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J Bacteriol. Apr 2007; 189(8): 3256–3270.
Published online Feb 16, 2007. doi:  10.1128/JB.01768-06
PMCID: PMC1855848

Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363[down-pointing small open triangle]


Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category “carbohydrate metabolism and transport,” by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the “lateral gene transfer hot spot” in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.

Lactococcus lactis, a mesophilic fermentative bacterium producing lactic acid from sugar (hexose) fermentation, is an important industrial microorganism with extensive and diverse uses in food fermentation. Strains of L. lactis are used as defined mixtures or in undefined combinations with other lactic acid bacteria (LAB) in the production of fermented milk products. The organism has adapted to growth in milk under stringent human selection for better performance with respect to taste, flavor, and texture of dairy products, and this process continues today (57, 98, 99). In 1985, the “dairy streptococci” were reclassified into two L. lactis subspecies, Lactococcus lactis subsp. lactis (previously Streptococcus lactis) and Lactococcus lactis subsp. cremoris (previously Streptococcus cremoris), to distinguish them from the streptococci sensu stricto, which contain a number of notorious human pathogens (82, 83).

The strain used in this study, L. lactis subsp. cremoris MG1363, is the international prototype for LAB genetics, and the knowledge gained from fundamental research on this strain has been exploited for a wide variety of biotechnological applications. The large and unstable complement of plasmid DNA of the parent strain, L. lactis NCDO712, was eliminated by employing UV treatment and protoplast-curing strategies in the early 1980s (41). The resultant plasmid-free strain, L. lactis MG1363, is robust and genetically amenable, which has facilitated the analysis of introduced lactococcal and heterologous DNA. Sophisticated systems have been developed for the expression of proteins and peptides in this strain, and it has been used as a cell factory for a wide variety of heterologous products (e.g., antimicrobials, including bacteriocins [50], bacteriophage endolysins [75], and defensins [47]). A recent review provides a snapshot of this diversity involving the favored “NICE” system (for nisin-controlled protein overexpression) (25, 71), which can be used to express heterologous proteins (e.g., lysostaphin) up to industrial scale (72). The importance of these developments for the field of biotechnology and for microbiology research is enormous, as many of the tools initially developed for L. lactis, e.g., plasmids, integration systems, and the NICE expression system, have also been shown to be applicable in all other LAB (55) and several species of other industrially relevant bacterial genera, e.g., Bacillus and Clostridium, and also in human pathogens, e.g., Listeria, Enterococcus, and Streptococcus (14, 58, 79).

The GRAS (generally regarded as safe) status of L. lactis is a distinct advantage for its use in the production and secretion of therapeutic or vaccine proteins (62). While L. lactis is not a natural inhabitant of the gastrointestinal tract, it does survive gut passage. L. lactis MG1363 has been successfully used to pioneer the gut delivery of bioactive molecules, such as vaccine antigens and immune modulators. It is especially relevant that a contained mutant of the genetically modified strain expressing the cytokine interleukin 10 has been used in a human trial of patients with inflammatory bowel disease (88-90). A mucosal vaccine based on live L. lactis MG1363 expressing the E7 antigen and interleukin 12 was shown to protect mice against human papillomavirus type 16-induced tumors (6). Recently, L. lactis was used as a nonliving nonrecombinant delivery system for mucosal vaccination (3). Pneumococcal antigens were bound to the walls of pretreated lactococcal cells by means of the peptidoglycan-binding domain of the major lactococcal autolysin (87). Local and systemic immune responses were induced following intranasal immunization of mice.

The analysis and engineering of lactococcal metabolism has been focused on L. lactis MG1363 and has involved both homologous pathways and the introduction of heterologous genes to reroute metabolic flux (26, 85). The relative simplicity of the lactococcal metabolism lends itself to modeling, e.g., derivatives of the strain that overproduce B vitamins (92) or alanine (49) have been constructed through metabolic rerouting.

In summary, in the international scientific community, L. lactis MG1363 has deserved its status as the undisputed prototype LAB because of the extent and diversity of its combined genetic and biotechnological amenities. The L. lactis MG1363 genome sequence presented here is vital to both applied and fundamental research, allowing the application of powerful postgenomic techniques (e.g., proteomics, transcriptomics, and metabolomics) to further these studies.


Genome sequencing and assembly.

Genomic DNA from L. lactis subsp. cremoris MG1363 was used to construct a small insert library (1 to 3 kb) in the pGEM-T (Promega, Madison, WI) vector. DNA sequencing was performed using standard primers on automated sequencing machines (ABI373 and ABI3730 [Applied Biosystems, Foster City, CA] and Licor [LI-COR Biosciences]). Base calling was performed with the phred package (38), followed by assembly using the phrap package (http://www.phrap.org) in conjunction with the Staden package (86). The resulting draft assembly was then mapped on the genome of L. lactis IL1403 using the Projector software package (97) to order the contigs into large scaffolds. Standard PCR, followed by primer walk sequencing of the resulting products, was used to close the remaining 800 gaps, in combination with inverse PCR for the gaps for which no linkage information could be obtained. To verify the complete assembly, PCRs predicted to be separated by 9 kb were performed on the genome, covering the entire genome with an overlap of 500 bp. Virtual digests of the complete assembly were compared with available pulsed-field gel electrophoresis restriction maps of the genome of L. lactis MG1363 (61). All low-quality regions on the genome were resequenced until every base pair was at least of phred 30 quality, resulting in a final average quality of approximately phred 70 and fivefold coverage of the entire genome. Regions containing putative frameshifts and point mutations were resequenced to verify the fidelity of the sequence.

