Distinct contributions of the nisin biosynthesis enzymes NisB and NisC and transporter NisT to prenisin production by Lactococcus lactis

Abstract

Several Lactococcus lactis strains produce the lantibiotic nisin. The dedicated enzymes NisB and NisC and the transporter NisT modify and secrete the ribosomally synthesized nisin precursor peptide. NisB can function in the absence of the cyclase NisC, yielding the dehydrated prenisin that lacks the thioether rings. A kinetic analysis of nisin production by L. lactis NZ9700 demonstrated that the prenisin was released from the cell into the medium before the processing of the leader sequence occurred. Upon the deletion of nisC, the production of prenisin was reduced by 70%, while in the absence of nisB, the production of prenisin was nearly completely abolished. In cells lacking nisT, no secretion was observed, while the expression of nisABC in these cells resulted in considerable growth rate inhibition caused by the intracellular accumulation of active nisin. Overall, these data indicate that the efficiency of prenisin transport by NisT is markedly enhanced by NisB, suggesting a channeling mechanism of prenisin transfer between the nisin modification enzymes and the transporter.

FIG. 1.

Prenisin secreted by L. lactis NZ9700 is slowly processed into mature nisin. L. lactis NZ9700 cells were pulse-labeled with [³⁵S]methionine. Samples were taken at the indicated time points after the addition of [³⁵S]methionine. Cells were lysed, and medium components were precipitated with TCA. A fraction containing 10% of the lysed cells (A) and the complete precipitated medium fraction (B) were loaded onto a Tricine-SDS-16% polyacrylamide gel. Molecular sizes (MW) of marker proteins are indicated in between the panels; prenisin and mature nisin are indicated by black and white arrowheads, respectively. *, samples from a similarly labeled L. lactis NZ9800 culture.

FIG. 2.

Prenisin production by L. lactis NZ9000 cells expressing different combinations of the nisB, nisT, and nisC genes together with nisA. Cells expressing the indicated nisin genes in medium containing 0.1 mM unlabeled methionine were labeled with [³⁵S]methionine, and at the indicated time points, cell (left) and medium (right) fractions were isolated as described in the legend to Fig. 1. Molecular sizes (MW) of marker proteins are indicated on the right. Prenisin is indicated by black arrowheads, and an intracellular prenisin degradation product is indicated by white arrowheads.

FIG. 3.

Intracellular accumulation of prenisin and stability of secreted prenisin and derived peptides. (A) Intracellular prenisin accumulation in L. lactis NZ9000 cells lacking nisABTC or expressing different combinations of the nisB, nisT, and nisC genes together with nisA as indicated. Samples were taken prior to induction (0 h) and after 2 and 4 h of induction. Equal amounts of lysed cells were analyzed by Tricine-SDS-16% PAGE, followed by immunoblotting using antibodies directed against the leader sequence (lanes 4 to 21). Samples of about 2 μg each of purified unmodified prenisin (pn; lane 1), dehydrated prenisin (lane 2), nisin (lane 3), and fully modified prenisin (lane 22) were loaded as controls. Note that the antibody does not recognize the mature nisin. (B) Purified peptide at a concentration of ∼0.3 mg/ml was incubated at 30°C in filter-sterilized spent medium from an L. lactis NZ9000 culture. At the time points indicated, samples were taken and analyzed by Tricine-SDS-16% PAGE and Coomassie brilliant blue staining. MW, molecular size markers.

FIG. 4.

In the absence of nisT, the expression of nisABC inhibits the growth of L. lactis NZ9000. L. lactis NZ9000 strains containing different combinations of nisA or truncated nisA [nisA^trunc, encoding NisA(Δ23-34)] and the nisB, nisT, and nisC genes (as indicated) under the control of the nisin-inducible promoter were grown in CDM containing nisin at a concentration of 0 (white bars), 0.1 (lightest gray bars), 2 (medium gray bars), or 10 (darkest gray bars) ng/ml. The specific growth rate (μ) was determined from the increase of the OD₆₆₀ in the exponential growth phase. Each bar indicates the average specific growth rate of three independent cultures. The standard deviations are indicated by error bars.

FIG. 5.

Proposed channeling mechanism of prenisin transfer between the nisin modification enzymes NisB and NisC and the transporter NisT. (A) Prenisin is targeted to the NisBTC complex, dehydrated by NisB, cyclized by NisC, and subsequently secreted by NisT. Outside the cell, the mature prenisin is processed by NisP into the active nisin. CM, cell membrane. (B) In the absence of NisB, the production of prenisin is almost completely abolished, suggesting a role of NisB in membrane targeting and channeling of unmodified prenisin in the cell. Without this targeting, the unmodified prenisin is proteolyzed in the cell.