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 statement Specific 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.
Introduction
Horizontal gene transfer (HGT) fulfills an important role in the evolution of bacteria (1–4). In several species, an important mechanism for HGT is natural competence. This phenomenon is defined as a cellular state that enables internalization of exogenous DNA, followed by autonomous replication as a plasmid or incorporation into the chromosome via homologous recombination. Among Gram-positive bacteria, natural competence was first described in Streptococcus pneumoniae (5, 6). More recently, it was found that among lactic acid bacteria (LAB), the important yoghurt bacterium Streptococcus thermophilus can enter a state of natural competence upon culturing in chemically defined medium (7). When Gram-positive bacteria enter a state of natural competence, exogenous DNA translocates through the DNA-uptake machinery, a multiprotein complex comprising the proteins ComEA, ComEC, ComFA, ComFC and a nuclease (EndA in S. pneumoniae) encoded by the late competence (com) genes (8, 9). Other late competence genes encode for proteins that compose pili-like structures (ComGA-GG) or protect internalized DNA against degradation (SsbA, SsbB, DprA and RecA) (8, 9). Expression of these genes is positively regulated by the competence master-regulator ComX, that acts as an alternative sigma factor (10–12). In S. thermophilus, expression of comX is initiated upon formation of the quorum sensing ComRS complex comprising the pheromone-like peptide ComS and transcriptional regulator ComR, encoded by the comRS operon (13, 14). Addition of a synthetic peptide that resembles the active competence pheromone has proven a successful strategy to induce natural competence in several bacterial species. For example, addition of a synthetic ComS peptide to S. thermophilus cultures in the early logarithmic phase of growth enabled the activation of natural competence and highly efficient DNA transformation (13, 15). Analogously, other streptococci including S. pneumoniae utilize the comCDE regulatory module to control natural competence, involving the competence-stimulating peptide (CSP, encoded by comC) and a two-component system (encoded by comD and comE, (16, 17)), and the addition of synthetic CSP leads to development of natural competence in this species.
Strains of Lactococcus lactis are of great importance in the dairy industry, primarily in the production of cheese and butter(milk) (18). So far, a comRS- or comCDE-like system has not been identified in L. lactis. Nevertheless, complete sets of late competence genes appear to be present in several L. lactis genomes (19, 20, 21 and this study). In addition, increased expression of competence genes has been observed in L. lactis subsp. lactis IL1403 and KF147 under specific conditions that included carbon starvation (22, 23). Unfortunately, in neither of these strains, or any other L. lactis strain, natural competence development could be experimentally established (19, 23). As an alternative route to establish natural competence, overexpression of comX has been employed, aiming to enhance expression of the complete late competence regulon. Such an approach has been successful in S. thermophilus (24), but failed in L. lactis IL1403 (19). Nevertheless, the observation that complete sets of late competence genes are apparently present in some of the L. lactis genomes (25, 26) and that their expression can be induced under specific conditions (22, 23), deserves a more dedicated bioinformatic and experimental effort.
Here, we present a comparative genomics analysis of 43 L. lactis genomes to assess their potential to enter a state of natural competence. Moreover, by employment of controlled expression of ComX we demonstrate enhanced expression of the late competence regulon and concomitant induction of natural competence, which was only successful in strains predicted to encode a complete late competence machinery. The discovery of natural competence in L. lactis will enable transfer of genetic information in a non-GMO manner, resulting in the improvement of the industrial performance of strains of this species and the enhancement of fermented product quality.
Results
Genomic analyses show complete sets of competence genes in several L. lactis strains
To evaluate whether L. lactis strains possess the genetic capacity to enter a state of natural competence, late-competence associated genes were initially identified in the L. lactis KF147 genome by using the known competence genes of the naturally competent Streptococcus thermophilus LMD-9 (7, 13). This strain was selected for this primary analysis based on previous work that reported that many of the late competence genes were induced in this strain under starvation, non-growing conditions (23). Similar proteins (both in length and sequence) were identified to be encoded within the KF147 genome for all selected late competence genes of S. thermophilus LMD-9, corroborating that a complete competence gene-set is present in this L. lactis strain (Table S2).
