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Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. Key words: L. lactis subsp. cremoris, L. lactis subsp. lactis, L. lactis subsp. lactis biovar diacetylactis, dairy starter cultures, PFGE, chromosome size. includes two subspecies (subsp. lactis and subsp. cremoris) Introduction and one biovar (subsp. lactis biovar diacetylactis). The lactis Lactococcus lactis can be isolated from various environ- and cremoris phenotypes are differentiated on the basis of ments but is predominantly studied because of its role as arginine utilization, growth temperature, and salt tolerance, the main constituent of many industrial and artisanal starter whereas the biovar diacetylactis strains have the additional cultures used for the manufacture of a vast range of fer- ability to metabolize citrate. Numerous studies including mented dairy products including fermented milks, sour DNA–DNA hybridization, 16S rRNA, and gene sequence cream, soft and hard cheeses, and lactic casein (Ward analysis have demonstrated the existence of two main gen- et al. 2002). For large-scale commercial production, the otypes. These two genotypic groups have also been called starter cultures used are commonly defined strains, which L. lactis subsp. lactis and L. lactis subsp. cremoris, but unfor- have been selected for their desirable properties especially tunately the genotype and phenotype designations do not in relation to acid production, flavor development, and necessarily correspond, thus introducing a degree of confu- bacteriophage resistance (Limsowtin et al. 1996). sion into the taxonomy of this species (Tailliez et al. 1998). The taxonomy of L. lactis has changed many times but is currently phenotypically based (Schleifer et al. 1985; van An extensive study of 102 L. lactis isolates of dairy and plant Hylckama Vlieg et al. 2006; Rademaker et al. 2007) and origin using various genomic fingerprinting methods and ª The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/ 2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 729 Kelly et al. GBE multilocus sequence analysis has clearly demonstrated much of the detailed biochemical and genetic knowledge that two major lineages exist (Rademaker et al. 2007). of L. lactis is based (Bolotin et al. 2001; Wegmann et al. One of these comprises those strains with a L. lactis subsp. 2007). Both IL1403 and MG1363 belong to L. lactis subsp. cremoris genotype and includes strains with both lactis and lactis phenotypically, but the parent strain of IL1403 cremoris phenotypes. The other comprises those strains (CNRZ157) has a citrate permease plasmid and is able to me- with a L. lactis subsp. lactis genotype that includes strains tabolize citrate placing it with L. lactis subsp. lactis biovar with the lactis phenotype as well as biovar diacetylactis. diacetylactis, whereas MG1363 has a lactis phenotype Comparative genome hybridization (CGH) using 39 L. lactis and a cremoris genotype. The third genome-sequenced strains of dairy or plant origin (Bayjanov et al. 2009) provides strain (SK11) has been used as a cheese starter culture further evidence confirming the unusual taxonomic and belongs to the subgroup of strains with both the subsp. structure in this species. As a result, it is necessary to specify cremoris genotype and phenotype (Makarova et al. 2006). a genotype (cremoris or lactis) and a phenotype (cremoris, The fourth genome is from a L. lactis subsp. lactis strain of diacetylactis, or lactis) to adequately describe individual plant origin (KF147), and a partial sequence is also available strains. for a second plant strain (KF282) (Siezen et al. 2008, 2010). Strains that show both the subsp. cremoris genotype and Comparison of the genomes from plant and dairy isolates phenotype cluster closely together and form a definite sub- has highlighted the differences in gene content that can oc- group that shows limited diversity relative to the other cur between individual strains in the same species (Siezen L. lactis strains examined (Rademaker et al. 2007; Taı¨bi et al. 2008) and shows that sequencing of one representa- et al. 2010). These L. lactis subsp. cremoris strains are fa- tive genome does not give a complete picture of the genetic vored for use as defined strain starter cultures for Cheddar repertoire of a species. Attempts to describe this intraspecies cheese production because they are less likely to cause bit- diversity have led to the terms species genome (Lan and terness and other flavor defects (Heap 1998). The citrate- Reeves 2000) and pangenome (Medini et al. 2005) being metabolizing biovar diacetylactis strains contribute to the defined to cover all the genes present in the characterized flavor and aroma profile of a range of fermented dairy strains of a species. Under both definitions, the genome has products and are also a component of the starter blends a core of genes responsible for the basic aspects of the bi- used for lactic casein manufacture (Heap and Lawrence ology of the species and a set of auxiliary or dispensable 1984). These strains have long been distinguished taxonom- genes that contribute to species diversity and may provide ically (Kempler and McKay 1981), but with the description a selective advantage in certain environments. As a result, of the genus Lactococcus (Schleifer et al. 1985), they were there is much to learn about diversity at the intraspecies incorporated into L. lactis subsp. lactis. Biovar diacetylactis level, and the aim of this work was to examine the chromo- dairy starter strains have been genotypically (Ko¨ hler et al. somal diversity of a large collection of L. lactis strains to 1991; Beimfohr et al. 1997) and phenotypically (Bachmann provide a framework for future comparative genomic work et al. 2009) distinguished from other L. lactis cultures, with this industrially important bacterial species. suggesting that these cultures may also form a separate subgroup. Both L. lactis subspecies have been isolated from a variety Materials and Methods of environmental sources but are most commonly associ- Bacterial Cultures and Growth Media ated with fresh or fermented plant material or with milk and milk products. Strains that show the lactis subspecies In initial screening, 558 L. lactis strains were examined by genotype can be readily isolated from these environments, pulsed-field gel electrophoresis (PFGE) analysis of SmaI di- whereas isolations of cultures with the cremoris subspecies gests of genomic DNA. These included 289 strains with genotype are comparatively rare (Klijn et al. 1995; Salama the cremoris phenotype and genotype (L. lactis subsp. et al. 1995). Attempts to isolate new cremoris or diacetylac- cremoris), 197 strains with the lactis phenotype (L. lactis tis phenotype strains from environmental sources have met subsp. lactis), and 72 strains that were able to metabolize with little success as wild-type strains of both subspecies citrate (L. lactis subsp. lactis biovar diacetylactis). The show the lactis phenotype (Klijn et al. 1995; Salama et al. cremoris phenotype strains and the citrate-metabolizing 1995; Ward et al. 1998). strains were all either used as or isolated from dairy starter Because of its industrial relevance, L. lactis has become cultures. The lactis phenotype strains included cultures with the best studied of the lactic acid bacteria and regarded both cremoris and lactis genotypes and came from diverse as a model organism for this bacterial group, although most origins. These included dairy starter cultures and individual work has been focused on a small number of laboratory strains isolated from raw milk, pasture, soil, plant material, strains of dairy origin. Complete genome sequences have the rumen, and insect gut. Some of these have been been published for four strains. These include the two described elsewhere (table 1), and the strains isolated from plasmid-cured strains (IL1403 and MG1363) on which plant sources were included in the L. lactis diversity study 730 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE Table 1 Sizes of I-CeuI Restriction Fragments, Total Chromosome Size (kb), Genotype, and Origin of Lactococcus lactis Cultures Strain Ce1 Ce2 Ce3 Ce4 Ce5 Ce6 Total (kb) Genotype Origin (Reference) 1. L. lactis subsp. cremoris LW1477 1,340 540 220 75 45 22 2,242 cremoris Dairy starter culture, ScrFI producer KH 1,380 530 240 95 45 38 2,328 cremoris Dairy starter culture (1) AM2 1,440 520 240 75 45 38 2,358 cremoris Dairy starter culture (1,2) 112 1,440 530 250 75 45 22 2,362 cremoris Dairy starter culture LW1494 1,440 530 240 75 45 45 2,375 cremoris Dairy starter culture HP 1,900 65 260 80 45 28 2,378 cremoris Dairy starter culture (1,2) 2188 1,900 80 250 80 45 38 2,393 cremoris Dairy starter culture 166 1,440 530 260 80 45 38 2,393 cremoris Dairy starter culture (2) KF322 1,440 460 340 75 45 38 2,398 cremoris Isolated from mixed strain dairy starter culture 2128 1,440 560 230 90 45 35 2,400 cremoris Dairy starter culture FG2 1,800 380 70 80 45 35 2,410 cremoris Dairy starter culture (3) LW1499 1,440 530 300 75 45 22 2,412 cremoris Dairy starter culture LW1489 1,480 530 260 85 45 22 2,422 cremoris Dairy starter culture AM1 1,520 530 240 75 45 38 2,448 cremoris Dairy starter culture (1,2) SK11 1,520 530 240 75 45 38 2,448 cremoris Dairy starter culture, phage-resistant AM1 (1,2) 448 1,440 560 300 75 45 22 2,442 cremoris Dairy starter culture (2) BK5 1,520 520 250 80 45 38 2,453 cremoris Dairy starter culture (1,2) LW1492 1,480 560 280 90 45 22 2,477 cremoris Dairy starter culture 168 1,520 600 240 90 45 48 2,543 cremoris Dairy starter culture E8 1,520 610 250 75 45 52 2,552 cremoris Dairy starter culture (1,2) 2. L. lactis subsp. lactis biovar diacetylactis CNRZ157 1,440 540 240 80 45 20 2,365 lactis Dairy starter culture (4) LW1807 1,380 570 270 80 45 20 2,365 lactis Dairy starter culture LW1688 1,380 570 270 80 45 20 2,365 lactis Dairy starter culture LW3081 1,440 540 240 95 45 20 2,380 lactis Dairy starter culture D10 1,380 570 300 80 45 20 2,395 lactis Dairy starter culture LW1690 1,380 600 270 80 45 20 2,395 lactis Dairy starter culture LW3079 1,900 115 240 80 45 35 2,415 lactis Isolated from mixed strain dairy starter culture LW1503 1,440 600 240 95 45 20 2,440 lactis Dairy starter culture LW1811 1,440 600 240 95 45 22 2,442 lactis Dairy starter culture DRC3 1,440 600 240 95 45 22 2,442 lactis Dairy starter culture (5) DRC1 1,440 600 270 80 45 20 2,455 lactis Dairy starter culture (2,5) LW3074 1,440 600 270 80 45 20 2,455 lactis Dairy starter culture LW3087 1,440 600 270 95 45 20 2,470 lactis Dairy starter culture LW2333 1,440 650 240 95 45 22 2,492 lactis Dairy starter culture DRC2 1,640 440 270 80 45 20 2,495 lactis Dairy starter culture (2,5) LW1505 1,440 650 240 95 45 35 2,505 lactis Dairy starter culture LW840 1,540 570 270 05 45 20 2,540 lactis Dairy starter culture D6 1,540 600 285 80 45 20 2,570 lactis Dairy starter culture LW3076 1,540 600 285 80 45 20 2,570 lactis Dairy starter culture LW3077 1,540 600 285 80 45 35 2,585 lactis Isolated from mixed strain dairy starter culture 3. L. lactis subsp. lactis (dairy cultures) LW1509 1,520 530 270 80 90 25 2,515 cremoris Dairy starter culture MG1363 1,640 530 240 80 50 25 2,565 cremoris Dairy starter culture, plasmid-free NCDO712 (6) NCDO712 1,640 530 270 80 50 25 2,595 cremoris Dairy starter culture (6) LW1515 1,640 530 290 75 50 20 2,605 cremoris Dairy starter culture GL17 1,640 550 300 110 50 25 2,675 cremoris Dairy starter culture IL1403 1,440 540 240 80 45 20 2,365 cremoris Dairy starter culture, plasmid-free CNRZ157 (4) LW1444 1,480 530 220 90 45 35 2,400 lactis Dairy starter culture BA2 1,440 550 260 90 45 20 2,405 lactis Dairy starter culture LW1448 1,540 500 240 80 50 22 2,432 lactis Dairy starter culture KF324 1,540 500 240 90 45 22 2,437 lactis Dairy starter culture ATCC7962 1,540 530 230 80 45 20 2,445 lactis Dairy starter culture, nisin producer NCDO895 1,440 550 230 170 45 20 2,455 lactis Dairy starter culture, nisin producer LW1512 1,440 550 300 90 45 35 2,460 lactis Dairy starter culture C10 1,520 530 240 90 45 35 2,460 lactis Dairy starter culture Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 731 Kelly et al. GBE Table 1 Continued Strain Ce1 Ce2 Ce3 Ce4 Ce5 Ce6 Total (kb) Genotype Origin (Reference) LW1449 1,440 650 220 120 45 20 2,495 lactis Dairy starter culture ML8 1,540 530 260 90 45 35 2,500 lactis Dairy starter culture (1,2) NCDO1404 1,620 530 200 80 50 20 2,500 lactis Dairy starter culture, nisin producer LW2004 1,540 540 260 100 45 20 2,505 lactis Dairy starter culture (7) LW1514 1,540 600 260 100 45 22 2,567 lactis Dairy starter culture U 1,640 550 260 110 45 28 2,633 lactis Dairy starter culture 4. L. lactis subsp. lactis (wild-type cultures) KW8 1,440 570 270 95 45 22 2,442 cremoris Kaanga wai (fermented corn) (8) KW2 1,590 520 240 80 48 22 2,500 cremoris Kaanga wai (fermented corn) (8) KF343 1,700 540 220 90 48 28 2,626 cremoris Cow’s