Bioinformatics analyses.

The finished L. lactis MG1363 sequence was annotated using the GenDB 2.2 Annotation Tool (70). Putative protein coding sequences (CDSs) were determined using REGANOR5 (68), based on the combined CDS predictions of CRITICA (4) and Glimmer (80). Putative ribosomal binding sites and tRNA genes were identified with RBSfinder (91) and tRNAscan-SE (65), respectively. An automatic functional annotation was followed by a manual annotation of each predicted gene. Pseudogenes were identified through FastA analysis of the intergenic regions versus the nonredundant nucleotide sequence database and a comparison of predicted gene products with respective proteins in the nonredundant peptide sequence database. Similarity searches were performed using BLASTN and BLASTP (2) against the nonredundant nucleotide and protein databases, respectively, provided by the National Center for Biotechnology Information. Additionally, a BLASTP search was performed against the KEGG database (54), and PSI-BLAST (2) was used to perform similarity searches against the SWISS-PROT (8) database. Analysis of protein families was done using the HMMer package (36) for searches against the Pfam (5) and TIGRFAM (46) databases. Furthermore, functional motifs and protein domains were automatically assigned by InterProScan queries against InterPro (73). RPSBlast was used to search the conserved domain database CDD (67). Searches against TransTerm (13) were applied on intergenic regions to predict transcription terminators. Additionally, SignalP (35), helix-turn-helix (29), and TMHMM (59) were applied. Finally, each gene was functionally classified by assigning a “clusters of orthologous groups” (COG) number and corresponding COG category (94) and gene ontology numbers (48) based on the best BLASTP results versus COG and InterPro results, respectively. Similarity searches using BLASTP were performed against the toxin and virulence factor database MvirDB to assess the GRAS status of L. lactis MG1363 (103).

Predicted CDSs were manually reviewed, and alterations were made on the basis of the presence of potential ribosomal binding sites, sequence alignments, and available data in the literature. Potential alien genes on the L. lactis genome sequence were identified using the program SIGI (for score-based identification of genomic islands) (69), based on the scoring of codon frequencies combined with cluster analysis. Alien genes are defined as those that are potentially acquired through horizontal gene transfer (HGT). This program has also been used to analyze the genome sequence of Bacillus licheniformis DSM13 (101) and the spread of the ycdB gene from Lactococcus to Salmonella (10). In order to identify secreted proteins, all putatively expressed proteins of L. lactis MG1363 were analyzed using the SignalP algorithm, and potential transmembrane domains were determined using the TMHMM (v2.0) algorithm. Proteins containing a signal peptide and a single transmembrane domain were further screened for the presence of the following protein retention signals: a lipobox (for lipid attachment to the membrane), a prepilin motif (for the formation of [pseudo]pili, which would indicate that the protein remained anchored to the cell envelope), peptidoglycan- or choline-binding domains for noncovalent binding to the cell wall, and an LPXTG motif for covalent attachment to the cell wall mediated by sortase.

Carbon utilization.

L. lactis MG1363 and L. lactis IL1403 were tested on Biolog Phenotype MicroArray plates for carbon catabolism (7).

Nucleotide sequence accession number.

The whole genome sequence of L. lactis MG1363 (http://www.cebitec.uni-bielefeld.de/groups/brf/cooperations/LlactisMG1363.html) has been deposited in the EMBL/GenBank databases with the accession number AM406671.


General genome features.

The circular chromosome of L. lactis MG1363 contains 2,529,478 bases (2.53 Mb), with an average GC content of 35.8% (Table (Table1).1). The Oriloc software (39) was used for the identification of the putative oriC and ter regions. The dnaA gene located in the putative oriC region is preceded by an AT-rich region (71%) containing seven DnaA boxes, four on the forward strand and three on the reverse strand. Therefore, the first base pair of dnaA was designated position 1 on the circular chromosome of L. lactis MG1363. The putative terminus is at position 1255251 (Fig. (Fig.1).1). A total of 2,517 predicted CDSs were identified, the majority (79%) of which were on the leading strand of chromosome replication. For 1,574 (62%) of the deduced CDS products, a general or specific function was predicted. A comparison with the genome sequences of L. lactis IL1403 (11) and L. lactis subsp. cremoris SK11 (66) revealed that there is approximately 85% DNA sequence identity between the CDSs present in both L. lactis MG1363 and L. lactis IL1403 and 97.7% between those in the two subspecies cremoris strains, L. lactis MG1363 and L. lactis SK11. The 16S rRNA sequences of L. lactis MG1363 and L. lactis IL1403 diverge by 0.33%, suggesting that they deviated approximately 17 million years ago (10). Using BLASTN at a cutoff of 1e−20, 465 of the L. lactis MG1363 genes do not have a corresponding hit in L. lactis IL1403 and 346 are not present in the chromosome of L. lactis SK11 (but 19 of these are present on plasmids in the latter), while 213 are absent in both L. lactis IL1403 and L. lactis SK11.

FIG. 1.
Genome atlas of the chromosome of L. lactis MG1363. The first base pair of dnaA in the oriC region is designated position 1, and the putative terminus is at position 1255251. The inversion relative to L. lactis IL1403 is indicated. Starting from the inside, ...
General genome features

Eighty-one pseudogenes were detected in L. lactis MG1363 (Table (Table2),2), representing around 3% of the total number of genes, which is well within the 1% to 5% described for other bacterial genomes (64). Most of the pseudogenes (a total of 32) originate from genes involved in transposition, followed by genes with putative transport functions (a total of 11) and regulatory roles (a total of 6). If transposon-related genes are ignored, most of the pseudogenes (a total of 31) arose from the inactivation of single-copy genes. The decay of genes following possible duplication events contributed 14 pseudogenes. Although L. lactis MG1363 hosts a large number of insertion sequence (IS) elements (see below and Table Table3),3), only three pseudogenes (bglR, fadA, and malQ) are the result of IS insertions.

