Conservation of key elements of natural competence in Lactococcus lactis ssp

Conservationof keyelementsof natural competence inLactococcus lactis ssp.Sandra Wydau1, Rozenn Dervyn1, Jamila Anba2, S. Dusko Ehrlich1 & Emmanuelle Maguin1

1Genetique Microbienne and 2Unite d’Ecologie et de Physiologie du Tube Digestif, INRA, Domaine de Vilvert, Jouy-en-Josas Cedex, France

Correspondence: Emmanuelle Maguin,

Unite de Genetique Microbienne, INRA,

Domaine de Vilvert, 78352 Jouy-en-Josas

Cedex, France. Tel.: 133 1 34 65 25 18;

fax: 133 1 34 65 25 21; e-mail:

[email protected]

Received 23 September 2005; revised 22

December 2005; accepted 2 January 2006.

First published online 16 February 2006.

doi:10.1111/j.1574-6968.2006.00141.x

Editor: Andre Klier

Keywords

sigma factor; regulation; late competence

genes; genetic diversity; comX, dprA.

Abstract

Natural competence is active in very diverse species of the bacterial kingdom and

probably participates in horizontal gene transfer. Recently, the genome sequence of

various species, including Lactococcus lactis, revealed the presence of homologues

of competence genes in bacteria, which were not previously identified as naturally

transformable. We investigated the conservation among lactococcal strains of key

components of the natural competence process in streptococci: (i) comX which

encodes a sigma factor, allowing the expression of the late competence genes

involved in DNA uptake, (ii) its recognition site, the cin-box and (iii) dprA which

encodes a protein shown to determine the fate of incoming DNA. The comX

and dprA genes and the cin-box appeared conserved among strains, although

some L. lactis ssp. lactis strains presented an inactivated dprA gene. We

established that ComX controls the expression of the late competence genes in

L. lactis. In conclusion, our work strongly suggests that ComX has the same role in

streptococci and L. lactis, i.e. the regulation of late competence genes. It also

allowed the identification of a set of L. lactis strains and the construction of a comX

overexpression system, which should facilitate the investigation of the natural

competence activity in lactococci.

Introduction

Natural competence is a cellular state allowing the uptake of

exogenous DNA by bacteria and its integration in the

chromosome. This process is active in diverse species of the

bacterial kingdom and participates in intra- and probably

also inter-species gene transfer (Dreiseikelmann, 1994; Lor-

enz & Wackernagel, 1994; Cvitkovitch, 2001; Claverys &

Martin, 2003). Natural competence has been reported in

several streptococci, e.g. in the Streptococcus mitis and

Streptococcus mutans groups, and studied in detail in Strep-

tococcus pneumoniae (Havarstein et al., 1997; Cvitkovitch,

2001).

In streptococci, natural competence is controlled by a

quorum-sensing system, which involves the production, the

export-maturation and the detection by a two-component

system (ComD, ComE) of a competence stimulating peptide

(CSP) (Pestova et al., 1996). The phosphorylated response

regulator, ComE�P, stimulates the transcription of the

genes responsible for CSP synthesis, export and detection,

and also of the comX1 and comX2 genes that encode

two homologous sigma factors (Lee & Morrison, 1999).

Each of these sigma factors can initiate the next step of the

competence process by switching on the expression of the

late competence genes and stimulating the expression of

competence-associated genes (Lee & Morrison, 1999). In

order to stimulate gene expression, ComX interacts with the

RNA polymerase core enzyme and the resulting holoenzyme

recognizes a specific sequence, the cin-box located upstream

of late competence genes (Lee & Morrison, 1999; Opdyke

et al., 2001). In this system, ComX has a key role, linking the

regulatory module constituted by the early competence

genes to the DNA uptake module encoded by the late

competence genes. The ComX-regulated genes are directly

involved in the uptake of exogenous DNA (Claverys &

Havarstein, 2002), in its protection from intracellular nu-

cleases by the DprA protein (Berge et al., 2003), and in its

incorporation in the chromosome by homologous recombi-

nation (Mortier-Barriere et al., 1998).

Lactococcus lactis is a gram-positive lactic acid bacterium

(LAB), which is closely related to the streptococci (Stiles &

Holzapfel, 1997). The two major L. lactis subspecies, L. lactis

ssp. lactis and L. lactis ssp. cremoris, are industrially impor-

tant as they are used as starters for the production of

fermented dairy food (Stiles & Holzapfel, 1997). Lactococcus

lactis was never identified as a species possessing a natural

FEMS Microbiol Lett 257 (2006) 32–42c� 2006 Federation of European Microbiological SocietiesNo claim to original French government works

competence pathway and to the best of our knowledge,

natural transformation was never observed in L. lactis

strains. However, the analysis of the complete genome

sequence of IL1403, an L. lactis ssp. lactis strain, revealed

the presence of orthologs of several genes involved in the

process of natural competence in various bacteria (Bolotin

et al., 1999, 2001; Claverys & Martin, 2003). Based on

sequence homology, a comX gene and all the late compe-

tence genes required for the formation of the DNA entry

pore [comC (also referred to as CCL in S. pneumoniae),

comEA, comEC, comFA, comFC and comGA to comGD] were

identified (Bolotin et al., 1999, 2001). Comparison of the

regions located upstream of these late competence genes

allowed the identification of a conserved sequence which

may constitute a recognition site of the ComX-RNA poly-

merase holoenzyme (Bolotin et al., 1999, 2001). These

genetic features of IL1403 raise the question whether L. lactis

can activate a natural competence system which may con-

tribute to genetic transfer in dairy food ecosystems (Gasson,

2000; Jonas et al., 2001; Kharazmi et al., 2002; van den Eede

et al., 2004).

