Identification of a nisI promoter within the nisABCTIP operon that may enable establishment of nisin immunity prior to induction of the operon via signal transduction

Abstract

Certain strains of Lactococcus lactis produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons: nisABTCIP, encoding production and immunity (nisI); nisRK, encoding regulation; and nisFEG, also involved in immunity. Transcription of nisABTCIP and nisFEG requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both Lactococcus and Enterococcus backgrounds revealed that nisI mRNA was present under conditions when no nisA transcription was occurring, suggesting an internal promoter within the operon. The nisA transcript was significantly more stable than nisI, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from nisI. Fusing this region to a lacZ gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the nisI promoter was mapped at bp 123 upstream of the nisI translation start codon. Ordered 5' deletions revealed that transcription activation depended on sequences located up to bp -234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this nisI promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.

FIG. 1.

(A) Detection of the nisI and nisA transcripts in Enterococcus sp. strain N12b by RT-PCR. R/T, reverse transcriptase. Reaction mixtures without reverse transcriptase were included as negative controls. (B) Detection of the nisI and nisC transcripts in Enterococcus sp. strain N12b by RT-PCR. −, cultures grown without added nisin; +, cultures grown with added nisin to induce nisA transcription; M, 1-kb DNA ladder.

FIG. 2.

(A) Measurement of nisA and nisI mRNA stabilities by Northern dot blot hybridization. The specificity of both probes has previously been demonstrated (10, 32). Total RNA was extracted at 0, 5, 10, 15, 20, and 30 min and hybridized with nisA and nisI DNA probes. (B) nisA mRNA secondary structure predicted with GeneQuest (DNAStar, Inc., Madison, WI).

FIG. 3.

(A) Bioassay to detect nisin, using Micrococcus luteus as the indicator strain. i, supernatant from L. lactis ATCC 11454 grown overnight at 30°C; ii, supernatant from L. lactis ATCC 11454 grown overnight at 40°C; iii, supernatant from L. lactis ATCC 11454 grown at 40°C for 25 generations; (iv) supernatant from the 25-generation culture in spot which was subinoculated and grown overnight at 30°C. (B) Northern hybridization of total RNA isolated from L. lactis ATCC 11454 grown at 30°C and at 40°C for 25 generations with either a nisA or a nisI probe. Lanes 1, 1 μg RNA was spotted; lanes 2, 5 μg RNA was spotted.

FIG. 4.

Graphical representation of the nisI promoter fusion plasmid, pDOL0620.

FIG. 5.

(A) Mapping of the nisI TSS in pDOL0620 by using the modified primer extension-based RACE methodology. Lane 1, nisI cDNA product; lane 2, negative control, where no reverse transcriptase was added. The TSS is indicated by +1, located 123 bp upstream from the nisI translation start codon (in boldface). (B) Sequence of the upstream region of the nisI promoter. The two poly(A) tracts are in boldface and indicated by arrows. The CA doublets at the 5′ ends of the poly(A) tracts are underlined.

FIG. 6.

Ordered 5′ deletion mutants of the nisI promoter fragment in pDOL0620. The poly(A) tracts are boxed. Plasmids exhibiting promoter activity are indicated by + or −.

FIG. 7.

Transcriptional organization of the complete nisin gene cluster, showing the two nisin-inducible promoters and the two constitutive promoters.