Pseudogenes in L. lactis MG1363 and putative functions of their unimpaired counterpartsa
IS elements of L. lactis MG1363, L. lactis IL1403, and L. lactis SK11

IS elements, HGT, and alien genes.

L. lactis MG1363 carries 11 different IS elements involving a total of 67 kb of DNA (Table (Table3).3). Several of these elements are contained within sequences not present in L. lactis IL1403 (Fig. (Fig.1).1). A rather remarkable feature is apparent in their distribution: nearly one-fifth of the IS elements are concentrated in a specific region of 59 kb, while three novel IS species are present only in this part of the chromosome. This “integration hot spot” also carries possible plasmid inserts (see below). The presence of the four IS elements, previously described only on plasmids, and plasmid-derived genes in the integration hot spot show that there has been a high level of gene exchange with the plasmid population of the parental strain in the past. Two ISs are unique to L. lactis MG1363: IS712 and IS1675 are not present in L. lactis IL1403 and L. lactis SK11. A striking feature in the last strain is that two-thirds of its 130 IS elements are IS981 (30 copies) and IS982 (55 copies).

Many of the IS elements in L. lactis MG1363 are now inactive, as shown by the fact that 42% of the pseudogenes were originally involved in transposition. Despite the fluidity of IS movement within the genome, there are very few (three, as mentioned above) direct effects on gene integrity. Insertion of IS elements can also result in the activation of downstream genes. Examples in L. lactis are those identified after a spontaneous IS insertion event, such as constitutive nisin production (27, 28), and those resulting from IS insertion under a strong selection pressure, such as the IS activation of the alternative ldhB gene in ldhA knockout mutant strains, enabling them to grow much better. More than one IS element has been shown to be capable of this activation, as independent strains have been isolated (IS981 [12] and IS905 [our unpublished results]).

The sex factor is a unique mobile genetic element present on the L. lactis MG1363 chromosome (Fig. (Fig.1)1) (reviewed in reference 42). It can conjugate into L. lactis IL1403, due to the presence of a potential attB site between guaC and xpt (positions 1159525 to 1159548 in the L. lactis IL1403 genome) (reference 42 and our unpublished results). The genes surrounding the putative attB site in L. lactis IL1403 are the same as those flanking the sex factor in L. lactis MG1363 (i.e., those surrounding the attB site in L. lactis MG1363, if the sex factor was lost spontaneously or temporarily excised). The L. lactis IL1403 genes are colinear in this region with those of L. lactis MG1363. There is no indication of the presence of a sex factor-related element at this position in L. lactis IL1403, nor does L. lactis SK11 contain a sex factor.

Although natural competence in L. lactis MG1363 has not been described, the organism is capable of acquiring novel DNA through conjugation or sex factor-mediated transfer or via bacteriophage transduction (reviewed in references 40, 42, and 43). Analyses of codon or amino acid sequences, as well as phylogenetic analyses, have been used to predict chromosomal regions obtained through HGT. In L. lactis MG1363, 7.1% (174/2,576) of the genes were defined as alien, i.e., acquired through HGT (using the SIGI software based on codon usage and cluster analysis), which compares well with the 10% obtained for Lactobacillus plantarum WCFS (56), defined as acquired by HGT using base composition analysis, but slightly lower than that predicted by SIGI for Streptococcus pneumoniae TIGR4 (13.0%), both Streptococcus agalactiae and Streptococcus pyogenes (10.9%), Listeria innocua (10.6%), Listeria monocytogenes (9.0%), and, in particular, L. lactis IL1403 (8.9%) (69).

Lactococcus is viable in a number of diverse environments, is plant associated, and is found in the gastrointestinal tracts of animals, insects, and humans, and also in fermented foods and feeds of dairy, meat, and plant origin, bringing it into close proximity to a wide variety of other microorganisms with a large reservoir of genes for potential gene transfer (63). There is evidence that gene transfer can occur within the intestinal tract of mice (51). The ycdB gene has an unknown function and is widespread in bacteria (some enteric bacteria have two copies) and some eukaryotes. Analysis suggested that the gene was transferred from L. lactis to Salmonella (37), and more recent research has shown that this region of DNA has been transferred between IL-like and MG-like lactococcal strains, possibly by a conjugal process (10).

L. lactis MG1363 has been used extensively as a model for future use of LAB in medical applications, e.g., as a (live) oral vaccine (6, 3, 87). These developments are fueled by the fact that L. lactis is innocuous and has an extensive record as a GRAS organism. From the genome sequence, there is evidence for past and potential for future gene transfer in L. lactis MG1363. In a preliminary search, we examined the presence in L. lactis MG1363 of proteins with homology to known virulence factors, using the MvirDB database (103). A number of hits against MVirDB were detected at a cutoff of 1e−20, but apart from genes that specify proteins involved in general metabolic pathways, only fbpA, encoding a putative fibronectin-binding protein, and a phage-encoded putative extracellular endonuclease (ps111) have been implicated in virulence in other organisms. FbpA might, by analogy to the activity of the protein in Streptococcus gordonii, be involved in adhesion to cell surfaces (18). On the other hand, fibronectin-binding proteins may also be important for probiotic action and might not be virulence factors per se (1). The absence of obvious virulence genes, such as those encoding hemolysins or toxins, corroborates the GRAS status of L. lactis MG1363. Clearly, the availability of the genome sequence will add another layer of safety to future studies using L. lactis MG1363 for medical purposes.