Two observations questioned the functionality of natural

competence in Lactococcus lactis. The first observation is that

orthologs of the early competence genes (the ABC transpor-

ter encoding comA and comB genes, the two-component

system encoding genes comD and comE, and the CSP

encoding gene) responsible for the regulation by the CSP,

were not clearly identified in IL1403 (Bolotin et al., 2001).

The strain does, however, possess multigenic families

encoding ABC transporters (at least 60 ABC transporters

in IL1403) and two-component systems (seven complete

systems and an additional response regulator) (Bolotin

et al., 2001). In IL1403, Redon et al. (2005) observed a

slight progressive overexpression of comX during glucose

starvation and proposed that specific starvation condi-

tions may lead to the induction of natural competence in

L. lactis.

The second observation is that the IL1403 dprA gene (also

referred to as smf in several microorganisms as Bacillus

subtilis) is inactivated by a frameshift resulting in a pre-

mature stop at nt 100 (protein of 33 amino acids). In several

bacteria (Karudapuram et al., 1995; Ando et al., 1999;

Smeets et al., 2000; Berka et al., 2002; Friedrich et al., 2002;

Ogura et al., 2002), a determining role for DprA in the fate

of the exogenous DNA taken up during competence was

demonstrated. In an S. pneumoniae strain mutated for dprA,

the incoming DNA is degraded and the nucleotides are

randomly incorporated in the chromosome, while in a wild-

type background the incoming DNA fragments are inte-

grated in the chromosome by homologous recombination

(Berge et al., 2003). Since DprA activity is crucial for the

outcome of the natural transformation process and its

contribution to horizontal gene transfer, we investigated

whether L. lactis dprA1 strains could be identified in a

collection of strains.

In order to evaluate if natural competence may allow the

development of a general genetic tool for the modification

of lactococcal strains, we investigated the conservation of

two other key elements of the natural competence system,

comX and the cin-box, in lactococci. Finally, as a prelude to a

study on the functionality of natural competence in L. lactis,

we overexpressed two alleles of the comX gene in IL1403 to

test whether they regulate the expression of late competence

genes.

Materials and methods

Bacterial strains and culture conditions

For this study, 31 lactococcal strains (Table 1) were chosen

in the INRA national collection (INRA collection, URLGA,

Jouy-en-Josas, France). These strains were classified in the

different lactococcal subspecies (ssp. lactis, ssp. lactis biovar.

diacetylactis and ssp. cremoris) according to their pheno-

types (growth at 40 1C or in the presence of 4% NaCl and

arginine or citrate metabolism, J. C. Ogier, pers. comm.).

The strain VI 7101 is a derivative of IL1403, which

contains the nisR and nisK genes integrated in the chromo-

somal histidine biosynthesis operon (S. Calero and P.

Renault, pers. comm.). Strain VI 7238 is a comX-deleted

mutant of VI 7101 which resulted from a double crossing-

over (DCO) using plasmid pVI6237 (carrying a 2 kb DNA

insert constituted of the upstream (1 kb) and downstream

(1 kb) regions of comX fused together). The DCO was

performed as previously described for L. lactis (Biswas

et al., 1993) and the mutated strain was identified among

the ery-sensitive clones by PCR with the S99 and S102

primers (Table 2). The chromosomal structure of VI 7238

was then confirmed by Southern hybridization of digested

chromosomal DNA (HindIII, XmnI and EcoRI) using a PCR

fragment generated with primers S124 and S185 (Table 2) as

a probe. The VI 7293, VI 7247 and VI 7298 strains resulted

from the transformation of VI 7238 with plasmids pVI6253

(pnisA), pVI6213 (pnisA:comXIL) and pVI6255 (pnisA::-

comXMG), respectively. The pnisA promoter was induced by

the addition of nisin (0.8 ng mL�1, Sigma, Lyon, France) in

exponential phase cultures (OD600 � 0.1) growing in SA

medium (Jensen & Hammer, 1993) at 30 1C. Otherwise,

strains were propagated at 30 1C in M17 (Terzaghi &

Sandine, 1975) supplemented with 0.5% glucose. Erythro-

mycin (ery) was added at 5mg mL�1.

DNA extraction and PCR amplification

Chromosomal DNA was extracted as previously described

(Biswas et al., 1993). PCR DNA amplifications were per-

formed with the ExTaq DNA polymerase (Takara, St Germain

FEMS Microbiol Lett 257 (2006) 32–42 c� 2006 Federation of European Microbiological SocietiesNo claim to original French government works

33Natural competence in L. lactis

en laye, France) and the appropriate primers (Table 2) as

recommended by the supplier. For the comX and dprA

genes, primers homologous to the intergenic regions flank-

ing the gene of interest in the IL1403 genome sequence were

designed. These primers allowed to amplify fragments from

all the strains of the lactis subspecies (as determined by the

16S analysis, Table 1) but not from the strains belonging to

the cremoris ssp. For the cremoris ssp. strains (as determined

with the 16S analysis), an internal fragment of the gene of

interest was first amplified and sequenced (comX, a�300 bp

fragment obtained with the primer pairs S141–S143 or

S142–S143 and dprA, a 703 bp fragment obtained with

S183–S184). Then, the upstream and downstream regions

were amplified using primers homologous to this internal

fragment (comX, S143 or S141 and dprA, S183 or S184) and

the flanking genes (comX, S125, S126 or S127 and dprA, S98

or S186). The promoter region of the comG operon was

amplified using primers homologous to the upstream (S149,

S152 or S150) and downstream (S150, S154 or S155)

sequences.