Subsequently, the identified KF147 competence protein sequences were used as a reference set for identification and comparison to the orthologous groups (OGs) of genes encoded by 42 other L. lactis strains (20, 21, 25–34). Full length protein sequence identity relative to the KF147 OGs was calculated for all 42 L. lactis strains (Fig. 1). This analysis revealed considerable genomic decay in several of the strains of both the lactis and cremoris subspecies. Moreover, for the OGs that were intact, there was a clear distinction between the levels of identity observed for strains belonging to the subspecies lactis (that includes strain KF147) and cremoris (Fig. 1), exemplifying the genetic distinction between these two subspecies (35). Among the strains belonging to the cremoris subspecies, only strains KW2 and KW10 appeared to encode full length homologues of all the late competence proteins selected in KF147, albeit with identity scores ranging from 56 to 99% (Fig. 1). Notably, when the late competence gene-set of the KW2 strain was used to determine full-length protein sequence identity levels, instead of those of strain KF147, it was apparent that late competence proteins displayed a high degree of conservation within the cremoris subspecies, but were distinct from their orthologues in the lactis subspecies (Fig. S1). Among the 28 strains belonging to the subspecies lactis, 9 appeared to encode a full set of late competence proteins.
The genomic decay within these late competence genes in the subspecies cremoris strains displayed several conserved disruptive mutations in specific genes, including IS982 insertions in comEC (strains SK11, A76 and UC509.9) and comGA (strains SK11, and A76), although with some variation with respect to the precise position of insertion (Fig. 1 and Fig. S2). Various strains of the cremoris subspecies contained conserved premature stop codons within one or more of their late competence genes, suggesting that these strains derive from a common ancestor, in which conserved and strain specific mutations have shaped the decay pattern of the late competence genes. For example, strains SK110, AM2, SK11, A76, UC5099, B40, FG2, HP and LMG6897 share similar mutational events in comEA, comEC, comFA and comGD, whereas strains N41, NCDO763 and MG1363 harbor common mutations in comEC (Fig. S3). In contrast, the disruptive mutations observed in the late competence genes of strains of the subspecies lactis appeared more scattered (Fig. 1), suggesting that degenerative mutations accumulated more recently in this subspecies. Nevertheless, several strains (KF282, KF24, N42, CV56, ML8, KLDS, UC317, KF67) contain a (remnant of a) prophage insertion within the comGC gene (Fig. S4). Remarkably, these phage sequences are always inserted at the same position within the comGC sequence, suggesting site-specific integration at a conserved sequence element within the comGC gene.
In summary, these findings indicate that in the majority of L. lactis strains one or more late competence functions are compromised, suggesting that these strains are not able to develop a state of natural competence. The analysis also implies that in some strains, including L. lactis KF147, the genetic capacity to enter a state of naturally competence appears to be intact. Finally, it is noteworthy that within the present panel of strains, there are no dairy isolates that appear to encode a complete set of intact late competence proteins, which may reflect the high-level of genome decay that has been reported for strains in the milk environment before (36–38).
Moderate overexpression of the late competence regulon regulator ComX results in a state of natural competence in L. lactis KF147
In order to test whether the identified competence machinery can be activated and is functional, we set out to overexpress the predicted competence regulator ComX. From the subset of strains predicted to harbor a complete set of competence genes, L. lactis KF147 harbors a chromosomal copy of nisRK but does not produce nisin (39), allowing nisin-inducible comX expression by cloning of this gene under control of PnisA in pNZ8150 ((40). This comX expression strategy led to a dose-dependent inhibition of growth (Fig. 2A), which was not observed in the control strains harboring pNZ8150 (Fig. S5; (40)), or pNZ8040, a vector enabling nisin-inducible expression of pepN (Fig. S5; (41)). Hence, the observed growth retardation is not caused by the addition of nisin or the overexpression of proteins as such but is specifically caused by the presence of ComX.