Prophages and prophage remnants.

The L. lactis MG1363 chromosome harbors six regions that represent bacteriophage-related sequences (Fig. (Fig.1).1). Two sites appear to contain complete prophage genomes, designated phiT712 (42,085 bp) and MG-3 (44,200 bp). The remaining bacteriophage sequences, designated MG-1 (19,053 bp), MG-2 (6,019 bp), MG-4 (18,029 bp), and MG-5 (10,598 bp), appear to represent remnant or satellite phages. Together, the bacteriophage sequences encompass approximately 5.5% of the L. lactis MG1363 genome, representing a large portion of the observed genomic differences between L. lactis MG1363, L. lactis SK11, and L. lactis IL1403. The latter finding is a clear indication that lysogenic bacteriophages contribute significantly to genome variability within this species. The six prophage sequences occupy various positions on the L. lactis MG1363 genome, and their insertion did not appear to have resulted in gene interruptions. Comparative analyses between the six prophages present in L. lactis IL1403 and those found in L. lactis MG1363 revealed only one common integration site, i.e., that of MG-1 and bIL310, two phages that also display the highest level of homology and synteny. A similar phage is not present in L. lactis SK11 (66). Interestingly, the integration site of phage MG-3 is the same as that of the L. lactis SK11 phage in the intergenic region between CDSs LACR_2145 and LACR_2146. However, there is sparse and low similarity between the two phage sequences, suggesting the presence of an insertion site that can be the target of different phages.

It is noteworthy that two of the L. lactis MG1363 phages (phiT712 and MG-4) contain sequences that are homologous to different plasmid-encoded abortive infection systems (reviewed in reference 17), which, together with a gene encoding a putative extracellular endonuclease found in prophage MG-1, seem to endow the host with a beneficial lysogenic conversion property. Moreover, prophages MG-2 and MG-3 carry tRNA genes (i.e., tRNALys and tRNAArg), which may increase the translational efficiency of the bacterial host. All L. lactis MG1363 prophages display limited similarity with DNA prophage sequences contained in L. lactis SK11. Their sequence similarity is mainly restricted to presumed integrase genes, to genes encoding structural proteins (e.g., phiT712), or to a putative DNA replication region (e.g., MG-4 and MG-3).

L. lactis MG1363 is a “phage-cured” strain; induction of prophages in L. lactis MG1363 has never been reported in the literature. It is worth noting that the largest prophage genomes in L. lactis MG1363 (MG-1, MG-3, and phiT712) contain IS712 elements, which are unique to L. lactis MG1363. It is not known whether their presence has any effect on the inducibility of the phages themselves. The phage genomes in L. lactis SK11 and L. lactis IL1403 are not interrupted by IS elements, except for phage bIL311 in L. lactis IL1403, which contains two IS983 elements (16).

An in-depth analysis of the phages from all three strains will be presented elsewhere (M. Ventura, A. Zomer, C. Canchaya, O. P. Kuipers, J. Kok, and D. van Sinderen, unpublished data).

Integration hot spot and chromosomal inversion.

A 59-kb DNA region (nucleotides 647,000 to 706,000) contains genes that have been reported to be plasmid encoded, e.g., two remnants of plasmid replication protein genes, genes of the HsdRMS restriction/modification system, and the opp-pepO operon (96). This region also contains three repeat sequences (Fig. (Fig.2),2), one of which is highly similar to a binding site for a recombinase of the resolvase family (PFAM PF00239) and the resolvase gene itself (tnpR). The homologous resolvase (CAA36950.1) in Staphylococcus aureus is involved in integration and excision of DNA (78). The repeat sequences are specifically found on plasmids and other bacterial genomes, including the “lateral gene transfer hot spot” in the genome of Streptococcus thermophilus (9). A number of such integration (and subsequent partial deletion) events could explain the large number of plasmid genes in this area of the genome. The parental strain of L. lactis MG1363, L. lactis NCDO712 (23), contains only one chromosomal opp gene cluster, coding for the oligopeptide transport system. This cluster is located in the same position as the opp-2 genes in L. lactis MG1363. In addition, strain NCDO712 contains an opp-pepO gene cluster on a plasmid of at least 40 kb (60). The chromosomally located opp-2 system in L. lactis MG1363 is inactive, since some of the opp-2 genes contain frameshift mutations (81). From the genome comparison with L. lactis IL1403, it can be concluded that opp-2 is the original copy of this locus. In L. lactis MG1363, the oppD1 promoter is regulated by CodY (24). A clustalW analysis of the upstream region of oppD1, oppD2, and oppD of L. lactis IL1403 showed marked differences (data not shown). Interestingly, the CodY binding site is missing in the region upstream of oppD2, as well as in oppD in L. lactis IL1403, indicating that L. lactis MG1363 and L. lactis IL1403 should differ with respect to the regulation of their active opp systems. It is noteworthy that an oppA1 mutant is not able to grow on the peptide Leu-enkephalin (96), showing that the apparently intact oppA2 gene does not complement this mutation. This could be due to a lack of transcription of the respective gene or the fact that OppA1 and OppA2, although the same size, show only 88% sequence identity. The fortuitous insertion, during plasmid curing of L. lactis NCDO712 (41), of the plasmid-derived opp genes in the integration hot spot gives the resulting plasmid-free strain, L. lactis MG1363, a growth advantage in mixed cultures in milk: although the strain cannot produce oligopeptides from milk casein due to the lack of proteinase (caseinase) activity, it can efficiently utilize the oligopeptides produced by proteinase-positive strains in the starter culture.