16S analysis

In order to characterize the 16S sequence of the 31 lacto-

coccal strains selected from the INRA collection, an internal

fragment (343 bp) of the 16S ribosomal RNA genes was

amplified using the Y1 and Y2 primers described by Ward

et al. (1998) and chromosomal DNA as templates. The PCR

products were then digested by CfoI and MboII to determine

to which subspecies the strain belonged (Ward et al., 1998).

DNA sequencing and analysis

PCR generated fragments were sequenced using the Big Dye

Terminator v3.1 (Applied Biosystems, Foster City, CA).

Reaction products were analysed using a capillary sequencer

(3700 DNA Analyser, ABI PRISM). Sequences were analysed

using the GCG package (University of Wisconsin) and were

aligned using CLUSTALW (Infobiogen, http://www.infobio-

gen.fr/) (Thompson et al., 1994), the percentage of diver-

gence between the alleles was calculated by MEGA 2.1

(Kumar et al., 2001) and the data of the phylogenetic

analysis were represented using TREEVIEW (Page, 1996).

All sequences were deposited in the EMBL database under

the following accession numbers: comX genes AJ890847,

AJ890848, AJ890849, AJ890850, AJ890851, AJ890852,

AJ890853, AJ890854, AJ890855, AJ890856, AJ890857,

AJ890858, AJ890859, AJ890860, AJ890861, AJ890862,

AJ890863, AJ890864, AJ890865, AJ890866, AJ890867,

AJ890868, AJ890869, AJ890873, AJ890874, AJ890875,

AJ890876, AJ890878, AJ890879, AJ890881, dprA genes

AJ890918, AJ890919, AJ890920, AJ890921, AJ890922,

AJ890923, AJ890924, AJ890925, AJ890926, AJ890927,

AJ890928, AJ890929, AJ890930, AJ890932, AJ890933,

AJ890934, AJ890935, AJ890936, AJ890937, AJ890938,

AJ890944, AJ890945, AJ890946, AJ890947, AJ890948,

AJ890949, AJ890950, AJ890951, AJ890952, AJ890953.

Construction of plasmids

pVI6237 is a derivative of the thermosensitive pG1host9

plasmid (Maguin et al., 1996) which carries a �2 kb

SpeI–XhoI insert (a fusion of two 1 kb fragments

Table 1. Strains of the INRA collection used in this study

Strains

Subspecies according to the

Phenotype 16S analysis�

RAPD Group 1

IL1403 lactis lactis

IL581w lactis cremoris

IL584 lactis lactis

IL1321 lactis lactis

A13 lactis lactis

NCDO2146 lactis lactis

IL1306 lactis bv. diacetylactis lactis

DRC1 lactis bv. diacetylactis lactis

A15w cremoris lactis

A11 lactis lactis

A17 lactis lactis

A26 lactis lactis


A7 lactis lactis

A8 lactis bv. diacetylactis lactis

A27 lactis bv. diacetylactis lactis

RAPD Group 2

CNRZ359 cremoris cremoris

C7 cremoris cremoris

AM1w cremoris lactis

AM2w cremoris lactis

RAPD Group 3

CO2 cremoris cremoris

CO4 cremoris cremoris

NCDO276w lactis bv. diacetylactis cremoris

SK1w lactis cremoris

MG1363 cremoris cremoris

A140w lactis cremoris



RAPD individual patterns




Groups 1, 2 and 3 correspond to the RAPD patterns the most frequently

identified in the CNRZ collection of lactococci (Tailliez et al., 1998).

Strains exhibiting specific RAPD patterns are referred to as RAPD

individual patterns.�The 16S analysis did not discriminate between Lactococcus lactis ssp.

lactis and L. lactis ssp. lactis biovar diacetylactis strains.wStrains indicated with were misclassified on the basis of their phenotypes.

Bv., biovariant.


