Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2

Abstract

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Keywords: Clp protease; ComX; Lactococcus; competence; lactic acid bacteria; natural DNA transformation; sigma factor.

FIG 1

Late com genes in the complete genomes of L. lactis subsp. cremoris strains MG1363, A76, SK11, UC509.9, and KW2. The strain origin is indicated above each name. The essential late genes for DNA transformation identified in S. pneumoniae (17) are highlighted in blue. Two genes of the comG operon, which are potentially essential for DNA transformation, are highlighted in gray. Gene-associated function in DNA transformation is indicated on the left. Reg., regulation. The complete and incomplete status of late genes is based on blastp and tblastn similarity searches using orthologues of S. pneumoniae TIGR4 and S. thermophilus LMD-9 and default parameters. Symbols and abbreviations: + (green), presence of a complete gene; * (yellow), incomplete gene due to nucleotide change, insertion, or deletion resulting in a premature stop codon; Tn (red), gene disrupted by the insertion of at least one transposon.

FIG 2

Activation of the late promoter P_comGA by constitutive comX overexpression. (A) Maximum specific luciferase (Lux) activity (RLU OD₆₀₀⁻¹) emitted by eight independent clones (clones 1 to 8) of the ComX⁺ strain [BLD101(pGIBLD001), P_comGA-luxAB] compared to the control strain (Ctl⁻) carrying the empty vector [BLD101(pG⁺host9)]. (B) Kinetics of specific Lux activity (solid lines) during growth (OD₆₀₀, dotted lines) for the control strain (Ctl⁻, black lines) and three selected ComX⁺ clones [BLD101(pGIBLD001), clones [cl.] 2, 4, and 5; gray lines]. Cells were grown in CDMG. The results of one representative experiment of two independent replicates are shown.

FIG 3

Upregulated late com genes preceded by a predicted Com-box upon constitutive comX expression. (A) Genetic organization of 11 loci (I to IX), including all the essential late genes for DNA transformation which are upregulated in clone 2 of the ComX⁺ strain [BLD101(pGIBLD001)], based on transcriptomic analyses. Pentagons represent open reading frames with respect to their sizes and orientations. Red-outline pentagons indicate genes essential for genetic transformation. Red- and yellow-filled pentagons represent, respectively, genes with an FC ≥ 2 and an FC between ≥1.5 and <2 as reported in the RNA sequencing analysis (see Table S2 in the supplemental material). The gene symbols and locus tag number are indicated above and below the pentagons, respectively. Black arrows topped with black-outlined squares indicate the position of predicted Com-boxes; horizontal arrows indicate the orientation of the motif. (B) Consensus of the predicted Com-box motif (27 bp) of strain KW2 (see Table S3) based on the sites identified in the 11 loci reported in panel A.

FIG 4

DNA transformation of the strA1 allele upon constitutive comX expression. (A) Transformation rate (white bars) and maximum specific luciferase (Lux) activity (black diamonds, RLU OD₆₀₀⁻¹, as reported in Fig. 2) of eight clones (clones 1 to 8) of the ComX⁺ strain [BLD101(pGIBLD001), P_comGA-luxAB] compared to the negative-control strain (Ctl⁻) carrying the empty vector [BLD101(pG⁺host9)]. (B) Transformation rate (white bars) and maximum specific luciferase (Lux) activity (black diamonds, RLU OD₆₀₀⁻¹) of four clones (clones 2-1 to 2-4) of the ComEC⁻ ComX⁺ strain [BLD102(pGIBLD001), P_comGA-luxAB] compared to the positive [Ctl⁺, BLD101(pGIBLD001) clone 2] and negative [Ctl⁻, BLD101(pG⁺host9)] control strains. Transformability was assessed according to the standard protocol described in Materials and Methods using strA1-carrying PCR products as donor DNA. ND, transformation rate below the detection level of spontaneous Str^r mutants (<10⁻⁷). The results of one representative experiment of two independent replicates are shown.

FIG 5

Activation of the late promoter P_comGA by inducible comX expression. (A) Kinetics of specific Lux activity during growth (OD₆₀₀) of strain BLD101(pGIFPT001) grown in CDMG, CDMX, M17G, and M17X. The symbols for OD₆₀₀ in CDMG (OD CDMG), CDMX (OD CDMX), M17G (OD M17G), and M17X (OD M17X) are long black dashes, long gray dashes, short black dashes, and short gray dashes, respectively. The symbols for Lux activity in CDMG (Lux CDMG) and M17G (Lux M17G) are solid black lines; the symbols in CDMX (Lux CDMX) and M17X (Lux M17X) are circles and squares, respectively. One representative experiment of three independent replicates. (B) Kinetics of specific Lux activity (solid lines) during growth (OD₆₀₀, dashed lines) of five randomly selected clones of strain BLD101(pGIFPT001) grown in M17G and M17X. The symbols for OD₆₀₀ in M17G (OD M17G) and M17X (OD M17X) are black and gray dashes, respectively. The symbols for Lux activity in M17G (Lux M17G) and M17X (Lux M17X) are gray and black solid lines, respectively. (C) Maximum specific luciferase (Lux) activity (RLU OD₆₀₀⁻¹) emitted by strains BLD101(pG⁺host9) (Ctl⁻, negative control, white bars), BLD101(pGIFPT001) (P_xyl, gray bars), and BLD101(pGIBLD001) (clone 2; P₃₂, black bars) grown in CDMG, CDMX, M17G, and M17X. Mean values (n = 3) ± the standard deviations are indicated.

FIG 6

Impact of the inactivation of members of the MecA-Clp machinery on P_comGA activation. The maximum specific luciferase (Lux) activity (RLU OD₆₀₀⁻¹) of the reporter strain BLD101 (Ctl) and its isogenic mutant strains (MecA⁻, ClpC⁻, ClpP⁻, ClpE⁻, and ComX⁻ [used as a negative control]), cured or not from plasmid pGIFPT001, were determined. Noncured (NC) strains grown in CMDG and CDMX are represented in white and black, respectively. Cured (C) strains obtained by temperature upshift and grown in CDMG are in gray. Mean values (n = 3) ± the standard deviations are indicated.