FIG. 2.
Integration hot-spot repeat sequences. The underlined sequences indicate inverted repeats. The asterisk indicates the fact that the transposase gene of IS905 is a pseudogene. (A) Sequence present in at least 27 plasmids from L. lactis subsp. lactis, ...

The chromosomes of L. lactis MG1363, L. lactis SK11, and L. lactis IL1403 show extensive gene synteny when a large chromosomal inversion, described previously (61), is taken into account (Fig. (Fig.3A).3A). The L. lactis MG1363 integration hot spot is located exactly at the left-hand end of this inversion region. The inversion occurs between the nucleotide positions 647123 and 1873134 and, with a total of 1,226,011 bp, represents 48% of the genome. The flanking regions in the three strains are identical, suggesting that the inversion in L. lactis MG1363 took place relatively recently, long after the subspecies division. L. lactis SK11 has the same genome organization as L. lactis IL1403, with the exception of a small 73-kb inversion near terC (see below). Analysis of the genetic loci at the left and right borders of the large inversion in L. lactis MG1363 (Fig. (Fig.3B)3B) suggests that the following sequence of events has led to the inversion. (i) A copy of IS904 transposed into a copy of ISLL6 in one of the border locations. (ii) A subsequent duplication and transposition of this region occurred, most likely involving a plasmid-encoded ISLL6 transposase, as there is no functional copy of ISLL6 left on the chromosome of L. lactis MG1363. (iii) Finally, a flip between the two divergently oriented duplicated sequences took place through homologous recombination. The inversion disturbs the symmetry between oriC and ter only marginally, by shifting terC a further 9.4 kb away from the symmetry center (Fig. (Fig.1).1). Since several PCR and Southern hybridization approaches failed to show the reversal of the 1.2-Mb segment, it is assumed that the inversion is stable and that L. lactis MG1363 cells in a culture all carry a genome with the structure published in this paper (data not shown). A similar inversion event was described previously (22) for L. lactis NCDO763, another derivative of L. lactis NCDO712 (60). This event concerned another inactive IS element, one that is also located in the integration hot-spot region of the L. lactis MG1363 chromosome. In this case, two inactivated copies of IS905 were involved, and the subsequent inversion comprised 56% of the genome. The inversion in NCDO763 results in the reinstatement of the original IL1403-like orientation of the central regions (regions A and B in Fig. Fig.3B)3B) on either side of ter. The left-hand border region (region C) between ISLL6::IS904A and IS905A, as seen in L. lactis MG1363, remains in place, and the DNA region (region D), originally to the right of the L. lactis MG1363 inversion region between the ISLL6::IS904A composite transposon and the IS905B transposon, has now been inverted and transposed next to region C. In L. lactis NCDO763, the DNA rearrangement has resulted in moving the ter site 200 kb to the right, affecting the symmetry between the origin and the terminus loci. The right side of the inversion in L. lactis MG1363 around position 1867516 contains the right junction, as well as the xis, int, and ardA genes of transposon Tn5276, indicating that this transposon has been deleted from the chromosome (Fig. (Fig.3B).3B). Since the orientations of the CDSs of L. lactis MG1363 are similar to those of its parent, L. lactis NCDO712 (60), the inversion must have occurred before the latter strain was deposited in the National Collection of Dairy Organisms, Reading, United Kingdom in 1954 (23) and might have resulted from the integration of foreign or plasmid DNA. This implies that the integration hot spot was not caused by the plasmid curing of L. lactis NCDO712 during the process of L. lactis MG1363 construction. The hot-spot region, together with the conjugative sex factor, may have provided the ancestor of L. lactis MG1363 with flexibility in the uptake and maintenance of DNA.

FIG. 3.
Chromosomal inversions in L. lactis. (A) Dot plot of the nucleotide sequences of the chromosomes of L. lactis IL1403 (red) and L. lactis SK11 (black) (both on the y axis) against that of L. lactis MG1363 (x axis). (B) Chromosomal inversions in L. lactis ...

A 73-kb chromosomal inversion in L. lactis SK11 relative to L. lactis IL1403 and L. lactis MG1363 is present in a region near terC, between the L. lactis SK11 nucleotide positions 1202292 and 1275464 (Fig. (Fig.3A).3A). This inversion has been facilitated through homologous recombination between two divergently oriented IS981 elements. This region in the three lactococcal strains contains genes usually found on plasmids: an integrase-recombinase gene (PF00589) and various IS elements. In L. lactis IL1403, it carries the citrate utilization genes. The region in L. lactis MG1363 contains a duplication of cspD (see below); two copies of ceo, a gene that is also present on the sucrose transposon Tn5306 (30); an additional recombinase-encoding gene; and a putative arsenic resistance gene cluster. The region in L. lactis SK11 is very similar to that of L. lactis MG1363, but it lacks some of the genes present in L. lactis MG1363. The presence of these and other possibly plasmid-derived sequences is suggestive of a plasmid insertion in the ancestor of all three Lactococcus strains, followed by differential reductive evolution events.

Correlation of genotypes with the classical lactococcal subspecies phenotypes.