34 S. Wydau et al.

Table 2. Oligonucleotides used in this study

Primer Localization Orientation Sequence 50 to 30

comX sequence

S57 Downstream of comX Reverse TCTTCTCTTATCAAAAAACTCCC

S125 16S Reverse AGAGCCGCTTTCGCCAC

S126 ezrA Forward TTAGCTGAACAATTGATTCAATATG

S127 16S Reverse CCTGAGCCAGGATCAAACTCTC

S141 comX Forward TAGTTATGAAATTAATGAAACAAATTCG

S142 comX Forward CATTAGAACATGGAAAATAGAGGATTAT

S143 comX Reverse GTAGTAAGTCTTGATAATGTGCGCTC

dprA sequence

S58 Upstream of dprA Forward AAATTGCTGACAAAGCTGTCAG

S59 Downstream of dprA Reverse TAGCGAAGTGGTTTTTCTCCG

S97 dprA Forward ATCTTGCTAAAAACCAACTCATAC

S98 topA Reverse TTGTTTTACTTTTAGTTGAAGTTGG

S183 dprA Forward TGGCTCAAGTCAAATCAATCCC

S184 dprA Reverse TAAAATATCTTGAGCTTGATAGAC

S186 aldR Forward GTCCTTATTGAAATAGAAGTCATTG

cin-box sequence

S149 polC Forward GAATGCCTGATGATAATCAATTAG

S150 comGA Reverse TTTCCTCTATTTCATACCAACAAG

S152 polC Forward TAGAGCGTGGTTTCACTTTTGG

S153 polC Reverse CTTAATTGATTATCATCAGGCATTC

S154 comGA Reverse CGTTGACTTGTAACTGAATAAAATC

S155 comGA Forward TTGTTGGTATGAAATAGAGGAAAG

Gene expression

S50 comX Forward CATGGAAAATAGAGGATTATCTTC

S51 comX Reverse GCGATACCGTTGCATGCGAG

S192 comX Forward GGTGGTCATATGAAACCAATCATCAGAAAAT

S193 comX Reverse GGTGGTCCGCGGTTAATCATCATCTCGAGAAAAT

S19 comGA Reverse GGGTTCTTCTATATTGATAACTTG

S21 comGA Forward TTAGAAGTTGGCTTAGCACTAC

S22 comGB Forward TTTGGTAGAAGTGCATGGTAAC

S23 comGB Reverse TGAGCAATTAGATTTCCCCATTC

S24 comGC Forward GCCGTCAGAAAGAGCTAAAAG

S25 comGC Reverse TTTTTGAGTAATCATCCCTGCAC

S26 comGD Forward CATTTACTTTACTAGAGTCTCTTC

S27 comGD Reverse TCTTTAACTGCTACCTCCTTTGG

S28 comEA Forward GTGCGGTAACAAAGCCTAATG

S29 comEA Reverse CCGAAAATCGATGATGTCTTGAG

S30 comEC Forward GAATGGCTAATTCATGAGGTTG

S31 comEC Reverse AGCTGGTCGATTTGACTTACTC

S32 comFA Forward TGTTGGTCTAGCTAGTCCAAG

S33 comFA Reverse TGAAAACGACGCGGGAGAAAC

S34 comFC Forward CGTGGTTTCAATCAAGTGACTG

S35 comFC Reverse ATGCGTGATATAAGGTGGTACC

S36 comC Forward TGGCTCACATTGCCGTTATTG

S37 comC Reverse TTGAATTTTTCAGCTAAGACGGC

S42 dprA Forward GAGAGAAGAGTATAAAATATACCC

S43 dprA Reverse TAATGGCAGAGAGATGACTTGC

S44 coiA Forward TCTCAAAGCTTGCGACTTATGG

S20 coiA Reverse AGGGATTTTAATCTCATCTTCTGAGC

S38 radA Forward TTGGTCGAAGTGACTAATCCC

S39 radA Reverse CAACCGCAACCGCGAGGTC

S266 radC Forward TTCTTCAACATTTTGAAACTTTGG

S267 radC Reverse CAGTTGCCTGATTAACCGCC

S72 recA Forward ATGCAAAAGCGCTCGGTGTC

S73 recA Reverse TCTTGTTATCTCCAGAACCTTC

S40 recQ Forward CAGGACGGGACGGGCTTG



corresponding to the upstream and downstream regions of

comX and amplified with primers S99–S135 and S136–S102,

respectively). pVI6213 is a derivative of the pJIM2246 vector

(Renault et al., 1996) which contains between its XhoI and

SacI sites a DNA insert containing 3 successive transcription

terminators (T3) followed by a fusion between the inducible

promoter pnisA and the comX gene of IL1403 (comXIL). This

XhoI-SacI insert was generated as described below. (i) The

pnisA::comXIL fusion was amplified by PCR using the S106

and S107 primers and as template the chromosomal DNA of

VI 7145, which contains the pnisA::comXIL fusion. This PCR

product was cloned in the pGEM-T vector (Promega,

Charbonnieres, France) leading to pVI6205. (ii) The

EcoRV-SacI fragment of pVI6205 (containing the fusion)

was cloned in pVI6207 (pBluescriptSKII containing T3)

leading to pVI6212 which contains the T3::pnisA::comXIL

fusion on an XhoI–SacI fragment. In order to generate

pVI6253 (pJIM2246::T3::pnisA), the comXIL gene was deleted

from pVI6213 by SacI and NcoI digestions followed by a

Klenow-T4 DNA polymerase treatment and self-ligation.

Finally, plasmid pVI6255 (pJIM2246::T3::pnisA::comXMG)

was obtained after SacI and NcoI digestions of pVI6253 and

ligation with a SacI–NcoI PCR fragment containing comXMG

(amplified from the MG1363 chromosomal DNA with

primers S260 and S261).

Extraction of total RNAs

A volume of 100 mL of cultures at OD600 � 0.1 were

centrifuged (5 min, 9800 g at 4 1C) and the cell pellet was

washed with cold TE. The pellet was resuspended in 500mL

of cold water and transferred in a 2 mL screw-cap micro-

centrifuge tube containing 0.6 g of glass beads (Sigma),

200 mL of Macaloid (2%) (Sambrook et al., 1989), 500 mL of

phenol–chloroform pH 4.7 and 25mL of SDS (20%). Cells

were disrupted by shaking in a Fastprep machine (BIO101)

for 40 s at speed 5.5. After centrifugation at 12 000 g for

15 min (4 1C), the aqueous supernatant, which contains the

RNA, was treated with phenol-chloroform pH 4.7, precipi-

tated with ethanol and resuspended in 30 mL of water.

