Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

Abstract

In biotechnological work horses like Streptococcus thermophilus and Bacillus subtilis natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis natural competence has not been established to date. However, in silico analysis of complete genome sequences of 43 L. lactis strains revealed complete late-competence gene-sets in 2 L. lactis subspecies cremoris strains (KW2 and KW10) and 8 L. lactis subspecies lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast, comX expression in L. lactis NZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147, abolished the comX driven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to other L. lactis strains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

“…However, DNA transfer between two L. lactis species by conjugation was developed [15] and a non-conjugative vertical gene transfer has been observed as well (L Morawska, OP Kuipers, personal communication). Recently, the induction of competence genes created functional competence in L. lactis [16]. Different gene modification systems have enabled efficient chromosomal mutations by using recombineering [17].…”

Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria

“…On the one Table 1 Natural competence in lactobacilli. The established genes encoding the DNA uptake machinery of Lactococcus lactis KF147 [12 ] were used to identify homologous genes in Lactobacillus plantarum WCFS1, identifying a complete late competence geneset in this species. Subsequently, the L. plantarum competence genes were used to search the genomes of a set of other Lactobacillus genomes (initially targeting the NCBI-reference genome for each species).…”

High-throughput Genome Editing Tools for Lactic Acid Bacteria: Opportunities for Food, Feed, Pharma and Biotech

“…In natural competence, exogenous DNA translocates through a native, genetically encoded DNA uptake machinery formed by a multiprotein complex (Blokesch 2016). It is well-known in Streptococcus (Gardan 2009;Muschiol 2015), but only recently identified and achieved in Lactococcus strains (David 2017;Mulder 2017).…”