L. lactis strains used in the dairy industry are divided into the two subspecies lactis and cremoris. Historically, these have been distinguished based on (industrially relevant) phenotypic properties, such as the ability of L. lactis subsp. lactis strains to metabolize arginine and maltose and to grow at 40°C and in the presence of 4% NaCl, in contrast to the L. lactis subsp. cremoris strains (95). Thus, L. lactis MG1363 was classified initially as L. lactis subsp. lactis. More recently, on the basis of thorough molecular-genetic analyses, including DNA-DNA hybridization, 16S rRNA and gene sequencing, and PCR-based typing methods, L. lactis MG1363 was reclassified as L. lactis subsp. cremoris. However, it represents an atypical example of the subspecies, as, e.g., it contains an active ADI pathway for arginine degradation (44, 98). In this respect, L. lactis subsp. cremoris SK11 is a “true” cremoris strain. The arginine deiminase-negative phenotype of L. lactis SK11, however, is attributable to a single-base-pair deletion at position 675 of the arcA gene, creating a frameshift and subsequent pseudogene in L. lactis SK11. Orthologs of all other ADI pathway genes are present in L. lactis SK11. The use of maltose as a carbon source can be correlated with the genomic sequences of the L. lactis strains MG1363, IL1403, and SK11 (see below). Other phenotypic traits are more difficult to identify from the genome, as they are multifactorial.

Carbon utilization.

Forty-seven of the genes present in L. lactis MG1363, but not in L. lactis IL1403, belong to the COG functional category “carbohydrate metabolism and transport” (94), by far the largest category of novel genes in this strain relative to L. lactis IL1403. Seventeen genes of this category are unique in L. lactis MG1363 compared to L. lactis SK11 (Table (Table4)4) . Both L. lactis MG1363 and L. lactis IL1403 were tested on Biolog Phenotype MicroArray plates for carbon catabolism (7). The results obtained suggest that L. lactis MG1363 has a much greater capability of growing on various sugars, especially those found in plant material (Fig. (Fig.44 and Table Table4).4). For example, polyols, like mannitol and sorbitol, are utilized by L. lactis MG1363 but not by L. lactis IL1403, which has a frameshift in mtlA, encoding the mannitol-specific enzyme II component of the phosphotransferase system (PTS). Conversely, dye reduction on γ-cyclodextrine and glycerol was higher for L. lactis IL1403 than for L. lactis MG1363. Indeed, L. lactis MG1363 contains a frameshift in glpF1, encoding a putative glycerol uptake facilitator. Although both strains contain a second glycerol uptake facilitator gene, glpF2, these results indicate that GlpF2 expression or activity might not be sufficient for growth on glycerol of L. lactis MG1363. The difference seen in γ-cyclodextrine usage cannot be fully explained. The L. lactis MG1363 genome does contain putative genes for the breakdown of polysaccharides with 1,4-glucosidic bonds, such as maltose and cyclodextrin. The corresponding region in the chromosome of L. lactis IL1403 contains a number of hypothetical genes not present in L. lactis MG1363 that may explain the observed differences between the two strains. L. lactis SK11 does not ferment maltose. As Table Table44 shows, this strain lacks malG and malF, genes encoding the maltose permease.

FIG. 4.
Growth of L. lactis on sugars. Utilization by L. lactis MG1363 (open bars) and L. lactis IL1403 (black bars) of various sugar sources as measured by a significant (<60) difference in dye intensity development (indicated in arbitrary units on the ...
Genes present in L. lactis MG1363, but not in L. lactis IL1403 or L. lactis SK11, belonging to the COG functional category “carbohydrate metabolism and transport”

L. lactis strains MG1363, SK11, and IL1403 all carry genes encoding the potential lactate dehydrogenases LdhA, LdhB, and LdhX and a malate/lactate dehydrogenase (HicD in L. lactis IL1403). L. lactis MG1363 and L. lactis SK11 do not contain the cit operon for citrate utilization, which is present in L. lactis IL1403. L. lactis MG1363, like L. lactis IL1403, contains an incomplete tricarboxylic acid cycle and carries several genes necessary for aerobic respiration. Unlike L. lactis MG1363 and L. lactis IL1403, the genes for citrate synthetase (gltA), aconitase (citB), and malolactic enzyme (mleS) are pseudogenes in L. lactis SK11. All three lactococcal strains have the men and cytABCD operons (menaquinone synthesis and cytochrome d biogenesis) and the hemH, hemK, and hemN genes for the late steps of heme synthesis. Duwat et al., Rezaïki et al., and Vido et al. (34, 77, 102) have demonstrated that L. lactis MG1363 is capable of biphasic fermentative and respiratory modes of growth under respiratory conditions, leading to a greater growth yield and better long-term survival. Their physiological and biochemical analyses confirm the L. lactis MG1363 genomic information.

There are many indications that the ancestor of L. lactis MG1363 occupied a plant-associated niche. L. lactis MG1363 still retains the ability to metabolize plant-derived sugars as carbon sources and has cell surface proteins (csc clusters of CscA, -B, -C, and -D) that are present in other gram-positive bacteria found in a plant environment (see “The lactococcal cell envelope and secretome” below). Adaptation to milk has included changes in metabolic activity, including the inactivation of several of the amino acid biosynthetic pathways (100). The ancestor of L. lactis MG1363 was obviously flexible in its ability to acquire DNA from other bacteria, in particular, plasmids and other mobile elements (see above). This property has contributed to the organism's capacity to adapt from a plant-associated niche to survival in the milk environment, e.g., through the acquisition of plasmids specifying the proteinase (caseinase) PrtP and enzymes and transport proteins for the utilization of the milk sugar lactose (43).