Dnase treatment and RT-PCR

In all, 5mg of total RNAs diluted in water (15mL final) were

incubated 3 min at 95 1C and 10 min on ice. 3mL of Dnase

(2 unitsmL�1, Ambion, Huntington, UK) and 2mL of buffer

were added, and the mixture was incubated 1.5 h at 37 1C. To

synthesize cDNAs 1mg of total RNA was incubated 10 min at

25 1C and 5 min on ice with 1mL of dNTPs (10 mM,

Promega) and 1mL of random primer (500 ngmL�1, NEB,

Beverly, MA) in a final volume of 13mL. A volume of 1mL

of reverse transcriptase (M-MLV Reverse transcriptase kit,

Invitrogen Cergy-Pontoise, France), 4mL of 5� buffer, 2mL

of DTT (0.1 mM) were then added and the mixture was

incubated 1 h at 37 1C and 15 min at 75 1C. The PCR

amplification (final volume 50mL) was performed from

cDNA (1mL) using appropriate primers (Table 2), Extaq

polymerase (Takara), dNTPs and the reaction buffer as

recommended by the supplier (Takara). The amplification

was carried out as follows: 5 min at 94 1C, (30 s at 94 1C, 30 s

Primer Localization Orientation Sequence 50 to 30

S41 recQ Reverse TCGGACGACACAAGATAGGAC

S264 ssbA Forward GTCCGTGTGACCTTGGCAG

S265 ssbA Reverse CTCGTAAAGCGATTGTTGCCC

S268 yqfG Forward TGCCTCCTATGCTGGCTTAG

S269 yqfG Reverse TTGGAAATTCTATACTACTTATGG

S272 ywgA Forward TTAAAACGACTTTACCGTGTGG

S273 ywgA Reverse GTTTTTTGTAAGAATATTTATGACCTC

S52 hu Forward CTAACAAACAAGATCTTATCGCTG

S53 hu Reverse GAACAACTGTAGCAGCGATTTTG

Plasmid construction

S99 Upstream of comX Forward GATATACTAGTGTTAGAGGTTATCAAGAAC

S102 Downstream of comX Reverse CTCAACTCGAGGTCTACCAGTTTCCAATG

S106 Upstream of pnisa Forward CATCAGATATCAACCAATCACGTCCGAG

S107 Downstream of comX Reverse TCACTGAGCTCAGTGATTGGGAATTCCTC

S124 Upstream of comX Forward ACGTTCATCAGTTCTCAAAG

S135 Upstream of comX Reverse AAACTCCCTAGAATTCCTATAGGTTTCATTTCTGAAAAAAG

S136 Downstream of comX Forward AAACCTATAGGAATTCTAGGGAGTTTTTTGATAAGAGAAG

S185 Upstream of comX Reverse GCTGCACCCGGTTCTAC

S260 comXMG Forward GGTGGTCCATGGAAAAGAGGAATATCAATGACAT

S261 comXMG Reverse ACCACCGAGCTCAGGGAGCTTTTTTTAATCATC

All the oligonucleotides were designed on the sequence of IL1403, except S183 and S184 which were designed on the sequence of MG1363.

Table 2. Continued


36 S. Wydau et al.

at 57 1C, 1 min at 72 1C) 30 cycles. Note that the expression of

ydbC was not monitored since the 2 pairs of oligonucleotides

that we tested did not allow PCR amplification from a DNA

template.

Results

Characterization of the lactococcal strains

We selected 31 lactococcal strains including the two best

characterized Lactococcus lactis strains, IL1403 (ssp. lactis)

and MG1363 (ssp. cremoris) in the INRA national collection.

These strains belong to the three major RAPD groups

defined for the L. lactis strains (Tailliez et al., 1998) and

according to their phenotypes, they belong to the two major

L. lactis subspecies used in dairy industry (ssp. lactis and ssp.

cremoris, Table 1).

In order to better characterize the chosen strains, their

16S sequences were analysed in a region which allowed to

distinguish between the lactis and cremoris ssp. (Ward et al.,

1998). Our data revealed that nine strains out of 31 (more

than 29%) were misclassified on the basis of their pheno-

types (Table 1). According to the 16S data, our set of strains

comprises 20 strains of the lactis ssp. and 11 of the cremoris

ssp. It is noteworthy that according to this new strain

classification, our sample of the RAPD group 3 contained

exclusively strains of cremoris spp.

Sequence analysis of the dprA gene

Sequence analysis revealed that in IL1403, the dprA gene

contained a premature stop codon at position 34; the

resulting truncated protein of 33 residues (instead of 282)

is likely to be inactive. As DprA is expected to play a crucial

role in natural transformation, its sequence was determined

in all of the other 30 strains.

In all the strains belonging to the lactis ssp. according

to the 16S analysis, two fragments (632 and 592 bp in

IL1403), which together contained the dprA gene were

amplified. For the remaining 11 strains of the cremoris ssp.,

unsuccessful amplification assays prompted us to change

our strategy. An internal fragment (703 bp) of dprA was

amplified and sequenced. The upstream and downstream

regions were then amplified using oligonucleotides located

in the internal sequenced region and in the dprA neighbour-

ing genes.

