Plantaricin NC8 (PLNC8), a coculture-inducible two-peptide bacteriocin fromLactobacillus plantarumNC8, 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 inL. plantarumC11. 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 ofplNC8IFin NC8 allowed constitutive bacteriocin production. Heterologous expression of this gene inLactococcus lactisMG1363 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 inL. plantarumNC8 via a quorum-sensing mechanism mediated by PLNC8IF.

Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed. We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI). The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2 ³ _5-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3 ² _3-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10 ^7.3 to 10 ^7.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10 ⁴ times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Dı́az, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459–4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides α and β, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus , which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr -like regulatory system for the production of plantaricin S is postulated.