milk KF355 1,700 500 270 80 48 38 2,636 cremoris Cow’s milk LW1190 1,700 500 270 80 48 38 2,636 cremoris Sheep’s milk (9) KF292 1,590 500 220 90 45 22 2,467 cremoris Soya sprouts, nisin producer (10) 511 1,640 470 220 90 45 20 2,485 lactis Rumen, nisin producer KF196 1,590 520 220 90 45 20 2,485 lactis Radish sprouts, nisin producer (10) KF363 1,640 470 240 90 45 20 2,505 lactis Soil KF201 1,640 470 240 80 45 38 2,513 lactis Sliced mixed vegetables (10) KF146 1,590 580 220 90 45 20 2,545 lactis Alfalfa and radish sprouts, nisin producer (10) LW1320 1,540 630 250 90 45 20 2,575 lactis Goat’s milk (9) KF181 1,590 520 320 80 45 22 2,577 lactis Alfalfa and onion sprouts (10) KF67 1,590 500 340 90 45 20 2,585 lactis Grapefruit juice, nisin producer (10) LW1,180 1,640 500 270 110 45 22 2,587 lactis Sheep’s milk (9) N1 1,760 450 240 75 45 20 2,590 lactis Moth larval midgut, nisin producer (11) KF5 1,590 520 290 120 45 28 2,593 lactis Alfalfa sprouts (10) KF165 1,740 500 220 80 45 20 2,605 lactis Mung bean sprouts, nisin producer (10) KF282 1,740 520 240 80 45 20 2,645 lactis Mustard and cress, nisin producer (10) KF147 1,740 520 270 90 48 20 2,688 lactis Mung bean sprouts (10) References: (1) Lawrence and Pearce (1972); (2) Jarvis and Wolff (1979); (3) Davidson et al. (1996); (4) Chopin et al. (1984); (5) Czulak and Hammond (1954); (6) Gasson (1983); (7) Ward et al. (2004); (8) Kelly et al. (1994); (9) Ward et al. (1998); (10) Kelly et al. (1998a); (11) Shannon et al. (2001). Cultures for which genome sequences are available and their parent strains are shown in bold. (Rademaker et al. 2007). Forty-eight of the lactis phenotype mixed with an equal volume of 2% (w/v) low melt agarose strains were nisin producers. Dairy starter strains were (Bio-Rad Laboratories). Embedded cells were lysed by treat- mainly from the culture collection of the Fonterra Research ment with lysozyme (1 mg/ml in EC buffer, 6 mM Tris–Cl:1 M Center, Palmerston North, New Zealand, with additional NaCl:100 mM ethylenediaminetetraacetic acid [EDTA]:1% cultures obtained from other culture collections or isolated [w/v] sarkosyl, pH 7.6) overnight at 37 C and proteinase from mixed strain dairy starters. K (0.5 mg/ml in lysis buffer, 50 mM Tris–Cl:50 mM EDTA:1% Lactococci were grown at 28 C in M17 broth (Merck) [w/v] sarkosyl, pH 8.0) for 24 h at 50 C. Agarose plugs con- (Terzaghi and Sandine 1975) supplemented with 0.5% w/ taining intact genomic DNA were washed three times with v glucose for growth of the plasmid-free dairy strains Tris–EDTA buffer (10 mM Tris–Cl:1 mM EDTA, pH 8.0) before (IL1403 and MG1363) and the wild-type strains. The stock storage in 10 mM Tris–Cl:100 mM EDTA (pH 8.0) at 4 C. cultures were maintained at 85 C in M17 broth supple- DNA embedded in agarose was digested for 16 h with mented with 20% (v/v) glycerol. Tests for arginine and cit- 1.0 U of ApaI, SmaI, or I-CeuI (New England Biolabs) in rate metabolism were used to confirm the phenotype of the 100 ll of restriction enzyme buffer, loaded into wells of strains, and strain genotypes were determined using the 1% (w/v) agarose gels (pulsed-field certified agarose, Bio- polymerase chain reaction (PCR) primers and conditions Rad), and run at 200 V for 20 h at 14 C in 0.5 Tris–borate described previously (Ward et al. 1998). buffer (Sambrook et al. 1989) using a CHEF DR III PFGE ap- paratus and model 1000 mini chiller (Bio-Rad). Pulse times used were 1–30 s for ApaIor SmaI and 5–60 s for I-CeuI. To Pulsed-Field Gel Electrophoresis determine the size of the largest fragments from I-CeuI di- Cells were harvested from 1.5 ml of an overnight culture by gests, gels were prepared from 0.8% chromosomal grade centrifugation (10,000  g, 10 min), washed twice with 1 M agarose (Bio-Rad) and run with the pulse time ramped be- NaCl:10 mM Tris–Cl (pH 7.6), and 300 ll aliquots were tween 150 and 400 s. Fragments smaller than 100 kb were 732 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE also resolved and measured using a FIGE Mapper electro- fragment by its average size before subjecting the values phoresis system (Bio-Rad). DNA was visualized by staining to pairwise F tests to test the equality of variances (Sokal with ethidium bromide and the image captured using and Rohlf 1981). a Gel Doc 1000 system (Eastman Kodak). Partial digestion with I-CeuI was used to establish the rrn chromosomal skel- Genomic Analysis eton as described by Liu et al. (1999). Genomic DNA pre- Genomic data for the four publicly available and completed pared from L. lactis subsp. lactis IL1403 and phage L. lactis genome sequences (GenBank accession numbers lambda concatamers or Saccharomyces cerevisiae pulsed- AM406671, CP000425, AE005176, and CP001834) were field gel (PFG) markers (New England Biolabs) were used downloaded from the National Center for Biotechnology In- as size standards. formation (NCBI) Web site (http://www.ncbi.nlm.nih.gov). Genome alignments of chromosomal sequences were per- Plasmid Analysis formed using Mauve software (Version 2.3.1) (Darling et al. Plasmid DNA was isolated by the method of Anderson and 2004). McKay (1983) and the size of individual plasmid bands de- termined following electrophoresis in 0.7% agarose gels in Phylogenetic Analysis Tris–acetate buffer (Sambrook et al. 1989) for 3 h at 4 V/cm Phylogenetic relationships were determined using a super- and staining as above. Strains were also screened for the tree approach. Protein coding gene sets for the four L. lactis presence of large linear plasmids by running undigested strains, three Streptococcus thermophilus strains (GenBank genomic DNA in PFGs as described above. accession numbers CP000023, CP000024, and CP000419), five Lactobacillus species (GenBank accession numbers DNA Methodology AL935263, CR936503, CR954253, CP000416, and To determine the relatedness of citrate-metabolizing strains CP000517), as well as two outgroups (Listeria species; Gen- based on the presence and chromosomal location of their Bank accession numbers AL591824 and AL592022) were prophage, the primers described by Chopin et al. (2001) downloaded from the NCBI Web site. The S. thermophilus, were used in PCR reactions to amplify the chromosome– L. delbrueckii subsp. bulgaricus and L. helveticus genomes prophage junction regions from IL1403 for use as probes were included for comparison because of their use as dairy for PFGs. Hybridizations were done using the North2South starter cultures. For a full description of the supertree meth- Direct HRP Labeling and Detection Kit (Pierce) using the con- odology used please refer to Fitzpatrick et al. (2006). ditions recommended by the manufacturer. To determine the presence of plasmids encoding citrate permease (citP), Results and Discussion plasmid gels were hybridized with a probe to the gene for citrate permease that was constructed by PCR amplification PFGE Patterns of L. lactis Subsp. cremoris Dairy from strain LW1503 using primers described by Klijn et al. Starter Cultures (1995). To determine if cultures, which failed to cut with PFGE analysis of SmaI digests of genomic DNA from the 289 SmaI, contained the ScrFI R/M system, PFGs were hybridized L. lactis subsp. cremoris strains showed that 230 (80%) with a probe specific for the scrFIAM methylase, which was could be linked into 12 groups of related strains. Represen- constructed by PCR amplification from strain UC503 using tatives of these groups are included in table 1. Three (E8, HP, primers described by Szatmari et al. (2006). and SK11) of the four strains compared by Taı¨bi et al. (2010) were representative of groups of strains, whereas the fourth Statistical Analysis (Wg2) had a unique PFGE profile. The observation that many Differences in average chromosome size among subspecific strains were related was not surprising because studies groups of L. lactis from each origin (dairy and wild type) based on phage host range had previously indicated that were tested using a one-way analysis of variance and fitting a relatively small number of significantly different cremoris a single factor comprising each genotype–phenotype origin starter strains exist (Lawrence and Pearce 1972; Lawrence combination. To test whether regions of the chromosome et al. 1978). The result is that many cremoris strains of di- differ in degree of variability, we compared the variances verse origin are unknowingly related and an example of this in the lengths of different chromosomal regions based on is shown in figure 1 where strains related to SK11 are com- I-CeuI fragments. The variance in the length of a given re- pared. SK11 was isolated in New Zealand as a phage- gion is expected to increase linearly with fragment size as- resistant derivative of strain AM1 (DRI 1962), which had suming that the number of insertions, duplications, and been obtained from Professor Collins from University of deletions per fragment increase with fragment length. California Davis and originally named LT8 (DRI 1960). Two Therefore, the variances in the sizes of the I-CeuI fragments other cultures were introduced to New Zealand at the same were standardized by dividing the variance of a given time, AM2 (FC4) and AM3 (4B) (DRI 1960; Collins 1961), Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 733 Kelly et al. GBE FIG.1.—PFGE patterns of SmaI-digested genomic DNA from Lactococcus lactis subsp. cremoris SK11 and related strains. The mixed strain starter isolates (MSS1–4) were isolated from various mixed strain dairy starter cultures. and all three had been isolated from commercial mixed fragment of chromosomal DNA from these strains hybrid- strain cultures. AM1 and AM2 were regarded as slower ized to a probe for the scrFIAM methylase gene. When starters and were found to make consistently good-flavored the histories of these strains were examined, most were Cheddar cheese (Martley and Lawrence 1972). Figure 1 found to have been isolated from mixed strain starter shows that AM1 and SK11 have the same PFGE SmaI digest cultures. pattern but that several other strains are similar. These include US3 and R6, which had both been in use as defined PFGE Patterns of Citrate-Utilizing Dairy Starter strain starters since the early 1950s (DRI 1951), strain 134 Cultures (originally described as a phage-resistant derivative of AM2, Limsowtin and Terzaghi 1976), and several isolates The PFGE patterns of SmaI-digested genomic DNA from 72 from mixed strain starter cultures. Cit strains showed that most strains had several bands in The predicted PFGE pattern from the sequenced SK11 common, and representatives are shown in figure 2A. The strain differs from that for SK11 shown in figure 1 but relatedness of these strains is supported by the presence of matches to that of strain 134. The major PFGE bands are prophage, and these were detected using probes to the very similar in these strains with the main difference being chromosome–prophage junctions in strain IL1403 (Chopin a deletion of ;20 kb from one of the largest PFGE bands et al. 2001). An example is shown in figure 2B where (a 293-kb doublet which separates into 293- and 273-kb the left hand junction fragment between the IL1403 bands). When the 273-kb SmaI fragment from the SK11 chromosome and the bIL309 prophage hybridizes strongly genome is compared with the corresponding region from with all the Cit strains. Six prophages have been identified MG1363, the only major difference is the presence of on the IL1403 chromosome (Bolotin et al. 2001), and junc- a 19-kb prophage sequence (MG-1) in MG1363 (Ventura tion fragments for five of these (bIL285, bIL309, bIL310, et al. 2007). A prophage (bIL310) is integrated in the same bIL311, and bIL312) could be detected in all strains. The genomic region in IL1403, and there is strong homology and sixth prophage (bIL286) was found only in IL1403 and synteny between bIL310 and MG-1. Consequently, it is likely CNRZ157 and may be less stable than the others as we iso- that a similar prophage has been lost from the sequenced lated a derivative of CNRZ157, which was spontaneously SK11 strain. cured of this prophage. In a study using minisatellite poly- DNA from 13 cremoris strains failed to cut with the morphism to distinguish closely related L. lactis strains, a se- enzyme SmaI, indicative of the presence of a restriction/ quence from within the bIL286 prophage was used as modification system operating in these strains. Of the a strain-specific minisatellite. This sequence was only found known lactococcal R/M systems, only the ScrFI methylase in the genome of IL1403 and not in nine other L. lactis ´ ´ potentially blocks the SmaI recognition site (Szatmari strains (Quenee et al. 2005). The observation by McGrath et al. 2006). Unlike most lactococcal R/M systems, ScrFI is et al. (2002) that the genomes of IL1403 and the citrate-uti- chromosomally encoded and a single 75-kb ApaI digest lizing strains IL409 (DRC1) and F7/2 contain identical 734 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.2.