The maximal dprA divergence was 21.7% and the sequence

analyses revealed two allelic types, one detected in all the

L. lactis ssp. lactis strains and the other in the ssp. cremoris

strains; they are referred to as the lactis- and the cremoris-

allelic types, respectively (Fig. 1a). The lactis- and cremoris-

type exhibited an intratype variation of 3.1% and 3.7%,

respectively. In the lactis-type, 10 genes (50%) presented a

premature stop codon that would result in truncated proteins

of 23 aa (A8), 33 aa (IL584, IL1403, DRC1), 167 aa (A7) or

193 aa (IL1306, A17, A15, AM2, IL1321). In all these cases,

the resulting protein is expected to be inactive. For the

cremoris-allelic type, all the predicted proteins had the full

size of 282 aa.

Complete dprA genes were identified in �68% of the

tested strains, and in both the lactis and cremoris ssp. Since

some strains contain an intact dprA gene, we were interested

to see if the upstream regulatory elements were conserved;

we therefore investigated the conservation of two other key

elements for natural competence, comX and the cin-box,

which regulate the expression of the late competence genes

in S. pneumoniae.

Genetic variability of the comX gene amonglactococci

As in the case of the dprA gene, the primers homologous to

the intergenic regions flanking the comX gene in IL1403 led

to a productive PCR only for the strains belonging to the

lactis ssp. Other primers were designed for amplification

from the cremoris strains (material and methods). The comX

comX

max div. = 27.5%

0.1

IL1403IL584DRC1AM1A11A13A26A8NCDO2118NCDO2633NCDO2146NCDO604A27A7A17IL1321IL1306A15AM2NCDO2091

CO4CO2C7CNRZ359IL578NCDO276MG1363SK1IL582IL581A140

(b)(a) dprA

max div. = 21.7%

0.1

A15A17AM2IL1321IL1306DRC1IL584AM1A11A26A13A8IL1403NCDO2146NCDO604NCDO2633NCDO2118A27A7NCDO2091

CO4CO2IL581C7CNRZ359IL578MG1363SK1A140NCDO276IL582

*******

**

*

Fig. 1. Neighbour-joining unrooted phylogenetic trees inferred from the

sequences of Lactococcus lactis genes. (a) dprA sequences. (b) comX

sequences. The multiple nucleotide sequences were compared by

CLUSTALW (Thompson et al., 1994). The tree representation was

obtained with TREEVIEW (Page, 1996). The maximal divergence (max.

div.) was calculated with MEGA 2.1 (Kumar et al., 2001). Asterisks (�)

indicate the strains in which the gene is inactivated.



gene was detected in the sequence of all of the 31 fragments.

The maximal divergence of the comX nucleotide sequences

was 27.5% and all genes were intact. Further sequence

comparison using CLUSTALW revealed two allelic types

(Fig. 1b).

One allelic type was detected in all the lactis ssp. lactis

strains (20 strains including IL1403); It exhibits a maximal

intratype divergence of 4.5%. In total the 20 predicted

proteins corresponding to this lactis-allelic type presented

eight positions with substitution and one with an insertion

(Fig. 2). However compared to the ComX of IL1403, the

most divergent proteins (strains NCDO2091 and A7) only

differed by four amino-acids: three substitutions (N38S,

D114E, Q129E) and one insertion (D40) for NCDO2091 and

four substitutions for A7 (N38S, L108S, Q129E, D145E). In the

other strains presenting this comX allelic type, the deduced

proteins only differed by one (six strains), two (four strains)

or three (two strains) amino acids from the IL1403 ComX.

The second allelic type was identified in the eleven L.

lactis ssp. cremoris strains (Fig. 1b); it has a maximal

intratype divergence of 1.4%. Consequently, the 11 pre-

dicted proteins corresponding to this allelic type are very

well conserved: only three positions with amino-acid sub-

stitutions (H54R, R67W and R130C) are observed (Fig. 2).

Comparison of the predicted proteins from the two comX

allelic types (represented by the sequences from IL1403 and

MG1363), revealed two clusters of amino-acid substitutions

(Fig. 2 grey boxes) in regions of the sigma factor which are

embedded in the functional domains 2.3 and 3.1 (Lonetto

et al., 1992; Wosten, 1998; Gruber & Gross, 2003). Since

both domains are proposed to be involved in the interaction

with DNA (opening of the DNA duplex and DNA binding,

respectively), we wondered whether this variability may be

correlated to any divergence of the sigma recognition site in

the promoters. We therefore determined the sequence of the

putative cin-box in these strains.

