Denis P. Twomey

Lantibiotics produced by lactic acid bacteria: structure, function and applications

Lantibiotics are a diverse group of heavily modified antimicrobial and/or signalling peptides produced by a wide range of bacteria, including a variety of lactic acid bacteria. Based on their diverse structures and mode of action, at least six separate lantibiotic subgroups can be suggested, but all subgroups are characterized by significant post-translational modifications, which include the formation of (β-methyl)lanthionines, among other unusual alterations. These small peptides are produced, modified, exported, sensed and combated by a complex set of proteins encoded by (usually) co-ordinately regulated operons. In some instances, the production and immunity have been shown to be auto-regulated by the mature lantibiotic. Since their discovery, interest in lantibiotics has been fuelled by their obvious potential as food-grade antimicrobials to improve food safety and quality; a potential which, to date, has been realised only by the longest characterised molecule, nisin. In addition, these peptides are often mooted as alternatives to antibiotics for some biomedical applications. The purpose of this paper is to review recent developments in our understanding of lantibiotic structure, molecular genetics and applications for this unusual class of bacteriocins.

Developing applications for lactococcal bacteriocins

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against Gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrat ed its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.

Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis

This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82. The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co‐integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain. Analysis of the regions involved in the co‐integration revealed that two novel hybrid hsdS genes had been formed during the co‐integration event. The HsdS chimeras had interchanged the C‐ and N‐terminal variable domains of the parent subunits, generating two new restriction specificities. Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co‐integration event is highly conserved among lactococcal type I hsdS determinants. Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes. This study demonstrates that, similar to previous observations in Gram‐negative bacteria, a Gram‐positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.

Molecular genetics of bacteriophage and natural phage defence systems in the genus Lactococcus

Abstract Bacteriophage infection of starter cultures used in a range of milk fermentation processes, particularly those involving Lactococcus lactis, poses a significant problem in industrial practice. The application of genetic and molecular technologies to the study of lactococcal bacteriophages has proven to be very rewarding in terms of understanding the nature of phage with respect to their physical and genetic organisation. The availability of the full genomic sequence of a number of phages provides an unambiguous basis for determining the relationship between them, for elucidating their evolutionary progression and will also yield strategies for obstructing successful phage proliferation on previously sensitive hosts. The genetic analysis of phage/host interactions has also highlighted the presence of natural defence systems (e.g. adsorption blocking, inhibition of phage DNA entry, restriction modification and abortive infection) in lactococci. A number of restriction modification systems and abortive infection mechanisms have been characterized at a molecular level and the genes involved have been cloned and sequenced. Plasmid-encoded phage resistance mechanisms can be exploited to generate strains which can successfully counter phage proliferation and will provide a basis for understanding the complex interactions between phages and their target hosts at a molecular level.

The lantibiotic lacticin 3147 produced in a milk-based medium improves the efficacy of a bismuth-based teat seal in cattle deliberately infected with Staphylococcus aureus.

A preparation of the bacteriocin lacticin 3147 (prepared from a demineralized whey protein fermentation liquor) was combined as a powder with a bismuth-based intramammary teat seal and evaluated for its potential as an antimicrobial in non-lactating cows. The lacticin/teat seal formulation enabled significant bacteriocin release from the seal without the requirement for a surfactant. Studies in vivo in lactating cows demonstrated that this formulation was effective in reducing bacterial recoveries (approximately 20-fold) from teats deliberately inoculated with Staphylococcus aureus after infusion. Moreover, this formulation also significantly reduced the numbers of Staph. aureus recovered from teats that were exposed to the challenge bacterium before the infusion of the teat seal preparation. The powdered preparation of lacticin 3147 did, however, cause some teat irritation as evidenced by associated rises in somatic cell count (SCC). However, this effect was short-lived and when the mean SCC readings pre-infusion and the final two readings post-infusion were compared, there was no significant difference in the immunological acceptance between treatments.

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2

ABSTRACT The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, “early”-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

ABSTRACT Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10−4 transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriTof pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.

Potential of the enterocin regulatory system to control expression of heterologous genes in Enterococcus

Aims: To exploit the enterocin regulatory system for regulated expression of genes in Enterococcus.