—(A) PFGE patterns of SmaI digests of genomic DNA from Cit Lactococcus lactis strains. (B) Southern blot of (A) hybridized with a PCR- amplified product of the left hand junction between the IL1403 chromosome and the prophage bIL309. prophage-encoded bacteriophage resistance genes is in for the synthesis of active citrate lyase, is missing from the agreement with our observation that these strains are other sequenced L. lactis strains (Wegmann et al. 2007; closely related and harbor related prophage. The gene Siezen et al. 2008). identified (sie ) showed 100% amino acid identity with IL409 orf2 of bIL309. PFGE Patterns of L. lactis Subsp. lactis Cultures The plasmid-free strain IL1403 was originally derived from the citrate-utilizing strain L. lactis subsp. lactis biovar PFGE was also used to compare 197 strains with the lactis diacetylactis CNRZ157 (IL594) following protoplast-induced phenotype (L. lactis subsp. lactis) made up of 110 dairy curing (Chopin et al. 1984; Bourel et al. 1996). Citrate uti- starter strains and 87 isolated from various sources. With lization requires citrate permease to transport citrate into the exception of a group of cultures used in lactic casein the cell and citrate lyase to initiate citrate breakdown (Drider manufacture that have been described previously (Ward et al. 2004). In lactococci, these activities are genetically et al. 2004), the L. lactis subsp. lactis strains showed much separate with citrate permease being plasmid encoded on more diversity than the other groups and the majority of an 8-kb plasmid (Kempler and McKay 1981), whereas strains gave unique PFGE patterns. Two of the sequenced L. lactis strains have the lactis phe- citrate lyase and other genes involved in citrate breakdown notype, MG1363 (Wegmann et al. 2007) and KF147 (Siezen are chromosomal (Bolotin et al. 2001). The distribution of et al. 2010). MG1363 was made plasmid free by UV treat- the chromosomal citrate-utilizing genes among different ment and protoplast-induced curing (Gasson 1983) and lactococcal strains is not known, but citrate lyase activity belongs to a group of related strains, which includes was not present in cell-free extracts of 24 dairy lactococcal NCDO712, C2, ML3, LM0230, and 952 (Davies et al. strains (Harvey and Collins 1961), and the gene cluster 1981; Lucey et al. 1993; Le Bourgeois et al. 2000). All have mae-maeP-citRCDEFXG, which includes the genes required Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 735 Kelly et al. GBE techniques developed for MG1363 have been difficult to transfer to dairy starter strains (Johansen 2003). KF147 is one of a group of L. lactis cultures isolated from minimally processed fruit and vegetable products (Kelly et al. 1998a) and has several novel properties not found in dairy starters. Selection of Bacterial Strains for Chromosomal Analysis Based on the SmaI PFGE patterns, 80 strains (table 1) were chosen as representative of the L. lactis species. These in- cluded 20 strains belonging to each of four groups chosen for comparison because of their origin or because of their use for different purposes in the dairy industry and also in- cluded the four strains whose genome sequence has been determined. These are 1) L. lactis subsp. cremoris dairy starter cultures, 2) L. lactis subsp. lactis biovar diacetylactis dairy starter cultures, 3) L. lactis subsp. lactis dairy starter cultures, and 4) wild-type L. lactis subsp. lactis strains. Based on PFGE patterns and strain history data, the cultures se- lected were believed to be unrelated to one another except for SK11, which is a bacteriophage-resistant derivative of L. lactis subsp. cremoris AM1, and IL1403 and MG1363, which are plasmid-free derivatives of L. lactis subsp. lactis biovar diacetylactis CNRZ157 and L. lactis subsp. lactis NCDO712, respectively. Characteristics of the L. lactis Chromosome and Chromosomal Rearrangements Chromosomal mapping has shown that L. lactis has a circular chromosome (fig. 3A) with six ribosomal operons that are transcribed divergently from the origin of chromosomal rep- lication (Davidson et al. 1996). Whereas it is expected that in any one strain, all six 16S rRNA copies will have the same nucleotide sequence, work by Pillidge et al. (2009) has high- lighted an additional level of complexity. A small number of L. lactis subsp. cremoris strains, some of which show PFGE patterns similar to SK11, appear to be genotypic hybrids and have both cremoris-like and lactis-like 16S rRNA types in their genome. Some L. lactis subsp. cremoris strains contain plasmids with a lactis-like 16S rRNA pseudogene, and it is proposed that these chimeric strains are the result of homol- FIG.3.—(A) Locations of I-CeuI recognition sites on the Lactococ- ogous recombination between the pseudogene and the cus lactis IL1403 chromosome. I-CeuI cleaves at sites within the six 23S corresponding chromosome gene (Pillidge et al. 2009). rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and Because plasmid DNA contributes to the PFGE patterns the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. (B) PFGE resulting from SmaI digests (Ward et al. 1993), the homing patterns of genomic DNA from L. lactis strains. endonuclease I-CeuI, which cuts only within the 23S rRNA gene (Liu et al. 1999), was used to produce a PFGE pattern a lactis phenotype but a cremoris genotype. NCDO712, C2, based on chromosomal DNA alone. Macrorestriction pat- and MG1363 have a chromosomally integrated sex factor terns produced by PFGE of I-CeuI digests of genomic not found in other lactococcal strains. Strains belonging DNA (fig. 3B) provide information on chromosomal size, to this group have been widely used as conjugation recip- the number and position of rRNA operons, and an indication ients, and large DNA fragments are known to be capable of chromosomal rearrangements or insertions and dele- of integration at several sites on the chromosome, but tions. All the 80 L. lactis strains examined gave six fragments 736 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.4.—Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome’s center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I-CeuI cut sites that indicate the locations of the 23S rRNA genes are shown above each strain. when their genomic DNA was digested with I-CeuI, indicat- Obis et al. (2001). The L. lactis subsp. lactis biovar diacety- ing that the copy number of the rRNA genes is conserved in lactis strain LW3079 isolated from a mixed strain starter cul- this species (table 1). These fragments were designated as ture has a rearrangement similar to that found in the HP-like Ce1–Ce6 following the nomenclature used by Le Bourgeois strains. These chromosomal changes have no observable et al. (1992). Partial digests were used to determine the or- effect on cell growth, morphology, or phenotype. der of the I-CeuI fragments, and for most strains, the relative With the availability of four different L. lactis genome se- size and order of the various fragments are the same as in quences, it is possible to get an indication of the larger scale IL1403, suggesting that chromosomal structure is conserved events that shape the lactococcal genome. Figure 4 com- in most cases. A minority of strains showed chromosomal pares the four genomes and shows that overall there is rearrangements, and these were of two types typified by a high degree of conservation. A large inversion involving strains HP and FG2. From the 289 cremoris strains investi- approximately half the chromosome has been described gated, 21 had PFGE patterns similar to HP and 15 had pat- in MG1363 (Daveran-Mingot et al. 1998), although this terns similar to FG2. The rearrangement in FG2 has been occurs within the Ce1 fragment and does not result in described previously during chromosome mapping studies a change to the I-CeuI pattern (fig. 4) or alteration in (Davidson et al. 1996). Curiously, strains HP and FG2 both chromosomal symmetry. Major genome insertions in these carry plasmids that specify the same uncommon type of cell strains are highlighted in figure 4 and are predominantly envelope proteinase (lactocepin) linked to a partially deleted associated with prophages, the integration of plasmid copy of abiB(Christensson et al. 2001). These two strains genes, polysaccharide biosynthesis, or the ability to metab- also cluster together and separate from strains SK11 and olize plant-derived carbohydrates. Prophages are an impor- AM2 in the CGH study reported by Bayjanov et al. tant feature, as phage resistance has been a major driver for (2009), and both HP and Z8 (a culture with the same atypical strain selection programs for dairy cultures, and phage chal- PFGE pattern as FG2) were the only L. lactis strains shown to lenge has been a continual selective stress in their environ- lack the busA operon in the osmolality study described by ment. Strain-specific genes of significance include the Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 737 Kelly et al. GBE Lactobacillus sakei (23K) Lactobacillus brevis (ATCC 367) Lactobacillus plantarum (WCFS1) Lactobacillus delbrueckii subsp. bulgaricus (ATCC 11842) Lactobacillus helveticus (DPC 4571) Streptococcus thermophilus (LMD-9) Streptococcus thermophilus (LMG 18311) Streptococcus thermophilus (CNRZ 1066) Lactococcus lactis subsp. lactis (MG1363) Lactococcus lactis subsp. cremoris (SK11) Lactococcus lactis subsp. lactis (KF147) Lactococcus lactis subsp. lactis (IL1403) Listeria monocytogenes (EGD-e) Listeria innocua (Clip11262) FIG.5.—maximum representation with parsimony (MRP) supertree for Lactococcus lactis and other lactic acid bacteria derived from 1,160 single gene families. Listeria species were selected as an outgroup. Bootstrap scores for all nodes are displayed. malate–citrate metabolism genes in IL1403 and the inte- gene families with the results shown in figure 5. This analysis grated sex factor in MG1363. KF147 also contains genes strongly supports the conclusion from the study by for nisin biosynthesis, although this strain does not produce Rademaker et al. (2007) that two main lineages exist in nisin (Kelly et al. 1998a; Siezen et al. 2008), and for nonri- L. lactis. These correspond to the two genotypes. Strains bosomal peptide and polyketide synthesis. The large inser- with a L. lactis subsp. cremoris genotype include strains with tion in the Ce3 fragment in KF147 (fig. 4) is a chromosomally both lactis (MG1363) and cremoris (SK11) phenotypes, integrated conjugative element that encodes the ability to whereas strains with the L. lactis subsp. lactis genotype metabolize alpha-galactosides such as melibiose and raffi- includes strains with the both diacetylactis (CNRZ157, the nose. Transfer of this element to a derivative of MG1363 parent strain of IL1403) and lactis (KF147) phenotypes. From and its integration at two different chromosomal sites have the supertree results, it appears that a similar situation may been described previously (Kelly et al. 1998b), and similar exist in S. thermophilus, but this awaits further study. conjugative elements were found in several L. lactis strains Differences in Chromosome Size between Sub- of plant origin. groups of L. lactis Phylogenetic Relationship between L. lactis Strains The average chromosome size of the 80 strains of L. lactis The availability of four complete genome sequences cover- was 2,483 kb, with the chromosomes of individual strains ing most genotype/phenotype combinations makes it pos- ranging in size from 2,242 to 2,688 kb. This variation in sible to produce a phylogeny truly representative of the chromosomal length (;20% of the size of the smallest entire genome. Supertree methods (Fitzpatrick et al. chromosome) is similar to that found in natural isolates of 2006) were used to derive phylogenies from 1160 single Escherichia coli (Bergthorsson and Ochman 1998). Table 2 Table 2 Mean Chromosome Lengths (kb) of Lactococcus lactis Strains Belonging to the Various Groups Genotype cremoris lactis lactis cremoris lactis cremoris Phenotype Cremoris Diacetylactis Lactis Lactis Lactis Lactis Source Dairy Dairy Dairy Wild type Wild type Dairy a b b c c c Mean chromosome length (kb) 2,412 2,457 2,471 2,568 2,568 2,591 Number of strains 20 20 15 5 15 5 Treatments that share the same letter are not significantly different at P , 0.05. 738 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.6.—(A) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis.(B) Relationship between variances of different I-CeuI fragments standardized by their average size and the average size of the corresponding fragments. shows the comparison between the mean chromosomal Variation among Chromosomal Regions lengths of L. lactis strains from different genotype– All the I-CeuI fragments show some degree of length phenotype origin combinations. The origin of the strains variation (table 1), although, except for the strains that show has the greatest influence on chromosome length with dairy chromosomal rearrangements, the sizes of the individual strains having smaller chromosomes than the wild-type fragments do not overlap. To test whether some strains. This smaller chromosome size may be the result chromosomal regions are more variable than others, we of a process of reductive genome evolution as a conse- compared the standardized variances for each fragment quence of the adaptation to growth in milk. Among the across strains in pairwise F tests. The four strains that dairy strains, those with the cremoris genotype and pheno- showed major chromosomal rearrangements (HP, 2188, type are significantly different and have the smallest FG2, and LW3079) were not included so this comparison chromosomes, whereas there is no significant difference was based on 76 strains. There were no significant differen- between strains with the diacetylactis and lactis phenotypes. ces when most of the fragments were compared against In this analysis, the five cremoris genotype/lactis phenotype each other; however, the largest fragment (Ce1) was signif- dairy strains grouped with the wild-type strains. This could icantly more variable (P  0.005), and it can be seen from be related to the small sample size or could indicate that figure 4 that the majority of chromosomal insertions are they are not as strongly adapted to the dairy environment found in this region. Ce5 was significantly less variable and are closer to strains of plant origin. That MG1363 has (P  0.001) than the other fragments. A feature of the size much greater ability than either IL1403 or SK11 to grow on variation in the I-CeuI fragments is a correlation between plant-derived carbohydrates (Wegmann et al. 2007) supports the latter option. the two largest fragments (fig. 6A). This suggests that Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 739 Kelly et al. GBE FIG.7.—Alignment of the Ce6 region of the chromosomes of Lactococcus lactis IL1403, KF147, MG1363, and SK11 and identification of the genes present. Insertions common to the cremoris strains MG1363 and SK11 are shown in red, and the genes found only in SK11 are shown in blue. The fusA pseudogene in SK11 is shown in mauve. chromosomal insertions and deletions in these regions are SK11 genome sequence, the fusA homologue (LACR_2595) not entirely independent events and that maintenance of is identified as a pseudogene. chromosomal symmetry may be an important consideration. The main difference in the genome sequences for this There is a significant correlation between the I-CeuI frag- region is a 15-kb insertion in SK11 between the ribosomal ment size and the standardized variance of each fragment protein S9 gene (rpsI) and the 16S rRNA gene. This insertion (fig. 6B) with the two smallest fragments showing the great- contains two genes with high homology to type III est departure from this relationship. The smallest fragment restriction–modification systems found in other lactic acid (Ce6) shows greater variance than expected, whereas the bacteria, several hypothetical proteins, an integrase, and variance in size of the second smallest fragment (Ce5) is less genes related to plasmid replication. It is probable that this than for any of the other fragments. The size of Ce5 is insertion increases the phage resistance of SK11, and it may strongly conserved (45–50 kb), and we have found only have been acquired by horizontal gene transfer, and one strain (LW1509) that contains a large insertion of subsequent chromosomal integration, of a small plasmid. DNA in this region. It is apparent that many dairy starter strains have an insertion of similar size in the Ce6 region (table 1), and it will be of interest to determine if similar genes are found Gene Content of Ce6 in other strains and whether they have an influence on cell Because Ce6 exhibits larger variation than expected for growth rate given their location close to the chromosomal a fragment of this size, we examined the genes present origin. in this region from the four available L. lactis genome se- quences (fig. 7). MG1363 and SK11 (both L. lactis subsp. Contribution of Extrachromosomal Elements to cremoris genotype) show two common insertions relative Genome Size to IL1403 and KF147. These are of genes of unknown func- tion between purR and fusA and between rpsL and dacA. In Many technologically important properties (lactose metab- IL1403, 5 of the 14 genes in this region are predicted to be olism, lactocepin proteinase, citrate permease, and bacte- highly expressed (Karlin et al. 2004). These are fusA riophage resistance) are plasmid encoded in L. lactis (predicted to be the most highly expressed gene in the strains used as dairy cultures and can be exchanged be- IL1403 genome) and four genes encoding ribosomal tween strains by conjugation and between replicons by proteins (rpsI, rplM, rpsG, and rpsL). Curiously, in the insertion sequence elements. Examination of plasmid gels 740 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.8.—(A) Plasmid profile of Cit Lactococcus lactis strains. Lane 1, Invitrogen supercoiled DNA ladder; lane 2, Invitrogen 1 kb DNA ladder. (B) Southern blot of (A) hybridized with a PCR-amplified product of the citP gene. Where two bands are present, they represent open and closed circular forms of the same plasmid. for 150 dairy starter cultures (90 L. lactis subsp. cremoris,30 instability has previously been reported for CNRZ157 L. lactis subsp. lactis, and 30 L. lactis subsp. lactis biovar di- (Chopin et al. 1984). acetylactis) gave an average of seven plasmids per strain (range 2–14) with up to 200 kb of plasmid DNA. By contrast, Origin of Defined Strain Dairy Starter Cultures in plant strains, the average was ,2 plasmids per strain (range 0–4). It was notable that small plasmids (,10 kb) Most of the L. lactis strains used in this study were dairy were prevalent in dairy cultures but rare in the plant strains. starter cultures and are representative of the cultures em- Intact genomic DNA was run on PFGs but showed no evi- ployed by the dairy industry since the concept of using de- dence of large linear plasmids in these strains. fined strain starters was developed in the 1930s (Limsowtin Plasmid profiles for 20 Cit strains are shown in figure 8A et al. 1996). During this period, the best cultures were freely and show that these strains have acquired a range of shared between laboratories, and this coupled with the re- different extrachromosomal elements. A plasmid-encoded peated isolation of individual strains with particular charac- citrate permease is required for citrate utilization in L. lactis, teristics from commercial mixed strain starters, and the and to determine its location, a Southern blot of the plasmid development of bacteriophage-insensitive cultures has re- gel was probed with a PCR-amplified product of the citP sulted in many closely related strains coexisting. Conse- gene (fig. 8B). The citrate permease plasmid is conserved quently, the relationship between strains is generally not in size (8 kb) in most strains, but three strains (D6, known, although it has been speculated that the pool of LW3076, and LW1503) have an enlarged citrate plasmid strains with certain properties is small (Lawrence et al. of ;15 kb. We observed that the plasmid complement 1978). Because of their differing histories of industrial use, even closely related strains may differ in some of their can be very unstable in some strains, and this spontaneous Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 741 Kelly et al. GBE important characteristics and in their plasmid complement S. thermophilus (Hols et al. 2005) dairy cultures. We con- (Ward et al. 2004). clude that dairy starter cultures generally and especially The aim of this work was to use PFGE to gain a measure strains with cremoris and diacetylactis phenotypes comprise of the chromosomal diversity present in a large collection of a specialized group of L. lactis strains. This is in accordance L. lactis cultures including both plant and wild-type strains, with the results from the recent studies of L. lactis with emphasis on the L. lactis strains with the cremoris and (Rademaker et al. 2007; Bayjanov et al. 2009) and fits with diacetylactis phenotypes that are of particular importance to knowledge of the origin of industrially used dairy starter cul- the dairy industry. The diacetylactis phenotype is unusual in tures. Consequently, the world’s dairy industry is based on that it depends on the inheritance of both plasmid and chro- the same small group of good starter strains, and these cul- mosomal components for citrate to be transported into the tures have transitioned from free-living organisms associ- cell and metabolized. The chromosomal genes are not ated with plant material. We hypothesize that these found in the other sequenced strains and show homology specialized dairy starters are no longer fit to survive outside with the plasmid-encoded citrate genes from Leuconostoc the dairy environment and have evolved to become essential and Weissella species isolated from the dairy environment. components of industrial processes. Whereas wild-type Therefore, it can be hypothesized that the diacetylactis strains may have genes that can be used to enhance the me- strains used as defined strain dairy starters represent a single tabolism of dairy strains (van Hylckama Vlieg et al. 2006), it is lineage within L. lactis in which both chromosomal unlikely that there is an environmental source for new dairy and plasmid elements are maintained and essential for an starter cultures similar to those currently in use. industrially significant phenotype. It should be noted that This analysis together with other studies (Nomura et al. this observation is restricted to the Cit strains used as de- 2006; Rademaker et al. 2007; Siezen et al. 2008; Bachmann fined strain dairy starter cultures. Wild-type L. lactis may et al. 2009; Bayjanov et al. 2009; Liu et al. 2010) begins to differ in their citrate metabolism, and strains with illustrate the genomic and phenotypic diversity present a 23-kb plasmid-encoding genes that match those found within L. lactis. Currently, too few genome sequences are in citrate-metabolizing leuconostocs have been isolated available to delineate sets of core and auxillary genes and from Algerian dromedary’s milk (Drici et al. 2010). describe the pangenome of L. lactis, but in the future, it will The cremoris phenotype is rather more complex to deter- be interesting to examine strains from other environments in mine because it is measured as negative attributes including more detail and to further define the genes necessary to the inability to metabolize arginine and inability to grow at make a good dairy starter culture. higher temperatures or at higher salt levels. From investiga- tions of the genome sequences, it appears that these prop- Funding erties have arisen through the accumulation of mutations and that they are a response to the nutrient rich milk envi- New Zealand Foundation for Research, Science, and ronment where certain gene functions are not longer re- Technology (Contract C10X0239). quired. The arginine deiminase–negative phenotype of SK11 correlates with a single base pair deletion (Wegmann Acknowledgments et al. 2007), whereas various defects including the absence of the busA operon have been shown to influence salt tol- We thank Zaneta Park for assistance with the statistical anal- erance (Obis et al. 2001). The taxonomy of L. lactis is cur- ysis and Dr David Fitzpatrick from the Genome Evolution rently based on phenotype, but as the genomic basis for Laboratory, National University of Ireland, Maynooth, Ire- these phenotypic differences is elucidated, a case could land, for assistance with the supertree phylogenetic analysis. be made for review and use of genotypic data to define We also wish to thank Howard Heap and the members of the two subspecies. the Microbial Fermentation Unit, Fonterra, Palmerston Comparison of our data with the analysis of the genome- North, for their support and advice on lactococcal dairy sequenced L. lactis strains strongly supports a plant- starter cultures. associated origin for dairy starter strains. A significant proportion of the KF147 genome is devoted to genes in- Literature Cited volved in the degradation and metabolism of plant-derived Anderson DG, McKay LL. 1983. 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J Bacteriol. 189:3256–3270. Siezen RJ, et al. 2010. Complete genome sequence of Lactococcus lactis subsp. lactis KF147, a plant-associated lactic acid bacterium. Associate editor: Takashi Gojobori J Bacteriol. 192:2649–2650. 744 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology and Evolution Oxford University Press

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

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Oxford University Press
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The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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1759-6653