To investigate the impact of elevated ComX levels on the expression level of the late competence genes, their transcript levels were determined by RT-qPCR on RNA derived from L. lactis KF147 harboring pNZ8150 or pNZ6200, either uninduced, or moderately or fully induced with nisin. In uninduced conditions, comX expression levels were 2.5- to 6-fold increased in L. lactis KF147 harboring pNZ6200 as compared to the pNZ8150 harboring cells, which is likely reflecting low-level ‘promoter leakage’ due to the presence of PnisA on a high-copy plasmid (Fig. 2B). Induction of comX expression in L. lactis KF147 harboring pNZ6200 with either 0.03 or 2 ng/ml nisin for 2 hours led to 15-20 and 1500-4000-fold induction of comX expression relative to the uninduced control of the same strain, respectively (Fig. 2B). Similarly, expression of the late competence genes comEA, comFA, and comGA was induced, illustrating the strongly enhanced expression of the late competence regulon as a consequence of the elevated levels of its regulator ComX (Fig. 2B). These induction conditions for the activation of late competence genes were employed to test whether the corresponding phenotype could also be observed, by adding pIL253 to the culture medium at the same timepoint that comX induction was initiated using a range of nisin concentrations. As expected, no pIL253 transformants were obtained for L. lactis KF147 harboring pNZ8150 under any of the conditions tested (data not shown). By contrast, pIL253 transformants were obtained for L. lactis KF147 harboring pNZ6200 following induction with nisin concentrations ranging from 0.005 to 0.1 ng/ml nisin, with an approximate transformation rate of 10−7−10−6 (transformants / total cell number μg plasmid DNA). The highest transformation rates were obtained after 0.03 ng/ml nisin induction (Fig. 2C). Both strain identity and pIL253 presence was confirmed by PCR in all transformants tested (Fig. 2D). Notably, full nisin-induction (2.0 ng/ml nisin) of comX expression in pNZ6200 harboring L.lactis KF147 did not result in any transformants. To check whether comX of the cremoris strain MG1363 is still functional, transformation of L. lactis KF147 harboring pNZ6201 upon nisin induction was also tested. Similar results were obtained when comX of L. lactis subsp. cremoris MG1363 was expressed in L. lactis subsp. lactis KF147 (Fig. 2E, F), indicating that comX derived from a cremoris strain is also fully functional. Taken together, these results demonstrate that activation of moderate expression, but not high-level expression, of endogenous comX in L. lactis KF147 elicits the natural competence phenotype in this strain. The observation that this does not occur at a high level of comX expression may be a consequence of the observed growth defect under these conditions, which may interfere with completion of the competence machinery assembly and/or recovery of potential transformants after plating. Such notion, is supported by the observation that expression of a heterologous copy of comX (derived from L. lactis subspecies cremoris) induced less severe growth defects upon maximal nisin induction, and still led to detectable natural competence development, albeit with reduced efficiency as compared to moderate nisin induction levels.
ComX-induced transformation in L. lactis depends on the late competence operon comEA-EC
The experiments above do not provide direct proof for a functional dependency of the observed transformation phenotype on the expression of the late competence genes, although this is likely, considering the fact that these genes encode the DNA-uptake machinery. Therefore, we constructed a comEA-EC negative derivative of L. lactis KF147 through the integration of a linear fragment harboring a tetracyclin-resistance encoding tetR flanked by regions homologous to the 5’- and 3’- regions surrounding the comEA-EC operon. The procedure for moderate comX induction was applied to transform this linear mutagenesis fragment to strain KF147 harboring pNZ6200. Integrants with the anticipated genotype (ΔcomEA-EC::tetR; Fig. 3A) were obtained with a similar efficiency as was observed for pIL253 transformation. Subsequent comX expression induction experiments in the ΔcomEA-EC::tetR derivative of strain KF147 (NZ6200) harboring pNZ6200. Full induction of comX expression led to a growth rate reduction in this strain (Fig. 3B) similar to what was observed for the parental KF147 strain. Importantly, nisin concentration-dependent comX overexpression and corresponding upregulation of expression of comFA and comGA, but not comEA, genes was similar to what was established in L. lactis KF147 harboring pNZ6200 (Fig. S6). However, in contrast to the parental strain KF147, transformation of NZ6200 harboring pNZ6200 with pIL253 did not yield any transformants (Fig. 3C), establishing the involvement of the comEA-EC operon in comX-induced competence in L. lactis KF147.
