Induction of plantaricin production in Lactobacillus plantarum NC8 after coculture with specific gram-positive bacteria is mediated by an autoinduction mechanism

Abstract

Plantaricin NC8 (PLNC8), a coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8, has recently been purified and genetically characterized. Analysis of an 8.1-kb NC8 DNA region downstream of the PLNC8 operon revealed the presence of at least four operons involved in bacteriocin production, showing high homology to the plantaricin cluster in L. plantarum C11. However, we found a three-component regulatory operon involving a quorum-sensing mechanism. Two of these components, the induction factor (PLNC8IF) and the histidine kinase, are novel, while the response regulator is identical to PlnD from C11. Homologous expression of plNC8IF in NC8 allowed constitutive bacteriocin production. Heterologous expression of this gene in Lactococcus lactis MG1363 produced supernatants which promoted bacteriocin production in NC8. Reverse transcription-PCR studies indicated that cocultivation of NC8 with inducing cells promoted transcription of the bacteriocin and regulatory operons in NC8. An identical result was obtained after addition of an external source of PLNC8IF. We propose that the presence of specific bacteria could act as an environmental signal that is able to switch on bacteriocin production in L. plantarum NC8 via a quorum-sensing mechanism mediated by PLNC8IF.

FIG. 1.

Genetic map of the PLNC8 gene cluster. (A) DNA fragments that were either cloned or PCR amplified and then subjected to total or partial DNA sequencing. (B) Genetic map of the PLNC8 gene cluster, showing the genes and ORFs that have been identified. Solid arrows, NC8 genes that share a high degree of homology with, or are virtually identical to, genes found in L. plantarum C11 (see Table 3). Open arrows, L. plantarum NC8 genes that have not been described elsewhere. Dotted arrows, genes that have been identified in NC8 by PCR amplification with specific primers. Numbered arrowheads represent primers used to amplify subsequently sequenced DNA fragments, as follows: 1, PlnM-for; 2, NC8-17; 3, PlnI-rev; 4, PlnH-rev (see Table 2). The bent arrow indicates the start point of the new sequence described in this work. Lollipops indicate the positions of putative promoter sequences. The 898-bp AccI/ClaI DNA fragment used as a probe is indicated. (C) Genetic map of the plantaricin gene cluster in L. plantarum C11, showing the genes that have been described previously (9, 11). Hatched arrows, genes that have not been found in L. plantarum NC8.

FIG. 2.

Diagrammatic representation of the construction of recombinant plasmids pSIG306 and pSIG308. Restriction enzymes used to digest parental molecules prior to ligation are indicated. Restriction sites of selected DNA regions are indicated: B, BamHI; E, EcoRI; K, KpnI. P1 and P2 stand for primers KpnNC8IF-for and EcoNC8IF-rev, respectively (see Table 2). The PLNC8IF protein sequence is shown, with the double-glycine motif (boldfaced) and the cleavage site in the native peptide (vertical arrow) indicated. The position of the lactococcal promoter P₅₉ is indicated. MCS, multicloning site.

FIG. 3.

Alignment of the promoter sequences of plNC8IF and plnA, showing conserved nucleotides (boldfaced) and significant features (L and R repeats; −35 and −10 sites) (boxes).

FIG. 4.

RT-PCR analysis of expression of the bacteriocin and regulatory operons in L. plantarum NC8 at the early-exponential phase of growth in MRS medium. Expression of the plNC8BA (A), plnEF (B), plnJK (C), and plNC8IF-plNC8HK-plnD (D) operons was analyzed. Lanes: 1, MW marker (1 kb plus DNA ladder; Gibco BRL); 2, pure culture of L. plantarum NC8; 3, pure culture of L. plantarum NC8 that had been induced with a PLNC8IF-rich, 10-fold-concentrated CFS from L. lactis MG1363(pSIG308); 4, L. plantarum NC8 culture that had been induced with heat-killed cells of L. lactis MG1363; 5, L. plantarum NC8 that had been cocultivated with L. lactis MG1363; 6, control PCR using total DNA from L. plantarum NC8 as the template. MWs of relevant bands of the marker are given on the left. Designations and sizes of the expected products are given on the right.