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10.1093/gbe/evq056
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Abstract

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. Key words: L. lactis subsp. cremoris, L. lactis subsp. lactis, L. lactis subsp. lactis biovar diacetylactis, dairy starter cultures, PFGE, chromosome size. includes two subspecies (subsp. lactis and subsp. cremoris) Introduction and one biovar (subsp. lactis biovar diacetylactis). The lactis Lactococcus lactis can be isolated from various environ- and cremoris phenotypes are differentiated on the basis of ments but is predominantly studied because of its role as arginine utilization, growth temperature, and salt tolerance, the main constituent of many industrial and artisanal starter whereas the biovar diacetylactis strains have the additional cultures used for the manufacture of a vast range of fer- ability to metabolize citrate. Numerous studies including mented dairy products including fermented milks, sour DNA–DNA hybridization, 16S rRNA, and gene sequence cream, soft and hard cheeses, and lactic casein (Ward analysis have demonstrated the existence of two main gen- et al. 2002). For large-scale commercial production, the otypes. These two genotypic groups have also been called starter cultures used are commonly defined strains, which L. lactis subsp. lactis and L. lactis subsp. cremoris, but unfor- have been selected for their desirable properties especially tunately the genotype and phenotype designations do not in relation to acid production, flavor development, and necessarily correspond, thus introducing a degree of confu- bacteriophage resistance (Limsowtin et al. 1996). sion into the taxonomy of this species (Tailliez et al. 1998). The taxonomy of L. lactis has changed many times but is currently phenotypically based (Schleifer et al. 1985; van An extensive study of 102 L. lactis isolates of dairy and plant Hylckama Vlieg et al. 2006; Rademaker et al. 2007) and origin using various genomic fingerprinting methods and ª The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/ 2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 729 Kelly et al. GBE multilocus sequence analysis has clearly demonstrated much of the detailed biochemical and genetic knowledge that two major lineages exist (Rademaker et al. 2007). of L. lactis is based (Bolotin et al. 2001; Wegmann et al. One of these comprises those strains with a L. lactis subsp. 2007). Both IL1403 and MG1363 belong to L. lactis subsp. cremoris genotype and includes strains with both lactis and lactis phenotypically, but the parent strain of IL1403 cremoris phenotypes. The other comprises those strains (CNRZ157) has a citrate permease plasmid and is able to me- with a L. lactis subsp. lactis genotype that includes strains tabolize citrate placing it with L. lactis subsp. lactis biovar with the lactis phenotype as well as biovar diacetylactis. diacetylactis, whereas MG1363 has a lactis phenotype Comparative genome hybridization (CGH) using 39 L. lactis and a cremoris genotype. The third genome-sequenced strains of dairy or plant origin (Bayjanov et al. 2009) provides strain (SK11) has been used as a cheese starter culture further evidence confirming the unusual taxonomic and belongs to the subgroup of strains with both the subsp. structure in this species. As a result, it is necessary to specify cremoris genotype and phenotype (Makarova et al. 2006). a genotype (cremoris or lactis) and a phenotype (cremoris, The fourth genome is from a L. lactis subsp. lactis strain of diacetylactis, or lactis) to adequately describe individual plant origin (KF147), and a partial sequence is also available strains. for a second plant strain (KF282) (Siezen et al. 2008, 2010). Strains that show both the subsp. cremoris genotype and Comparison of the genomes from plant and dairy isolates phenotype cluster closely together and form a definite sub- has highlighted the differences in gene content that can oc- group that shows limited diversity relative to the other cur between individual strains in the same species (Siezen L. lactis strains examined (Rademaker et al. 2007; Taı¨bi et al. 2008) and shows that sequencing of one representa- et al. 2010). These L. lactis subsp. cremoris strains are fa- tive genome does not give a complete picture of the genetic vored for use as defined strain starter cultures for Cheddar repertoire of a species. Attempts to describe this intraspecies cheese production because they are less likely to cause bit- diversity have led to the terms species genome (Lan and terness and other flavor defects (Heap 1998). The citrate- Reeves 2000) and pangenome (Medini et al. 2005) being metabolizing biovar diacetylactis strains contribute to the defined to cover all the genes present in the characterized flavor and aroma profile of a range of fermented dairy strains of a species. Under both definitions, the genome has products and are also a component of the starter blends a core of genes responsible for the basic aspects of the bi- used for lactic casein manufacture (Heap and Lawrence ology of the species and a set of auxiliary or dispensable 1984). These strains have long been distinguished taxonom- genes that contribute to species diversity and may provide ically (Kempler and McKay 1981), but with the description a selective advantage in certain environments. As a result, of the genus Lactococcus (Schleifer et al. 1985), they were there is much to learn about diversity at the intraspecies incorporated into L. lactis subsp. lactis. Biovar diacetylactis level, and the aim of this work was to examine the chromo- dairy starter strains have been genotypically (Ko¨ hler et al. somal diversity of a large collection of L. lactis strains to 1991; Beimfohr et al. 1997) and phenotypically (Bachmann provide a framework for future comparative genomic work et al. 2009) distinguished from other L. lactis cultures, with this industrially important bacterial species. suggesting that these cultures may also form a separate subgroup. Both L. lactis subspecies have been isolated from a variety Materials and Methods of environmental sources but are most commonly associ- Bacterial Cultures and Growth Media ated with fresh or fermented plant material or with milk and milk products. Strains that show the lactis subspecies In initial screening, 558 L. lactis strains were examined by genotype can be readily isolated from these environments, pulsed-field gel electrophoresis (PFGE) analysis of SmaI di- whereas isolations of cultures with the cremoris subspecies gests of genomic DNA. These included 289 strains with genotype are comparatively rare (Klijn et al. 1995; Salama the cremoris phenotype and genotype (L. lactis subsp. et al. 1995). Attempts to isolate new cremoris or diacetylac- cremoris), 197 strains with the lactis phenotype (L. lactis tis phenotype strains from environmental sources have met subsp. lactis), and 72 strains that were able to metabolize with little success as wild-type strains of both subspecies citrate (L. lactis subsp. lactis biovar diacetylactis). The show the lactis phenotype (Klijn et al. 1995; Salama et al. cremoris phenotype strains and the citrate-metabolizing 1995; Ward et al. 1998). strains were all either used as or isolated from dairy starter Because of its industrial relevance, L. lactis has become cultures. The lactis phenotype strains included cultures with the best studied of the lactic acid bacteria and regarded both cremoris and lactis genotypes and came from diverse as a model organism for this bacterial group, although most origins. These included dairy starter cultures and individual work has been focused on a small number of laboratory strains isolated from raw milk, pasture, soil, plant material, strains of dairy origin. Complete genome sequences have the rumen, and insect gut. Some of these have been been published for four strains. These include the two described elsewhere (table 1), and the strains isolated from plasmid-cured strains (IL1403 and MG1363) on which plant sources were included in the L. lactis diversity study 730 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE Table 1 Sizes of I-CeuI Restriction Fragments, Total Chromosome Size (kb), Genotype, and Origin of Lactococcus lactis Cultures Strain Ce1 Ce2 Ce3 Ce4 Ce5 Ce6 Total (kb) Genotype Origin (Reference) 1. L. lactis subsp. cremoris LW1477 1,340 540 220 75 45 22 2,242 cremoris Dairy starter culture, ScrFI producer KH 1,380 530 240 95 45 38 2,328 cremoris Dairy starter culture (1) AM2 1,440 520 240 75 45 38 2,358 cremoris Dairy starter culture (1,2) 112 1,440 530 250 75 45 22 2,362 cremoris Dairy starter culture LW1494 1,440 530 240 75 45 45 2,375 cremoris Dairy starter culture HP 1,900 65 260 80 45 28 2,378 cremoris Dairy starter culture (1,2) 2188 1,900 80 250 80 45 38 2,393 cremoris Dairy starter culture 166 1,440 530 260 80 45 38 2,393 cremoris Dairy starter culture (2) KF322 1,440 460 340 75 45 38 2,398 cremoris Isolated from mixed strain dairy starter culture 2128 1,440 560 230 90 45 35 2,400 cremoris Dairy starter culture FG2 1,800 380 70 80 45 35 2,410 cremoris Dairy starter culture (3) LW1499 1,440 530 300 75 45 22 2,412 cremoris Dairy starter culture LW1489 1,480 530 260 85 45 22 2,422 cremoris Dairy starter culture AM1 1,520 530 240 75 45 38 2,448 cremoris Dairy starter culture (1,2) SK11 1,520 530 240 75 45 38 2,448 cremoris Dairy starter culture, phage-resistant AM1 (1,2) 448 1,440 560 300 75 45 22 2,442 cremoris Dairy starter culture (2) BK5 1,520 520 250 80 45 38 2,453 cremoris Dairy starter culture (1,2) LW1492 1,480 560 280 90 45 22 2,477 cremoris Dairy starter culture 168 1,520 600 240 90 45 48 2,543 cremoris Dairy starter culture E8 1,520 610 250 75 45 52 2,552 cremoris Dairy starter culture (1,2) 2. L. lactis subsp. lactis biovar diacetylactis CNRZ157 1,440 540 240 80 45 20 2,365 lactis Dairy starter culture (4) LW1807 1,380 570 270 80 45 20 2,365 lactis Dairy starter culture LW1688 1,380 570 270 80 45 20 2,365 lactis Dairy starter culture LW3081 1,440 540 240 95 45 20 2,380 lactis Dairy starter culture D10 1,380 570 300 80 45 20 2,395 lactis Dairy starter culture LW1690 1,380 600 270 80 45 20 2,395 lactis Dairy starter culture LW3079 1,900 115 240 80 45 35 2,415 lactis Isolated from mixed strain dairy starter culture LW1503 1,440 600 240 95 45 20 2,440 lactis Dairy starter culture LW1811 1,440 600 240 95 45 22 2,442 lactis Dairy starter culture DRC3 1,440 600 240 95 45 22 2,442 lactis Dairy starter culture (5) DRC1 1,440 600 270 80 45 20 2,455 lactis Dairy starter culture (2,5) LW3074 1,440 600 270 80 45 20 2,455 lactis Dairy starter culture LW3087 1,440 600 270 95 45 20 2,470 lactis Dairy starter culture LW2333 1,440 650 240 95 45 22 2,492 lactis Dairy starter culture DRC2 1,640 440 270 80 45 20 2,495 lactis Dairy starter culture (2,5) LW1505 1,440 650 240 95 45 35 2,505 lactis Dairy starter culture LW840 1,540 570 270 05 45 20 2,540 lactis Dairy starter culture D6 1,540 600 285 80 45 20 2,570 lactis Dairy starter culture LW3076 1,540 600 285 80 45 20 2,570 lactis Dairy starter culture LW3077 1,540 600 285 80 45 35 2,585 lactis Isolated from mixed strain dairy starter culture 3. L. lactis subsp. lactis (dairy cultures) LW1509 1,520 530 270 80 90 25 2,515 cremoris Dairy starter culture MG1363 1,640 530 240 80 50 25 2,565 cremoris Dairy starter culture, plasmid-free NCDO712 (6) NCDO712 1,640 530 270 80 50 25 2,595 cremoris Dairy starter culture (6) LW1515 1,640 530 290 75 50 20 2,605 cremoris Dairy starter culture GL17 1,640 550 300 110 50 25 2,675 cremoris Dairy starter culture IL1403 1,440 540 240 80 45 20 2,365 cremoris Dairy starter culture, plasmid-free CNRZ157 (4) LW1444 1,480 530 220 90 45 35 2,400 lactis Dairy starter culture BA2 1,440 550 260 90 45 20 2,405 lactis Dairy starter culture LW1448 1,540 500 240 80 50 22 2,432 lactis Dairy starter culture KF324 1,540 500 240 90 45 22 2,437 lactis Dairy starter culture ATCC7962 1,540 530 230 80 45 20 2,445 lactis Dairy starter culture, nisin producer NCDO895 1,440 550 230 170 45 20 2,455 lactis Dairy starter culture, nisin producer LW1512 1,440 550 300 90 45 35 2,460 lactis Dairy starter culture C10 1,520 530 240 90 45 35 2,460 lactis Dairy starter culture Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 731 Kelly et al. GBE Table 1 Continued Strain Ce1 Ce2 Ce3 Ce4 Ce5 Ce6 Total (kb) Genotype Origin (Reference) LW1449 1,440 650 220 120 45 20 2,495 lactis Dairy starter culture ML8 1,540 530 260 90 45 35 2,500 lactis Dairy starter culture (1,2) NCDO1404 1,620 530 200 80 50 20 2,500 lactis Dairy starter culture, nisin producer LW2004 1,540 540 260 100 45 20 2,505 lactis Dairy starter culture (7) LW1514 1,540 600 260 100 45 22 2,567 lactis Dairy starter culture U 1,640 550 260 110 45 28 2,633 lactis Dairy starter culture 4. L. lactis subsp. lactis (wild-type cultures) KW8 1,440 570 270 95 45 22 2,442 cremoris Kaanga wai (fermented