Expansion of the natural competence phenotype to a broader set of L. lactis strains
In order to employ the comparative genomics analysis performed in this study as a predictor for competence potential, L. lactis subspecies cremoris strains KW2 (27), and NZ9000 (40, 42), and L. lactis subspecies lactis IL9000 (43) were tested for transformability upon comX induction. Strains NZ9000 and IL9000 are derivatives of MG1363 and IL1403, respectively, which contain the nisRK genes integrated in their chromosome, thereby allowing the use of the nisin controlled expression system. Strain KW2 does not harbor nisRK in its genome and to facilitate the use of the nisin induction system in this strain, pNZ9531 was first introduced into this strain, thereby expressing nisRK in this background from a low-copy plasmid vector that is compatible with the pNZ8150 backbone of the pNZ6200 and pNZ6201 vectors used for comX expression (44). The plasmids pNZ8150, pNZ6200 or pNZ6201 were transformed to electrocompetent cells of IL9000, NZ9000, and pNZ9531-harboring L. lactis KW2. These transformants were induced with 0, 0.03 or 2 ng/ml nisin, and the induction of the competence phenotype was evaluated in these strains by transformation with pIL253 (for NZ9000 and IL9000) or pNZ6202 (a tetracycline-selectable pIL253 derivative). As anticipated, none of the conditions employed allowed the activation of natural competence in strain NZ9000 (Table 1), which is in good agreement with the comEC and coiA mutations observed in its parental strain MG1363 (Fig. 1). In contrast, transformants were obtained for pNZ6200 and pNZ6201 harboring derivatives of IL9000, and when these plasmids were transformed to KW2 harboring pNZ9531 (Table 1). Notably, although the efficiency of transformation appeared to be the highest for the cells in which moderate comX expression was induced (i.e., 0.03 ng/ml), also under uninduced and upon high level induction of comX expression (i.e., 2 ng/ml) transformants were obtained. The observed transformation under uninduced conditions might be caused by the previously reported higher levels of ‘leakage’ of the nisA promoter activity in L. lactis strains harboring the nisRK expression vector pNZ9531 (44), whereas differential expression of nisRK and/or the dprA mutation in the IL9000 parental strain (IL1403; Fig. 1) may require lower levels of comX expression to activate competence, as the DprA function has been associated with competence shut-off (45, 46). The observation that high-level comX expression still allowed competence development in KW2 and IL9000 despite the growth-inhibitory consequences of this level of induction, which is in contrast with the results obtained for strain KF147, remains to be determined. Finally, in pNZ9531-harboring KW2, induced expression of the comX derived from L. lactis subspecies cremoris (i.e., as expressed from pNZ6201) allowed competence development, confirming the bidirectional functional exchangeability of the comX genes from these two L. lactis subspecies. These results confirm the predictions made by comparative genomics (Fig. 1) with respect to the capacity to develop a natural competence phenotype in L. lactis strains, and establish that strains of both subspecies have the capacity of natural competence which can be induced by controlled expression of the comX encoded regulator from either of the subspecies.