Sequence of the cin-box in various lactococcalstrains

In agreement with Bolotin et al. (2001), MEME (Bailey &

Elkan, 1994) generated a consensus sequence (GTTACAA-

TN9TTTTCGTATA, Fig. 3) from the upstream region of the

late competence operons comE, comF, comC, comG, several

competence associated genes dprA, coiA, radA, recQ, ssbA

and a gene of unknown function yqfG in IL1403. This

sequence is proposed as the cin-box required for ComX

recognition. Using PATSCAN (Dsouza et al., 1997) to search

the IL1403 genome, a similar sequence was also identified

upstream of the radC, recA, ydbC (encoding a conserved

hypothetical protein, COG 4443) and ywgA (encoding a

IL1403 MKPIVMKLMKQIRIRTWKIEDYLQEGMIILHLLLEEQNDGQKLHTKFKVKYHQRLIDELRRSYAKKRSHDHFIG

lactis-T -----------------E-------------------S--H--------------------------N------1 13 1 1

MG1363 ----IR--------KA-D----Y--------H----NHPSTNIY---------H------H------L----V-

cremoris-T -----------------------------------------------------R------------W-------4 1

IL1403 LDVYECSDWINSGDTSPDNEVVFNHLLAEVYEGLSAHYQDLLLRQMRGEELTRMQRYRLREKIKAILFSEDEE

lactis-T ---------------------------------S----E/N-------------E---------------E--1 1 1 7 1

MG1363 --I-------DA-GST-ES-L------------------E--V-----------E---------N----R-DD

cremoris-T -------------------------------------------------------C-----------------2

2.1 2.2 2.3 2.4

4.24.13.1

D

Fig. 2. Amino-acid sequences of ComX. The black lines shown above the protein sequences indicate the putative functional domains (2.1, 2.2, 2.3,

2.4, 3.1, 4.1 and 4.2) as defined for other sigma factors (Lonetto et al., 1992). The first protein sequence corresponds to the ComX protein of the IL1403

strain. The lactis-T line shows all the amino-acid differences observed in the lactis-allelic type. The third sequence indicates the differences in the

MG1363 ComX protein in comparison with the IL1403 protein; most of the substitutions indicated were found in all of the ssp. cremoris strains. The

cremoris-T line shows amino-acid differences (other than that indicated for the MG1363 ComX protein) observed in the cremoris-allelic type. The

numbers below the substitutions correspond to the number of strains in which the difference was observed. NCDO2091 is the strain exhibiting the aa

(D) insertion in position 40 of ComX.


38 S. Wydau et al.

conserved hypothetical protein [COG 2137] which may

belong to the RecX family [pfam02631]).

In order to evaluate the conservation of this putative cin-

box, the region corresponding to the 415 nucleotides up-

stream of the late competence comG operon in IL1403 was

sequenced in the thirty lactococcal strains. Since the cin-box

located upstream of ComGA slightly differs from the IL1403

consensus sequence, it seemed more likely to vary between

strains. Comparison of these sequences revealed a conserved

sequence identical to observed in the cremoris-allelic

type. Then numbers below the substitutions correspond

to the number of strains in which the difference was

observed the IL1403 putative cin-box located upstream of

the comG operon in all strains (Fig. 3). In conclusion,

although two types of ComX proteins were detected in the

lactococcal strains, an identical cin-box was revealed by this

analysis.

Overexpression of the comXIL and comXMG

genes

In order to test the functionality of two comX allelic types,

comXIL and comXMG were cloned under the control of a

nisin-inducible promoter (pnisA) and introduced in VI 7238,

an IL1403 derived strain deleted of the comX gene (by

double crossing-over) and carrying the nisR and nisK genes

(required for the nisin-inducible expression system). The VI

7238 strain containing the pnisA-vector (pVI6253) was used

as a control. After 30 min of incubation with nisin, total

RNA was extracted from the various cultures and the

expression of comX and late competence genes was mon-

itored using RT-PCR (Fig. 4).

In the presence of nisin, the comXIL and comXMG tran-

scripts were detected while they were absent in the control

strain (VI 7238/pVI6253). To check the functionality of the

comX gene products, the expression of all genes (except

ydbC) exhibiting a putative cin-box in their upstream

sequence was also monitored (Fig. 3). The expression of the

comG, comE, comF, comC, dprA, coiA, ssbA and yqfG genes

increased markedly after the over-expression of comXIL

as well as that of the comXMG gene, whereas their expression

remained barely detectable or low in the control

strain treated with nisin. The radA, radC, recA and ywgA

appeared already well expressed in the absence of ComX.

Consequently, an accurate measurement of mRNA or

cDNA amounts will be needed to determine if their

expression increase in the presence of ComX. It is worth

noting the similarity between the expression profiles after

induction of ComXIL and ComXMG, which suggests that

both are active.

pnisA

pnisA::comXIL

pnisA::comXMG

com

XM

G

com

XIL

com

GA

com

GB

com

GC

com

GD

com

EA

com

EC

com

FA

com

FC

com

Cdp

rAco

iAra

dAra

dCre

cAre

cQss

bAyq

fGyw

gAhu

Fig. 4. Expression of late competence genes without or with induction of comXIL or comXMG. Strains VI 7293 (pnisA), VI 7247 (pnisA::comXIL) and VI

7298 (pnisA::comXMG) were incubated 30 min with nisin. Total RNAs were then extracted and the expression of comXIL, comXMG and of most of the

genes preceded by a putative cin-box were evaluated by RT-PCR. The expression of hu was used as a control and serial dilutions were also used to ensure

that the amount of hu cDNA was similar in all samples (data not shown).