Amino acid-, vitamin-, and nucleic acid biosynthesis.

The six amino acids glutamate, leucine, isoleucine, valine, histidine, and methionine are essential for the growth of L. lactis (74, 76), while L. lactis MG1363 grows to an appreciable extent only when, in addition, one of the five amino acids asparagine, glutamine, alanine, arginine, and threonine is present (53). The many conflicting results on the minimal growth requirements of L. lactis may be attributed to the use of different formulations of chemically defined medium (CDM), as concentrations of CDM constituents, such as amino acids, can affect the growth rates of different L. lactis strains (44). For example, excess isoleucine results in blocking of several CodY-dependent amino acid biosynthetic pathways and subsequent growth inhibition (45). The L. lactis MG1363 genome contains homologues of genes with the potential for biosynthesis of five of the six essential amino acids. In both L. lactis MG1363 and L. lactis IL1403, leuA and leuB (leucine biosynthesis) are pseudogenes, while in L. lactis SK11, the genes are not mutated. Further differences between L. lactis MG1363, L. lactis SK11, and L. lactis IL1403 exist in the predicted biosynthetic pathways for the nonessential amino acids. L. lactis MG1363 harbors a frameshift mutation in the gene for glycerate kinase, the enzyme catalyzing the first step in serine biosynthesis from glycerate. A potential alternative route for serine biosynthesis by L. lactis MG1363 is from pyruvate. Threonine biosynthesis can potentially occur from aspartate or by condensation of glycine by threonine aldolase. Jensen et al. (52) observed in vivo activity of threonine aldolase in L. lactis MG1363, while 95% of the threonine was taken up from the medium. Growth of L. lactis IL1403 was reduced by 25% in the absence of threonine (19). A threonine aldolase gene is not present in the genome of L. lactis MG1363, L. lactis SK11, or L. lactis IL1403, implying that all threonine biosynthesis occurs through aspartate or that a cryptic gene specifies the activity measured by Jensen and coworkers (52). The gene encoding histidinol-phosphate aminotransferase, responsible for the conversion of phenylpyruvate or 4-hydroxy-phenylpyruvate to tyrosine and phenylalanine, respectively, is present in L. lactis MG1363 and L. lactis SK11 but absent in L. lactis IL1403. In the last strain, formation of phenylalanine and tyrosine is predicted to be catalyzed by alternative routes involving histidinol-phosphate aminotransferase and prephenate dehydratase, respectively. Lysine is synthesized from aspartate; clear homologues of the required aminotransferase (DapC) and the epimerase (DapF) are present in L. lactis MG1363 and L. lactis SK11 but absent in L. lactis IL1403. The absence in L. lactis IL1403 is unusual, since the intermediates of lysine biosynthesis are required for cell wall biosynthesis. Alanine racemase (Alr) may provide the necessary building blocks in L. lactis IL1403 and, for that matter, in L. lactis MG1363 and L. lactis SK11.

In silico analysis of the L. lactis MG1363 genome illustrates that nicotinate, pantothenate, biotin, pyridoxine, ubiquinone, and cobalamin cannot be synthesized by this strain, while it remains unclear if thiamine can be synthesized: putative enzymes for synthesis of thiamine phosphate from pyruvate are encoded by the genome of L. lactis MG1363, but a gene encoding thiamine kinase, necessary for the conversion of thiamine phosphate to thiamine, could not be identified. Both riboflavin and folic acid are not essential, as riboflavin/FMN and FAD are synthesized from GTP and folic acid from purine metabolism and phenylalanine biosynthesis (15, 93). Folic acid and vitamin B2 function as cofactors in the synthesis of purines and pyrimidines, and their presence in minimal medium lacking nucleic acids (19, 53) may be essential or stimulatory.

The L. lactis MG1363 genome carries genes encoding all enzymes required for the biosynthesis and metabolism of all purines and pyrimidines.

Gene regulation in L. lactis MG1363.

The genome of L. lactis MG1363 encodes 136 proteins with possible functions in gene regulation. Of these, 10 are unique to L. lactis MG1363 compared to L. lactis IL1403 or L. lactis SK11 (Table (Table5)5) Seven of the eight two-component regulatory systems (2CSs) present in L. lactis MG1363 have orthologs in L. lactis SK11 and L. lactis IL1403. The eighth 2CS (llmg_pseudo_52 and llmg_pseudo_53), which is absent from the L. lactis IL1403 genome, harbors frameshift mutations in both the histidine kinase and response regulator genes in L. lactis MG1363 and is unlikely to be functional in this strain. This 2CS is intact in L. lactis SK11.

Regulators present in L. lactis subsp. cremoris MG1363 but absent in L. lactis subsp. cremoris SK11 or L. lactis subsp. lactis IL1403

The lactococcal cell envelope and secretome.

L. lactis MG1363 is predicted to secrete around 184 (7.5%) proteins. Of these, 39 contain a consensus lipoprotein signal peptide and are expected to be attached to the cytoplasmic membrane, while 38 are (putative) cell wall-attached proteins, as they contain a cell wall-anchoring motif(s) leading to covalent (9 proteins) or noncovalent (29 proteins) attachment. Of the LPXTG motif-containing proteins of L. lactis IL1403 (12 proteins) and L. lactis MG1363 (9 proteins), 4 are common to both strains. L. lactis MG1363, L. lactis SK11, and L. lactis IL1403 all contain two putative sortase homologues, SrtA and SrtC, which are assumed to be distinguishable on the basis of their substrate specificities, namely, cleavage between the threonine and glycine residues in the sequence LP(KENQA)TG(EDS) (SrtA) or (LI)P(KSELAN)TG(GVTSA) (SrtC) (20). Interestingly, the LPXTG motif-containing proteins unique to L. lactis MG1363 are all SrtA targets.