Discussion
This study demonstrates that the L. lactis strains KF147, KW2 and IL1403 possess a functional DNA-uptake machinery, which can be activated by the ComX regulator. This implies that identification of a complete set of late competence genes through comparative genomics represents an appropriate approach to predict the capacity of a strain to enter a state of natural competence, and it seems likely that most, if not all, of the other strains identified here to encode complete gene sets can be made naturally competent via the same strategy of comX overexpression. It should be noted that the expression of a much larger set of over 100 genes is regulated upon addition of the competence pheromone in streptococci (11, 47, 48). For instance, development of natural competence usually occurs in concert with increased expression of proteins involved in DNA recombination, thereby facilitating integration of acquired DNA (49), a feature that has been observed in this study for L. lactis KF147 as well suggesting expression of such proteins in L. lactis KF147 upon competence development. Nevertheless, we show that the dedicated assessment of only the canonical late competence genes is a valid predictor for competence potential in L. lactis strains.
It is commonly assumed that the L. lactis ancestor strain prior to subspeciation into the lactis and cremoris subspecies originates from a plant-associated niche and that strains adapted to increase their fitness in the nutritionally rich dairy environment (39, 50, 51). Remarkably, none of the dairy isolates of L. lactis that were analyzed here appears to encode a complete set of late-competence genes, suggesting that during the adaptation to the dairy niche there is no significant environmental fitness benefit associated with the capacity to become naturally competent. This may relate to a real lack of fitness benefit of this phenotype within the dairy environment or may be due to highly consistent suppression of the phenotype during growth in milk, thereby preventing the possible fitness benefit to become apparent, which may allow the decay of encoding genes without apparent fitness cost for the bacteria. The latter scenario appears to be in agreement with the observed activation of the expression of late competence genes in L. lactis upon carbon starvation conditions (22, 23), that are not likely to occur within the dairy niche, as it is very rich in lactose. The genomic decay events associated with dairy-derived L. lactis strains include prophage disruption of the comGC locus in strains of the subspecies lactis (27, 52), and insertion of IS982 into several com genes in strains of the cremoris subspecies (39, 53–55). Notably, the phylogenetic relatedness of L. lactis subspecies cremoris strains predicted on basis of competence gene-decay events, displayed a topology that was remarkably similar to that observed for the core-genome relatedness of these strains (Fig. S3). Importantly, typical dairy environment associated lactic acid bacteria quite commonly display genomic decay as a consequence of the adaptation to this nutritionally rich environment (36–38). For example, loss of function events have been observed in S. thermophilus, Lactobacillus helveticus and Lactobacillus bulgaricus upon prolonged culturing in milk, with mutations accumulating in genes encoding transport, energy metabolism and virulence associated functions, implying that these functions do not contribute to fitness in the dairy niche (36, 38, 56, 57). Analogously, experimental evolution of L. lactis KF147 to enhanced fitness and growth in milk was shown to be associated with suppression of gene repertoires associated with the import and utilization of a variety of typically plant-environment associated carbon sources, as well as mutations leading to functional reconstitution and elevated transcription of the peptide import system (opp) of this strain [52]. Paradoxically, dairy strains of S. thermophilus still possess the genetic and phenotypic capacity to develop natural competence (13, 24, 38, 58), suggesting that competence development in this species contributes to fitness in this habitat. Contrary to L. lactis, where carbon starvation has been associated with induction of late competence expression (22, 23), similar conditions have not been implied in competence regulation in S. thermophilus. This may suggest that S. thermophilus actively expresses the competence phenotype in the dairy environment, which may contribute to this species’ fitness in the milk environment.
In nature, natural competence in bacteria is commonly a transient phenotypic state with a small window of opportunity to take up DNA (59), of which activation and shut-down are subject to subtle regulation (45, 46) to prevent futile activation of the costly process and to sustain genomic stability. Analogously, optimal induction in L. lactis was achieved with a moderate level of ComX induction, whereas high-level induction of this regulator failed to lead to competence development (strain KF147), or led to significantly reduced levels of transformation (strains KW2, and IL9000). Moreover, high level comX expression was consistently associated with reduced growth efficiency of the strains used in this study, which is illustrative of the tight connection between competence and growth (60). Previous comX expression studies using L. lactis IL1403, the parental strain of IL9000, failed to elicit natural competence (19), which may have been due too inappropriate expression levels of comX, or might have been caused by the fact that the endogenous comX gene of IL1403 was used which contains an alternative start codon and appears to be truncated.