corn) (8) KW2 1,590 520 240 80 48 22 2,500 cremoris Kaanga wai (fermented corn) (8) KF343 1,700 540 220 90 48 28 2,626 cremoris Cow’s milk KF355 1,700 500 270 80 48 38 2,636 cremoris Cow’s milk LW1190 1,700 500 270 80 48 38 2,636 cremoris Sheep’s milk (9) KF292 1,590 500 220 90 45 22 2,467 cremoris Soya sprouts, nisin producer (10) 511 1,640 470 220 90 45 20 2,485 lactis Rumen, nisin producer KF196 1,590 520 220 90 45 20 2,485 lactis Radish sprouts, nisin producer (10) KF363 1,640 470 240 90 45 20 2,505 lactis Soil KF201 1,640 470 240 80 45 38 2,513 lactis Sliced mixed vegetables (10) KF146 1,590 580 220 90 45 20 2,545 lactis Alfalfa and radish sprouts, nisin producer (10) LW1320 1,540 630 250 90 45 20 2,575 lactis Goat’s milk (9) KF181 1,590 520 320 80 45 22 2,577 lactis Alfalfa and onion sprouts (10) KF67 1,590 500 340 90 45 20 2,585 lactis Grapefruit juice, nisin producer (10) LW1,180 1,640 500 270 110 45 22 2,587 lactis Sheep’s milk (9) N1 1,760 450 240 75 45 20 2,590 lactis Moth larval midgut, nisin producer (11) KF5 1,590 520 290 120 45 28 2,593 lactis Alfalfa sprouts (10) KF165 1,740 500 220 80 45 20 2,605 lactis Mung bean sprouts, nisin producer (10) KF282 1,740 520 240 80 45 20 2,645 lactis Mustard and cress, nisin producer (10) KF147 1,740 520 270 90 48 20 2,688 lactis Mung bean sprouts (10) References: (1) Lawrence and Pearce (1972); (2) Jarvis and Wolff (1979); (3) Davidson et al. (1996); (4) Chopin et al. (1984); (5) Czulak and Hammond (1954); (6) Gasson (1983); (7) Ward et al. (2004); (8) Kelly et al. (1994); (9) Ward et al. (1998); (10) Kelly et al. (1998a); (11) Shannon et al. (2001). Cultures for which genome sequences are available and their parent strains are shown in bold. (Rademaker et al. 2007). Forty-eight of the lactis phenotype mixed with an equal volume of 2% (w/v) low melt agarose strains were nisin producers. Dairy starter strains were (Bio-Rad Laboratories). Embedded cells were lysed by treat- mainly from the culture collection of the Fonterra Research ment with lysozyme (1 mg/ml in EC buffer, 6 mM Tris–Cl:1 M Center, Palmerston North, New Zealand, with additional NaCl:100 mM ethylenediaminetetraacetic acid [EDTA]:1% cultures obtained from other culture collections or isolated [w/v] sarkosyl, pH 7.6) overnight at 37 C and proteinase from mixed strain dairy starters. K (0.5 mg/ml in lysis buffer, 50 mM Tris–Cl:50 mM EDTA:1% Lactococci were grown at 28 C in M17 broth (Merck) [w/v] sarkosyl, pH 8.0) for 24 h at 50 C. Agarose plugs con- (Terzaghi and Sandine 1975) supplemented with 0.5% w/ taining intact genomic DNA were washed three times with v glucose for growth of the plasmid-free dairy strains Tris–EDTA buffer (10 mM Tris–Cl:1 mM EDTA, pH 8.0) before (IL1403 and MG1363) and the wild-type strains. The stock storage in 10 mM Tris–Cl:100 mM EDTA (pH 8.0) at 4 C. cultures were maintained at 85 C in M17 broth supple- DNA embedded in agarose was digested for 16 h with mented with 20% (v/v) glycerol. Tests for arginine and cit- 1.0 U of ApaI, SmaI, or I-CeuI (New England Biolabs) in rate metabolism were used to confirm the phenotype of the 100 ll of restriction enzyme buffer, loaded into wells of strains, and strain genotypes were determined using the 1% (w/v) agarose gels (pulsed-field certified agarose, Bio- polymerase chain reaction (PCR) primers and conditions Rad), and run at 200 V for 20 h at 14 C in 0.5 Tris–borate described previously (Ward et al. 1998). buffer (Sambrook et al. 1989) using a CHEF DR III PFGE ap- paratus and model 1000 mini chiller (Bio-Rad). Pulse times used were 1–30 s for ApaIor SmaI and 5–60 s for I-CeuI. To Pulsed-Field Gel Electrophoresis determine the size of the largest fragments from I-CeuI di- Cells were harvested from 1.5 ml of an overnight culture by gests, gels were prepared from 0.8% chromosomal grade centrifugation (10,000  g, 10 min), washed twice with 1 M agarose (Bio-Rad) and run with the pulse time ramped be- NaCl:10 mM Tris–Cl (pH 7.6), and 300 ll aliquots were tween 150 and 400 s. Fragments smaller than 100 kb were 732 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE also resolved and measured using a FIGE Mapper electro- fragment by its average size before subjecting the values phoresis system (Bio-Rad). DNA was visualized by staining to pairwise F tests to test the equality of variances (Sokal with ethidium bromide and the image captured using and Rohlf 1981). a Gel Doc 1000 system (Eastman Kodak). Partial digestion with I-CeuI was used to establish the rrn chromosomal skel- Genomic Analysis eton as described by Liu et al. (1999). Genomic DNA pre- Genomic data for the four publicly available and completed pared from L. lactis subsp. lactis IL1403 and phage L. lactis genome sequences (GenBank accession numbers lambda concatamers or Saccharomyces cerevisiae pulsed- AM406671, CP000425, AE005176, and CP001834) were field gel (PFG) markers (New England Biolabs) were used downloaded from the National Center for Biotechnology In- as size standards. formation (NCBI) Web site (http://www.ncbi.nlm.nih.gov). Genome alignments of chromosomal sequences were per- Plasmid Analysis formed using Mauve software (Version 2.3.1) (Darling et al. Plasmid DNA was isolated by the method of Anderson and 2004). McKay (1983) and the size of individual plasmid bands de- termined following electrophoresis in 0.7% agarose gels in Phylogenetic Analysis Tris–acetate buffer (Sambrook et al. 1989) for 3 h at 4 V/cm Phylogenetic relationships were determined using a super- and staining as above. Strains were also screened for the tree approach. Protein coding gene sets for the four L. lactis presence of large linear plasmids by running undigested strains, three Streptococcus thermophilus strains (GenBank genomic DNA in PFGs as described above. accession numbers CP000023, CP000024, and CP000419), five Lactobacillus species (GenBank accession numbers DNA Methodology AL935263, CR936503, CR954253, CP000416, and To determine the relatedness of citrate-metabolizing strains CP000517), as well as two outgroups (Listeria species; Gen- based on the presence and chromosomal location of their Bank accession numbers AL591824 and AL592022) were prophage, the primers described by Chopin et al. (2001) downloaded from the NCBI Web site. The S. thermophilus, were used in PCR reactions to amplify the chromosome– L. delbrueckii subsp. bulgaricus and L. helveticus genomes prophage junction regions from IL1403 for use as probes were included for comparison because of their use as dairy for PFGs. Hybridizations were done using the North2South starter cultures. For a full description of the supertree meth- Direct HRP Labeling and Detection Kit (Pierce) using the con- odology used please refer to Fitzpatrick et al. (2006). ditions recommended by the manufacturer. To determine the presence of plasmids encoding citrate permease (citP), Results and Discussion plasmid gels were hybridized with a probe to the gene for citrate permease that was constructed by PCR amplification PFGE Patterns of L. lactis Subsp. cremoris Dairy from strain LW1503 using primers described by Klijn et al. Starter Cultures (1995). To determine if cultures, which failed to cut with PFGE analysis of SmaI digests of genomic DNA from the 289 SmaI, contained the ScrFI R/M system, PFGs were hybridized L. lactis subsp. cremoris strains showed that 230 (80%) with a probe specific for the scrFIAM methylase, which was could be linked into 12 groups of related strains. Represen- constructed by PCR amplification from strain UC503 using tatives of these groups are included in table 1. Three (E8, HP, primers described by Szatmari et al. (2006). and SK11) of the four strains compared by Taı¨bi et al. (2010) were representative of groups of strains, whereas the fourth Statistical Analysis (Wg2) had a unique PFGE profile. The observation that many Differences in average chromosome size among subspecific strains were related was not surprising because studies groups of L. lactis from each origin (dairy and wild type) based on phage host range had previously indicated that were tested using a one-way analysis of variance and fitting a relatively small number of significantly different cremoris a single factor comprising each genotype–phenotype origin starter strains exist (Lawrence and Pearce 1972; Lawrence combination. To test whether regions of the chromosome et al. 1978). The result is that many cremoris strains of di- differ in degree of variability, we compared the variances verse origin are unknowingly related and an example of this in the lengths of different chromosomal regions based on is shown in figure 1 where strains related to SK11 are com- I-CeuI fragments. The variance in the length of a given re- pared. SK11 was isolated in New Zealand as a phage- gion is expected to increase linearly with fragment size as- resistant derivative of strain AM1 (DRI 1962), which had suming that the number of insertions, duplications, and been obtained from Professor Collins from University of deletions per fragment increase with fragment length. California Davis and originally named LT8 (DRI 1960). Two Therefore, the variances in the sizes of the I-CeuI fragments other cultures were introduced to New Zealand at the same were standardized by dividing the variance of a given time, AM2 (FC4) and AM3 (4B) (DRI 1960; Collins 1961), Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 733 Kelly et al. GBE FIG.1.—PFGE patterns of SmaI-digested genomic DNA from Lactococcus lactis subsp. cremoris SK11 and related strains. The mixed strain starter isolates (MSS1–4) were isolated from various mixed strain dairy starter cultures. and all three had been isolated from commercial mixed fragment of chromosomal DNA from these strains hybrid- strain cultures. AM1 and AM2 were regarded as slower ized to a probe for the scrFIAM methylase gene. When starters and were found to make consistently good-flavored the histories of these strains were examined, most were Cheddar cheese (Martley and Lawrence 1972). Figure 1 found to have been isolated from mixed strain starter shows that AM1 and SK11 have the same PFGE SmaI digest cultures. pattern but that several other strains are similar. These include US3 and R6, which had both been in use as defined PFGE Patterns of Citrate-Utilizing Dairy Starter strain starters since the early 1950s (DRI 1951), strain 134 Cultures (originally described as a phage-resistant derivative of AM2, Limsowtin and Terzaghi 1976), and several isolates The PFGE patterns of SmaI-digested genomic DNA from 72 from mixed strain starter cultures. Cit strains showed that most strains had several bands in The predicted PFGE pattern from the sequenced SK11 common, and representatives are shown in figure 2A. The strain differs from that for SK11 shown in figure 1 but relatedness of these strains is supported by the presence of matches to that of strain 134. The major PFGE bands are prophage, and these were detected using probes to the very similar in these strains with the main difference being chromosome–prophage junctions in strain IL1403 (Chopin a deletion of ;20 kb from one of the largest PFGE bands et al. 2001). An example is shown in figure 2B where (a 293-kb doublet which separates into 293- and 273-kb the left hand junction fragment between the IL1403 bands). When the 273-kb SmaI fragment from the SK11 chromosome and the bIL309 prophage hybridizes strongly genome is compared with the corresponding region from with all the Cit strains. Six prophages have been identified MG1363, the only major difference is the presence of on the IL1403 chromosome (Bolotin et al. 2001), and junc- a 19-kb prophage sequence (MG-1) in MG1363 (Ventura tion fragments for five of these (bIL285, bIL309, bIL310, et al. 2007). A prophage (bIL310) is integrated in the same bIL311, and bIL312) could be detected in all strains. The genomic region in IL1403, and there is strong homology and sixth prophage (bIL286) was found only in IL1403 and synteny between bIL310 and MG-1. Consequently, it is likely CNRZ157 and may be less stable than the others as we iso- that a similar prophage has been lost from the sequenced lated a derivative of CNRZ157, which was spontaneously SK11 strain. cured of this prophage. In a study using minisatellite poly- DNA from 13 cremoris strains failed to cut with the morphism to distinguish closely related L. lactis strains, a se- enzyme SmaI, indicative of the presence of a restriction/ quence from within the bIL286 prophage was used as modification system operating in these strains. Of the a strain-specific minisatellite. This sequence was only found known lactococcal R/M systems, only the ScrFI methylase in the genome of IL1403 and not in nine other L. lactis ´ ´ potentially blocks the SmaI recognition site (Szatmari strains (Quenee et al. 2005). The observation by McGrath et al. 2006). Unlike most lactococcal R/M systems, ScrFI is et al. (2002) that the genomes of IL1403 and the citrate-uti- chromosomally encoded and a single 75-kb ApaI digest lizing strains IL409 (DRC1) and F7/2 contain identical 734 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.2.