisT. (a) SEC chromatogram of 10HNisT (WT, black line) displayed a homogeneous peak (E_SEC) at 13 ml on a Superose 6 10/300 GL column (V₀: void volume of the column). Inset: A typical colloidal Coomassie (cc) stained SDS-PAGE gel shows a protein band between 55 and 72 kDa marker protein bands (M). (b) Purification of the H-loop mutant 10HNisT_H551A (HA, dashed line) showed comparable results for SEC profile and SDS-PAGE gel (inset). (c) The specific ATPase rate (nmol·min⁻¹·mg protein⁻¹) of purified WT (black dot) and HA mutant (unfilled circle) was plotted against ATP concentration (mM) to determine kinetic parameters. The ATPase rate was fitted by Michaelis–Menten equation to determine V_max (nmol·min⁻¹·mg protein⁻¹), K_m (mM) and k_cat (min^-1). Activity assays were performed from five independent experiments with three replicates and are represented as means ± s.d. (n = 5).

Figure 4

Dependence of NisT ATPase rate on different substrates. The ATPase rate of purified 10HNisT was analyzed in the presence of different substrates. (a) The leader peptide of NisA (NisA_LP, black dots), (b) uNisA (grey dots), (c) dNisA (blue dots) and (d) mNisA (red dots) was used in various concentrations (µM) and the ATPase rate is shown as normalized ATPase rate (%). The basal ATPase rate (from 10HNisT without any substrate; compare Fig. 3) was set as 100% (dashed line) and further values were normalized accordingly. The assays were performed in at least four independent experiments and are represented as means ± s.d. (n = 4). The means were analyzed by a one-way ANOVA and the differences were not significant (p-value: ≥ 0.05). ns: not significant.

Figure 5

Influence of NisBC on the ATPase rate of NisT. The ATPase rate of purified 10HNisT was analyzed in the presence of the modification enzymes NisB and NisC, respectively. (a) ATPase rate of 10HNisT was plotted against variation of 10HNisT with the modification enzymes NisB, NisC and NisBC. It showed the normalized ATPase rate, in which the ATPase rate of 10HNisT was set to 100% (dashed line). (b) The substrate NisA_LP (black dots) and (c) mNisA (red dots) were used in the assay with 10HNisT in presence of NisBC. The normalized ATPase rate was plotted against various concentrations (µM), where the ATPase rate of 10HNisT plus NisBC was set to 100% (dashed line). All assay assays were performed in at least four independent experiments and are represented as means ± s.d. (n = 4). The means were analyzed by a one-way ANOVA and the differences were not significant (p-value: ≥ 0.05).

Figure 6

Pull-down assay of NisT with NisB and NisC. The interaction of 10HNisT with NisB and NisC was studied by a pull-down assay. The ABC transporter was immobilized on NTA-magnetic beads, the specific interaction partner were added and incubated. After six washing steps, the last washing step (W₆) and the EDTA elution fraction (E) were analyzed by Western blot with the specific antibodies (α-NDB, α-NisB or α-NisC). Western blots displayed the eluted bands for NisB, NisT and NisC without substrate (a) and with substrates mNisA or mNisA_CCCCA (b). The pull-down assay was repeated three times and showed similar results. M: marker protein bands; + : protein was used in assay; −: protein was not used in assay.