Taken together, this study shows that in L. lactis strains that encode complete late-competence gene-sets, a state of competence can be induced by controlled expression of comX, in particular moderate expression of this regulator appears to be effective in activation of this phenotype. Naturally competent L. lactis strains could internalize plasmid and linear DNA from their environment with a similar efficiency. The conditions that naturally activate comX expression and contribute to the regulation of competence development in L. lactis remain to be established. Unraveling the in situ control mechanisms of natural competence in L. lactis would offer opportunities to exploit this phenotype for strain improvement purposes in this industrially important species.
Materials & Methods
Bacterial strains, plasmids, and media
The strains used in this study can be found in Table S1. The publicly available draft genome sequences of 43 L. lactis strains (20, 21, 25–34) were used for comparative genomics of late competence genes, by employing OrthoMCL to obtain orthologous group (OG) sequences in order to construct an orthologous gene matrix (61, Wels et al., manuscript in preparation). L. lactis strains were routinely cultivated in M17 (Tritium, Eindhoven, the Netherlands) supplemented with 1% (w/v) glucose (Tritium, Eindhoven, the Netherlands), at 30 °C without agitation. For competence experiments, L. lactis strains were cultivated in chemically defined medium (CDM) (62, 63) supplemented with 1% (w/v) glucose (Tritium, Eindhoven, the Netherlands). Upon recovery after electro or natural transformation, L. lactis cells were cultivated in recovery medium (M17, supplemented with 1% glucose, 200mM MgCl2 and 20mM CaCl2). Escherichia coli TOP10 (Invitrogen, Breda, The Netherlands) was routinely cultivated in TY (Tritium, Eindhoven, the Netherlands) at 30 °C with agitation. Antibiotics were added when appropriate: 5.0 μg/ml chloramphenicol, 10 μg/ml erythromycin, 12.5 μg/ml tetracyclin.
DNA manipulations
Plasmid DNA from E. coli and L. lactis was isolated using a Jetstar 2.0 maxiprep kit (ITK Diagnostics bv, Uithoorn, The Netherlands). Notably, phenol chloroform extraction was performed prior to loading on the Jetstar column for plasmid isolation from L. lactis cultures (64). Primers were synthesized by Sigma-Aldrich (Zwijndrecht, The Netherlands). PCR was performed using KOD polymerase according to the manufacturer’s instructions (Merck Millipore, Amsterdam, The Netherlands). PCR products and DNA fragments in agarose gel were purified using the WizardR SV gel and PCR Clean-Up System (Promega, Leiden, The Netherlands). PCR-grade chromosomal DNA was isolated by using InstaGene™ Matrix (Bio-rad, Veenendaal, The Netherlands). Ligations were performed using T4 ligase and when applicable either transformed to electrocompetent E. coli TOP10 (Invitrogen, Breda, The Netherlands) or L. lactis NZ9000, IL9000, KW2 or KF147 (65).
Plasmid and mutant construction
To enable controlled expression of comX in L. lactis, the comX gene was amplified by PCR using the primer pairs C1-C2 or C3-C4, and L. lactis KF147 or MG1363 chromosomal DNA as a template, respectively. The resulting 502 bp comX amplicons were digested with KpnI (introduced in primers C2 and C4) and ligated into KpnI-ScaI digested pNZ8150 (40), yielding pNZ6200 and pNZ6201, respectively. These comX overexpression vectors were transformed into electrocompetent L. lactis KF147, NZ9000, IL9000 and KW2 (65). The natural competence potential in L. lactis strains was evaluated using pIL253 when possible but because of incompatibility of antibiotic resistance markers in strain KW2 an alternative plasmid was constructed in which the erythromycin resistance (eryR) gene was replaced by tetracyclin resistance (tetR) gene. To this end, a 1644 bp tetR amplicon was generated using primers C15 and C16 with pGhost8 (66) as a template, and cloned as a PstI-SacI fragment into similarly digested pIL253, yielding pNZ6202.