Recently, a gene cluster typically comprising the genes cscABCD, which is present only in a subgroup of gram-positive bacteria, has been described to be possibly related to association with plants (84). Four csc clusters are present in L. lactis MG1363, three of which are also present in L. lactis IL1403. The same four clusters are present in the L. lactis SK11 chromosome, while this strain carries an additional csc operon on one of its plasmids. The cluster extending from llmg_1503 to llmg_1507, which is not present in L. lactis IL1403, is involved in UV sensitivity in L. lactis MG1363 (33).

There has been a growing interest in the food industry in extracellular polysaccharide-producing LAB because of their influence on the textural characteristics of fermented food products. Although 28 putative glycosyltransferases are encoded by the L. lactis MG1363 genome, we could not identify a gene cluster with the typical genetic organization of a lactococcal extracellular polysaccharide operon (21). The glycosyltransferases are more likely to be involved in the decoration of the lactococcal cell wall with cell wall polysaccharides (WPS). In comparison with L. lactis IL1403, L. lactis MG1363 harbors at least 13 unique glycosyltransferase genes, suggesting that distinct differences exist in the compositions of the WPS of these two strains. These unique genes are organized in three clusters of three glycosyltransferase genes and one cluster of four genes (Table (Table6)6) . One cluster displays a distinctly lower GC content (29%) than that of the average L. lactis DNA, indicating an HGT event.

Glycosyltransferase gene clusters present in L. lactis MG1363 but not in IL1403

Recently, it has been demonstrated that the receptor binding proteins of lactococcal phages bind to WPS structures in the bacterial cell wall (31). On the basis of the C-terminal parts of their receptor binding proteins, the bacteriophages investigated could be grouped into L. lactis subsp. lactis- and L. lactis subsp. cremoris-infecting phages (32). Today, our understanding of the function and physiological relevance of WPS for the bacterial cell is very limited, but one of the obvious effects of distinct sets of glycosyltransferases may be the different susceptibilities of the bacteria to certain bacteriophages.


Despite the fact that L. lactis IL1403 carries 41 kb more bacteriophage-related sequences, the L. lactis MG1363 genome is significantly bigger (160 kb) than that of L. lactis IL1403. This is largely due to the presence of mobile genetic elements in L. lactis MG1363, namely, the unique sex factor and a larger number of insertion elements, and the integration hot-spot region. The latter chromosomal section enables L. lactis MG1363 to stably integrate laterally acquired DNA, as is documented by the large number of plasmid-related genes therein. Our findings show that the integration hot spot has played a key role in the evolution of the genome of L. lactis MG1363 and its related strains, influencing the genome content, as well as its overall structure, as demonstrated by the insertion sequence-mediated chromosomal inversion. It allowed L. lactis MG1363, e.g., to stably maintain a functional copy of the opp operon, an operon that is essential for growth in milk, thus contributing to the overall “fitness” of the strain. Forty-seven of the genes present in L. lactis MG1363 but absent in L. lactis IL1403 belong to the COG functional category “carbohydrate metabolism and transport.” Consequently, L. lactis MG1363 displays a greater capability to grow on various sugars, especially those found in plant material. The ability to metabolize plant-derived sugars and the fact that the L. lactis MG1363 genome carries four csc gene clusters associated with the utilization of complex plant polysaccharides point to a plant-associated biological niche for the ancestor of L. lactis MG1363.

The L. lactis MG1363 genome sequence presented here is vital to both applied and fundamental research into LAB and other low-percent GC gram-positive bacteria. This is especially important in view of current and future uses of L. lactis MG1363 in medical research, which today includes the delivery in the gastrointestinal tract of bioactive molecules and, importantly, the therapeutic use of a live L. lactis MG1363 derivative strain in a human trial study. As we now have the genome sequences of both Lactococcus subspecies, L. lactis subsp. cremoris strain MG1363, reported here, and L. lactis subsp. lactis IL1403 (11), and, very recently, L. lactis subsp. cremoris SK11 (66)—and with several genomes of other, plant-derived, strains to come (98)—these are promising times for in-depth comparative-genomics analyses of this economically important bacterial species.


Udo Wegmann, Michael J. Gasson, and Claire Shearman acknowledge funding by a CSG grant from the BBSRC research council. Mary O'Connell-Motherway and Douwe van Sinderen acknowledge the Higher Education Authority and Science Foundation Ireland for financial support (grants PRTLI Cycle 3 and 04/BRB0647, respectively). Carlos Canchaya is supported by an EMBARK postdoctoral fellowship. Aldert Zomer and Girbe Buist acknowledge funding by IOP Genomics grant IGE01018.

Jan Kok, Oscar P. Kuipers, Girbe Buist, and Aldert Zomer acknowledge Sacha van Hijum and Anne de Jong for their support and helpful discussions. Mary O'Connell-Motherway and Douwe van Sinderen acknowledge Sinead Leahy and Gerald Fitzgerald for their support and assistance.

The groups of Michael J. Gasson, Jan Kok/Oscar P. Kuipers, and Douwe van Sinderen contributed equally to the initiation, financing, and execution of these studies.


[down-pointing small open triangle]Published ahead of print on 16 February 2007.


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