—(A) PFGE patterns of SmaI digests of genomic DNA from Cit Lactococcus lactis strains. (B) Southern blot of (A) hybridized with a PCR- amplified product of the left hand junction between the IL1403 chromosome and the prophage bIL309. prophage-encoded bacteriophage resistance genes is in for the synthesis of active citrate lyase, is missing from the agreement with our observation that these strains are other sequenced L. lactis strains (Wegmann et al. 2007; closely related and harbor related prophage. The gene Siezen et al. 2008). identified (sie ) showed 100% amino acid identity with IL409 orf2 of bIL309. PFGE Patterns of L. lactis Subsp. lactis Cultures The plasmid-free strain IL1403 was originally derived from the citrate-utilizing strain L. lactis subsp. lactis biovar PFGE was also used to compare 197 strains with the lactis diacetylactis CNRZ157 (IL594) following protoplast-induced phenotype (L. lactis subsp. lactis) made up of 110 dairy curing (Chopin et al. 1984; Bourel et al. 1996). Citrate uti- starter strains and 87 isolated from various sources. With lization requires citrate permease to transport citrate into the exception of a group of cultures used in lactic casein the cell and citrate lyase to initiate citrate breakdown (Drider manufacture that have been described previously (Ward et al. 2004). In lactococci, these activities are genetically et al. 2004), the L. lactis subsp. lactis strains showed much separate with citrate permease being plasmid encoded on more diversity than the other groups and the majority of an 8-kb plasmid (Kempler and McKay 1981), whereas strains gave unique PFGE patterns. Two of the sequenced L. lactis strains have the lactis phe- citrate lyase and other genes involved in citrate breakdown notype, MG1363 (Wegmann et al. 2007) and KF147 (Siezen are chromosomal (Bolotin et al. 2001). The distribution of et al. 2010). MG1363 was made plasmid free by UV treat- the chromosomal citrate-utilizing genes among different ment and protoplast-induced curing (Gasson 1983) and lactococcal strains is not known, but citrate lyase activity belongs to a group of related strains, which includes was not present in cell-free extracts of 24 dairy lactococcal NCDO712, C2, ML3, LM0230, and 952 (Davies et al. strains (Harvey and Collins 1961), and the gene cluster 1981; Lucey et al. 1993; Le Bourgeois et al. 2000). All have mae-maeP-citRCDEFXG, which includes the genes required Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 735 Kelly et al. GBE techniques developed for MG1363 have been difficult to transfer to dairy starter strains (Johansen 2003). KF147 is one of a group of L. lactis cultures isolated from minimally processed fruit and vegetable products (Kelly et al. 1998a) and has several novel properties not found in dairy starters. Selection of Bacterial Strains for Chromosomal Analysis Based on the SmaI PFGE patterns, 80 strains (table 1) were chosen as representative of the L. lactis species. These in- cluded 20 strains belonging to each of four groups chosen for comparison because of their origin or because of their use for different purposes in the dairy industry and also in- cluded the four strains whose genome sequence has been determined. These are 1) L. lactis subsp. cremoris dairy starter cultures, 2) L. lactis subsp. lactis biovar diacetylactis dairy starter cultures, 3) L. lactis subsp. lactis dairy starter cultures, and 4) wild-type L. lactis subsp. lactis strains. Based on PFGE patterns and strain history data, the cultures se- lected were believed to be unrelated to one another except for SK11, which is a bacteriophage-resistant derivative of L. lactis subsp. cremoris AM1, and IL1403 and MG1363, which are plasmid-free derivatives of L. lactis subsp. lactis biovar diacetylactis CNRZ157 and L. lactis subsp. lactis NCDO712, respectively. Characteristics of the L. lactis Chromosome and Chromosomal Rearrangements Chromosomal mapping has shown that L. lactis has a circular chromosome (fig. 3A) with six ribosomal operons that are transcribed divergently from the origin of chromosomal rep- lication (Davidson et al. 1996). Whereas it is expected that in any one strain, all six 16S rRNA copies will have the same nucleotide sequence, work by Pillidge et al. (2009) has high- lighted an additional level of complexity. A small number of L. lactis subsp. cremoris strains, some of which show PFGE patterns similar to SK11, appear to be genotypic hybrids and have both cremoris-like and lactis-like 16S rRNA types in their genome. Some L. lactis subsp. cremoris strains contain plasmids with a lactis-like 16S rRNA pseudogene, and it is proposed that these chimeric strains are the result of homol- FIG.3.—(A) Locations of I-CeuI recognition sites on the Lactococ- ogous recombination between the pseudogene and the cus lactis IL1403 chromosome. I-CeuI cleaves at sites within the six 23S corresponding chromosome gene (Pillidge et al. 2009). rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and Because plasmid DNA contributes to the PFGE patterns the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. (B) PFGE resulting from SmaI digests (Ward et al. 1993), the homing patterns of genomic DNA from L. lactis strains. endonuclease I-CeuI, which cuts only within the 23S rRNA gene (Liu et al. 1999), was used to produce a PFGE pattern a lactis phenotype but a cremoris genotype. NCDO712, C2, based on chromosomal DNA alone. Macrorestriction pat- and MG1363 have a chromosomally integrated sex factor terns produced by PFGE of I-CeuI digests of genomic not found in other lactococcal strains. Strains belonging DNA (fig. 3B) provide information on chromosomal size, to this group have been widely used as conjugation recip- the number and position of rRNA operons, and an indication ients, and large DNA fragments are known to be capable of chromosomal rearrangements or insertions and dele- of integration at several sites on the chromosome, but tions. All the 80 L. lactis strains examined gave six fragments 736 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.4.—Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome’s center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I-CeuI cut sites that indicate the locations of the 23S rRNA genes are shown above each strain. when their genomic DNA was digested with I-CeuI, indicat- Obis et al. (2001). The L. lactis subsp. lactis biovar diacety- ing that the copy number of the rRNA genes is conserved in lactis strain LW3079 isolated from a mixed strain starter cul- this species (table 1). These fragments were designated as ture has a rearrangement similar to that found in the HP-like Ce1–Ce6 following the nomenclature used by Le Bourgeois strains. These chromosomal changes have no observable et al. (1992). Partial digests were used to determine the or- effect on cell growth, morphology, or phenotype. der of the I-CeuI fragments, and for most strains, the relative With the availability of four different L. lactis genome se- size and order of the various fragments are the same as in quences, it is possible to get an indication of the larger scale IL1403, suggesting that chromosomal structure is conserved events that shape the lactococcal genome. Figure 4 com- in most cases. A minority of strains showed chromosomal pares the four genomes and shows that overall there is rearrangements, and these were of two types typified by a high degree of conservation. A large inversion involving strains HP and FG2. From the 289 cremoris strains investi- approximately half the chromosome has been described gated, 21 had PFGE patterns similar to HP and 15 had pat- in MG1363 (Daveran-Mingot et al. 1998), although this terns similar to FG2. The rearrangement in FG2 has been occurs within the Ce1 fragment and does not result in described previously during chromosome mapping studies a change to the I-CeuI pattern (fig. 4) or alteration in (Davidson et al. 1996). Curiously, strains HP and FG2 both chromosomal symmetry. Major genome insertions in these carry plasmids that specify the same uncommon type of cell strains are highlighted in figure 4 and are predominantly envelope proteinase (lactocepin) linked to a partially deleted associated with prophages, the integration of plasmid copy of abiB(Christensson et al. 2001). These two strains genes, polysaccharide biosynthesis, or the ability to metab- also cluster together and separate from strains SK11 and olize plant-derived carbohydrates. Prophages are an impor- AM2 in the CGH study reported by Bayjanov et al. tant feature, as phage resistance has been a major driver for (2009), and both HP and Z8 (a culture with the same atypical strain selection programs for dairy cultures, and phage chal- PFGE pattern as FG2) were the only L. lactis strains shown to lenge has been a continual selective stress in their environ- lack the busA operon in the osmolality study described by ment. Strain-specific genes of significance include the Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 737 Kelly et al. GBE Lactobacillus sakei (23K) Lactobacillus brevis (ATCC 367) Lactobacillus plantarum (WCFS1) Lactobacillus delbrueckii subsp. bulgaricus (ATCC 11842) Lactobacillus helveticus (DPC 4571) Streptococcus thermophilus (LMD-9) Streptococcus thermophilus (LMG 18311) Streptococcus thermophilus (CNRZ 1066) Lactococcus lactis subsp. lactis (MG1363) Lactococcus lactis subsp. cremoris (SK11) Lactococcus lactis subsp. lactis (KF147) Lactococcus lactis subsp. lactis (IL1403) Listeria monocytogenes (EGD-e) Listeria innocua (Clip11262) FIG.5.—maximum representation with parsimony (MRP) supertree for Lactococcus lactis and other lactic acid bacteria derived from 1,160 single gene families. Listeria species were selected as an outgroup. Bootstrap scores for all nodes are displayed. malate–citrate metabolism genes in IL1403 and the inte- gene families with the results shown in figure 5. This analysis grated sex factor in MG1363. KF147 also contains genes strongly supports the conclusion from the study by for nisin biosynthesis, although this strain does not produce Rademaker et al. (2007) that two main lineages exist in nisin (Kelly et al. 1998a; Siezen et al. 2008), and for nonri- L. lactis. These correspond to the two genotypes. Strains bosomal peptide and polyketide synthesis. The large inser- with a L. lactis subsp. cremoris genotype include strains with tion in the Ce3 fragment in KF147 (fig. 4) is a chromosomally both lactis (MG1363) and cremoris (SK11) phenotypes, integrated conjugative element that encodes the ability to whereas strains with the L. lactis subsp. lactis genotype metabolize alpha-galactosides such as melibiose and raffi- includes strains with the both diacetylactis (CNRZ157, the nose. Transfer of this element to a derivative of MG1363 parent strain of IL1403) and lactis (KF147) phenotypes. From and its integration at two different chromosomal sites have the supertree results, it appears that a similar situation may been described previously (Kelly et al. 1998b), and similar exist in S. thermophilus, but this awaits further study. conjugative elements were found in several L. lactis strains Differences in Chromosome Size between Sub- of plant origin. groups of L. lactis Phylogenetic Relationship between L. lactis Strains The average chromosome size of the 80 strains of L. lactis The availability of four complete genome sequences cover- was 2,483 kb, with the chromosomes of individual strains ing most genotype/phenotype combinations makes it pos- ranging in size from 2,242 to 2,688 kb. This variation in sible to produce a phylogeny truly representative of the chromosomal length (;20% of the size of the smallest entire genome. Supertree methods (Fitzpatrick et al. chromosome) is similar to that found in natural isolates of 2006) were used to derive phylogenies from 1160 single Escherichia coli (Bergthorsson and Ochman 1998). Table 2 Table 2 Mean Chromosome Lengths (kb) of Lactococcus lactis Strains Belonging to the Various Groups Genotype cremoris lactis lactis cremoris lactis cremoris Phenotype Cremoris Diacetylactis Lactis Lactis Lactis Lactis Source Dairy Dairy Dairy Wild type Wild type Dairy a b b c c c Mean chromosome length (kb) 2,412 2,457 2,471 2,568 2,568 2,591 Number of strains 20 20 15 5 15 5 Treatments that share the same letter are not significantly different at P , 0.05. 738 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.6.—(A) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis.(B) Relationship between variances of different I-CeuI fragments standardized by their average size and the average size of the corresponding fragments. shows the comparison between the mean chromosomal Variation among Chromosomal Regions lengths of L. lactis strains from different genotype– All the I-CeuI fragments show some degree of length phenotype origin combinations. The origin of the strains variation (table 1), although, except for the strains that show has the greatest influence on chromosome length with dairy chromosomal rearrangements, the sizes of the individual strains having smaller chromosomes than the wild-type fragments do not overlap. To test whether some strains. This smaller chromosome size may be the result chromosomal regions are more variable than others, we of a process of reductive genome evolution as a conse- compared the standardized variances for each fragment quence of the adaptation to growth in milk. Among the across strains in pairwise F tests. The four strains that dairy strains, those with the cremoris genotype and pheno- showed major chromosomal rearrangements (HP, 2188, type are significantly different and have the smallest FG2, and LW3079) were not included so this comparison chromosomes, whereas there is no significant difference was based on 76 strains. There were no significant differen- between strains with the diacetylactis and lactis phenotypes. ces when most of the fragments were compared against In this analysis, the five cremoris genotype/lactis phenotype each other; however, the largest fragment (Ce1) was signif- dairy strains grouped with the wild-type strains. This could icantly more variable (P  0.005), and it can be seen from be related to the small sample size or could indicate that figure 4 that the majority of chromosomal insertions are they are not as strongly adapted to the dairy environment found in this region. Ce5 was significantly less variable and are closer to strains of plant origin. That MG1363 has (P  0.001) than the other fragments. A feature of the size much greater ability than either IL1403 or SK11 to grow on variation in the I-CeuI fragments is a correlation between plant-derived carbohydrates (Wegmann et al. 2007) supports the latter option. the two largest fragments (fig. 6A). This suggests that Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 739 Kelly et al. GBE FIG.7.—Alignment of the Ce6 region of the chromosomes of Lactococcus lactis IL1403, KF147, MG1363, and SK11 and identification of the genes present. Insertions common to the cremoris strains MG1363 and SK11 are shown in red, and the genes found only in SK11 are shown in blue. The fusA pseudogene in SK11 is shown in mauve. chromosomal insertions and deletions in these regions are SK11 genome sequence, the fusA homologue (LACR_2595) not entirely independent events and that maintenance of is identified as a pseudogene. chromosomal symmetry may be an important consideration. The main difference in the genome sequences for this There is a significant correlation between the I-CeuI frag- region is a 15-kb insertion in SK11 between the ribosomal ment size and the standardized variance of each fragment protein S9 gene (rpsI) and the 16S rRNA gene. This insertion (fig. 6B) with the two smallest fragments showing the great- contains two genes with high homology to type III est departure from this relationship. The smallest fragment restriction–modification systems found in other lactic acid (Ce6) shows greater variance than expected, whereas the bacteria, several hypothetical proteins, an integrase, and variance in size of the second smallest fragment (Ce5) is less genes related to plasmid replication. It is probable that this than for any of the other fragments. The size of Ce5 is insertion increases the phage resistance of SK11, and it may strongly conserved (45–50 kb), and we have found only have been acquired by horizontal gene transfer, and one strain (LW1509) that contains a large insertion of subsequent chromosomal integration, of a small plasmid. DNA in this region. It is apparent that many dairy starter strains have an insertion of similar size in the Ce6 region (table 1), and it will be of interest to determine if similar genes are found Gene Content of Ce6 in other strains and whether they have an influence on cell Because Ce6 exhibits larger variation than expected for growth rate given their location close to the chromosomal a fragment of this size, we examined the genes present origin. in this region from the four available L. lactis genome se- quences (fig. 7). MG1363 and SK11 (both L. lactis subsp. Contribution of Extrachromosomal Elements to cremoris genotype) show two common insertions relative Genome Size to IL1403 and KF147. These are of genes of unknown func- tion between purR and fusA and between rpsL and dacA. In Many technologically important properties (lactose metab- IL1403, 5 of the 14 genes in this region are predicted to be olism, lactocepin proteinase, citrate permease, and bacte- highly expressed (Karlin et al. 2004). These are fusA riophage resistance) are plasmid encoded in L. lactis (predicted to be the most highly expressed gene in the strains used as dairy cultures and can be exchanged be- IL1403 genome) and four genes encoding ribosomal tween strains by conjugation and between replicons by proteins (rpsI, rplM, rpsG, and rpsL). Curiously, in the insertion sequence elements. Examination of plasmid gels 740 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 Chromosomal Diversity in Lactococcus lactis GBE FIG.8.—(A) Plasmid profile of Cit Lactococcus lactis strains. Lane 1, Invitrogen supercoiled DNA ladder; lane 2, Invitrogen 1 kb DNA ladder. (B) Southern blot of (A) hybridized with a PCR-amplified product of the citP gene. Where two bands are present, they represent open and closed circular forms of the same plasmid. for 150 dairy starter cultures (90 L. lactis subsp. cremoris,30 instability has previously been reported for CNRZ157 L. lactis subsp. lactis, and 30 L. lactis subsp. lactis biovar di- (Chopin et al. 1984). acetylactis) gave an average of seven plasmids per strain (range 2–14) with up to 200 kb of plasmid DNA. By contrast, Origin of Defined Strain Dairy Starter Cultures in plant strains, the average was ,2 plasmids per strain (range 0–4). It was notable that small plasmids (,10 kb) Most of the L. lactis strains used in this study were dairy were prevalent in dairy cultures but rare in the plant strains. starter cultures and are representative of the cultures em- Intact genomic DNA was run on PFGs but showed no evi- ployed by the dairy industry since the concept of using de- dence of large linear plasmids in these strains. fined strain starters was developed in the 1930s (Limsowtin Plasmid profiles for 20 Cit strains are shown in figure 8A et al. 1996). During this period, the best cultures were freely and show that these strains have acquired a range of shared between laboratories, and this coupled with the re- different extrachromosomal elements. A plasmid-encoded peated isolation of individual strains with particular charac- citrate permease is required for citrate utilization in L. lactis, teristics from commercial mixed strain starters, and the and to determine its location, a Southern blot of the plasmid development of bacteriophage-insensitive cultures has re- gel was probed with a PCR-amplified product of the citP sulted in many closely related strains coexisting. Conse- gene (fig. 8B). The citrate permease plasmid is conserved quently, the relationship between strains is generally not in size (8 kb) in most strains, but three strains (D6, known, although it has been speculated that the pool of LW3076, and LW1503) have an enlarged citrate plasmid strains with certain properties is small (Lawrence et al. of ;15 kb. We observed that the plasmid complement 1978). Because of their differing histories of industrial use, even closely related strains may differ in some of their can be very unstable in some strains, and this spontaneous Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010 741 Kelly et al. GBE important characteristics and in their plasmid complement S. thermophilus (Hols et al. 2005) dairy cultures. We con- (Ward et al. 2004). clude that dairy starter cultures generally and especially The aim of this work was to use PFGE to gain a measure strains with cremoris and diacetylactis phenotypes comprise of the chromosomal diversity present in a large collection of a specialized group of L. lactis strains. This is in accordance L. lactis cultures including both plant and wild-type strains, with the results from the recent studies of L. lactis with emphasis on the L. lactis strains with the cremoris and (Rademaker et al. 2007; Bayjanov et al. 2009) and fits with diacetylactis phenotypes that are of particular importance to knowledge of the origin of industrially used dairy starter cul- the dairy industry. The diacetylactis phenotype is unusual in tures. Consequently, the world’s dairy industry is based on that it depends on the inheritance of both plasmid and chro- the same small group of good starter strains, and these cul- mosomal components for citrate to be transported into the tures have transitioned from free-living organisms associ- cell and metabolized. The chromosomal genes are not ated with plant material. We hypothesize that these found in the other sequenced strains and show homology specialized dairy starters are no longer fit to survive outside with the plasmid-encoded citrate genes from Leuconostoc the dairy environment and have evolved to become essential and Weissella species isolated from the dairy environment. components of industrial processes. Whereas wild-type Therefore, it can be hypothesized that the diacetylactis strains may have genes that can be used to enhance the me- strains used as defined strain dairy starters represent a single tabolism of dairy strains (van Hylckama Vlieg et al. 2006), it is lineage within L. lactis in which both chromosomal unlikely that there is an environmental source for new dairy and plasmid elements are maintained and essential for an starter cultures similar to those currently in use. industrially significant phenotype. It should be noted that This analysis together with other studies (Nomura et al. this observation is restricted to the Cit strains used as de- 2006; Rademaker et al. 2007; Siezen et al. 2008; Bachmann fined strain dairy starter cultures. Wild-type L. lactis may et al. 2009; Bayjanov et al. 2009; Liu et al. 2010) begins to differ in their citrate metabolism, and strains with illustrate the genomic and phenotypic diversity present a 23-kb plasmid-encoding genes that match those found within L. lactis. Currently, too few genome sequences are in citrate-metabolizing leuconostocs have been isolated available to delineate sets of core and auxillary genes and from Algerian dromedary’s milk (Drici et al. 2010). describe the pangenome of L. lactis, but in the future, it will The cremoris phenotype is rather more complex to deter- be interesting to examine strains from other environments in mine because it is measured as negative attributes including more detail and to further define the genes necessary to the inability to metabolize arginine and inability to grow at make a good dairy starter culture. higher temperatures or at higher salt levels. From investiga- tions of the genome sequences, it appears that these prop- Funding erties have arisen through the accumulation of mutations and that they are a response to the nutrient rich milk envi- New Zealand Foundation for Research, Science, and ronment where certain gene functions are not longer re- Technology (Contract C10X0239). quired. The arginine deiminase–negative phenotype of SK11 correlates with a single base pair deletion (Wegmann Acknowledgments et al. 2007), whereas various defects including the absence of the busA operon have been shown to influence salt tol- We thank Zaneta Park for assistance with the statistical anal- erance (Obis et al. 2001). The taxonomy of L. lactis is cur- ysis and Dr David Fitzpatrick from the Genome Evolution rently based on phenotype, but as the genomic basis for Laboratory, National University of Ireland, Maynooth, Ire- these phenotypic differences is elucidated, a case could land, for assistance with the supertree phylogenetic analysis. be made for review and use of genotypic data to define We also wish to thank Howard Heap and the members of the two subspecies. the Microbial Fermentation Unit, Fonterra, Palmerston Comparison of our data with the analysis of the genome- North, for their support and advice on lactococcal dairy sequenced L. lactis strains strongly supports a plant- starter cultures. associated origin for dairy starter strains. A significant proportion of the KF147 genome is devoted to genes in- Literature Cited volved in the degradation and metabolism of plant-derived Anderson DG, McKay LL. 1983. 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J Bacteriol. 189:3256–3270. Siezen RJ, et al. 2010. Complete genome sequence of Lactococcus lactis subsp. lactis KF147, a plant-associated lactic acid bacterium. Associate editor: Takashi Gojobori J Bacteriol. 192:2649–2650. 744 Genome Biol. Evol. 2:729–744. doi:10.1093/gbe/evq056 Advance Access publication August 13, 2010

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