A comEA-EC deletion derivative of L. lactis KF147 mutant was constructed using double cross over recombination. To construct the mutagenesis fragment, the 5’- and 3’- flanking regions of the comEA-EC operon were amplified using chromosomal DNA of strain KF147 as a template and primer pairs C7-C8, and C11-C12, respectively. The tetracylin resistance encoding gene tetR was amplified from pNZ7103 (67) using primers C9 and C10. SOEing PCR (68) was employed to join the three amplicons using the compatible sequence overhangs introduced by the primers in the individual PCR reactions (Table S1), and primers C7 and C12 for amplification in this PCR. The 6 kb SOEing amplicon was purified from 1% agarose gels and transformed to naturally competent L. lactis KF147 (see results section). The anticipated comEA-EC deletion in the resulting derivatives of L. lactis KF147 yielding L. lactis NZ6200 was confirmed by PCR using C13 and C14 primers.
Induction of competence in L. lactis
Cells harboring pNZ6200, pNZ6201 or pNZ8150 were grown overnight in GCDM with appropriate antibiotics, followed by subculturing (1:65) in the same medium to an OD600 of 0.3 at which Ultrapure Nisin A (Handary, Brussels, Belgium) was added to the media at a final concentration of 0.005, 0.01, 0.03, 0.05, 0.07, 0.1, 0.5 or 2ng/ml. In parallel, 1 µg of plasmid DNA was added. Samples were incubated for 2h at 30°C, after which 5ml recovery medium was added and incubation was continued for another 2 h. Bacteria were pelleted by centrifugation at 4000xg for 8 minutes and transformants were enumerated by plating of serial dilutions on GM17 plates. KF147 Transformants were subjected to PCR analysis to assess presence of the transformed plasmid with primers PS1 and PS2, whereas the strain-specific primers SS1+2 were used to confirm strain identity.
Analysis of competence gene expression
RNA was isolated from L. lactis cultures using the High Pure RNA isolation kit (Roche Diagnostics Nederland B.V., Almere, The Netherlands), including an on-column DNase treatment. Eluted RNA was again DNase (1 U; Thermo fisher scientific, Waltham, USA) treated for 45 min at room temperature to remove remaining DNA, followed by DNase inactivation by the addition of EDTA to a final concentration of 25mM, followed by heating at 75 °C for 15min. cDNA was prepared using 10 ng total RNA and random hexamer primers in the reverse transcriptase reaction (Applied Biosystems, Foster city, USA) according to the manufacturer’s protocol. Control RNA samples that were not reverse transcribed were included as negative control to ensure the absence of DNA contamination. Transcripts of competence genes were quantified using 2 μl cDNA and locus-specific primers for each competence associated target gene (Primers Q1-Q10, Table S1) in SYBR green-quantified PCR (Bio-rad, Veenendaal, The Netherlands). Transcript copy-numbers were calculated using a template standard curve and normalized to the housekeeping-control transcript of rpoA. These RT-qPCR analyses were performed in triplicate for each sample using the Freedom EVO 100 robot system (Tecan, Männedorf, Switzerland), and amplicon identities were verified using melting curve analysis. The non-parametric Mann-Whitney U-test (one-tailed) was used to determine whether gene expression levels were significantly different between uninduced and induced conditions (P-value<0.05).
Acknowledgements
We gratefully acknowledge Prof. dr. Jan Kok for providing L lactis strain IL9000. We would like to thank Sabri Cebeci and Koen Giesbers of NIZO for technical assistance. This work was carried out within the BE-Basic R&D Program, which was granted a FES subsidy from the Dutch Ministry of Economic affairs.