-NGTTACAAT-N -ATTTCGTATA -ATGGAAACTTT-N -TTTTCGTATA-N -ATGGTTACATT-N -TTTTCGTATA-N -ATGGTTACATT-N -TTTGCGTATA-N -ATG

GTTACAAA-N -TTTTCGTATA-NGTTACAAG-N -TTTACGTATA-N -ATG

comCcomEAcomFAcomGA

coiAdprA

GTGACAAA-N -TTTACGTATA-N -ATGssbA

GTGACAAA-N -TTTCCGTATA-N -ATGradCGTGACAAA-N -AAAACGTATA-N -TTGrecA

GTTACATT-N -TTTGCGTATA-N -AGTATGGTTACATT-N -TTTGCGTATA-N -AATATGstrain A7

Other Lactococcus lactis strains

29 strains

ydb GTGACAAA-N -TTTTCGTATA-N -ATGCGTAACAAT-N -TTTTCGTATA-N -ATGywgA

GTTACAAT-N -TTTTCGTATAcin-box

IL1403

-

GTGATTTT-N -TTTACGTATA-NrecQ -ATG

GTTACATT-N -TTTACGTATG-NradA -ATGGTTACAAA-N -TTTTCGTATA-N -ATGyqfG

ATG

Fig. 3. Comparison of the cin-box sequences. The upstream regions of

the comC, comEA, comFA, comGA, dprA, coiA, ssbA, yqfG, radA, radC,

recA, recQ, ydbC and ywgA genes of IL1403 and the consensus

sequence referred to as cin-box obtained with MEME (Bailey & Elkan,

1994). Dark grey or pale grey highlight the bases found in a

given position in at least 80% or at least 30% of the sequences. The

sequences identified upstream of comGA in the other L. lactis strains

studied are indicated below the cin-box. The G to A transition is only

observed in A7.



Discussion

Comparison of 16S analysis, dprA and comXsequences

The classification of the lactis strains used in this study on

the basis of their 16S rRNA revealed that more than 29% of

the selected strains had been misclassified on the basis of the

phenotypic tests classically used to distinguish the L. lactis

ssp. The comX and dprA sequences each revealed two allelic

types, which perfectly matched the 16S classification of

strains while only partially fitting the phenotypic or RAPD

groups. The mean nucleic divergence observed between the

lactis and cremoris alleles was 26.9% for comX and 20.9% for

dprA. This observation strongly suggests that the natural

competence genes were present in lactococci before the

divergence between the lactis and cremoris ssp., i.e. around

17 million years ago (Bolotin et al., 2004) since the rate

of divergence was estimated at �0.9% per million years

(Ochman et al., 1999).

Conservation of key elements of the naturalcompetence system

In all strains, intact comX genes were found and identical

cin-boxes were identified upstream of the comG operon.

Two types of ComX were distinguished, each being well

conserved among the strains of one ssp. The observation

that the same cin-box is conserved among all tested strains

suggests that these two types of ComX recognize the same

DNA sequence. We showed that in IL1403, both ComX

variants were active and allowed the expression of all the late

competence genes required for DNA uptake in other micro-

organisms. It establishes that comX is not degenerated in L.

lactis, suggesting that having an active ComX might be an

advantage for the strain for an as yet undiscovered reason.

Contrary to comX, dprA was inactivated in about 32% of the

strains all belonging to the ssp. lactis. Although the exact role

of DprA remains to be established in L. lactis, this high

occurrence of a mutated dprA gene may indicate that the

natural competence process is used for another purpose

than genetic diversity (i.e. natural transformation) such as a

competitive advantage as previously shown in Escherichia

coli (Finkel & Kolter, 2001). In a dprA background, the

natural competence could provide DNA, which will be

degraded leading to an increase of the intracellular nucleo-

tide pool. As free bases are present in limited amounts in

milk, one can propose that a dprA mutation might be

advantageous. In addition to DNA uptake, the natural

induction of competence may also allow the predation of

non-competent cells or of other species via the expression of

bacteriocin as being recently described for Streptococcus

pneumoniae (Guiral et al., 2005) and S. mutans (Kreth

et al., 2005).

For both the lactis and cremoris ssp., strains presenting a

complete dprA gene were also found. The L. lactis strains

presenting the full DprA (lactis-, cremoris-types) or a

truncated form would be useful to test whether natural

competence is functional in L. lactis and to investigate the

role of this protein in lactococci. We demonstrated in this

work that ComXMG (present in the cremoris ssp.) can

stimulate the expression of the late competence genes in a

ssp. lactis strain. Consequently a comX overexpression

system could be introduced in the various strains exhibiting

a complete or a truncated dprA gene (identified in this

work) to test whether the upregulation of comX could lead

to natural transformation. This system constitutes an inter-

esting alternative to the laborious approach that is to seek

conditions allowing the induction of competence in a given

species by testing different media, starvation conditions,

growth phases etc. This latter approach may be time

consuming since the conditions of induction of natural

competence vary between species (Dubnau, 1991).

In conclusion, this study revealed that the homologues of

three key elements of the natural competence process are

conserved among lactococci and it demonstrates for the first

time in lactococci the activity of the ComX–cin-box regula-

tory circuit. Strains with different allelic types of the comX

and dprA genes and the comX overexpression system con-

stitute promising tools to test the functionality of natural

transformation in this species.

Acknowledgements

We are grateful to D. Vailhen, A. Bolotin, M. van de Guchte,

P. Serror for helpful discussion and suggestions. S. Wydau is

the recipient of a fellowship from the Ministere de la

Recherche.

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42 S. Wydau et al.

Conservation of key elements of natural competence in Lactococcus lactis ssp

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