Vol. 173, No. 9 Physical Map of the Chromosome of Lactococcus lactis subsp. lactis DL11 and Localization of Six Putative rRNA Operons DEBRA L. TULLOCH,1 LLOYD R. FINCH,' ALAN J. HILLIER,2 AND BARRIE E. DAVIDSON'* Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052,1 and Commonwealth Scientific and Industrial Research Organization, Division of Food Processing, Dairy Research Laboratory, Highett, Victoria 3190,2 Australia Received 2 July 1990/Accepted 15 February 1991 A physical map of the chromosome of Lactococcus lactis subsp. lactis DLl1 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two- dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DLll chromosome. By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed- and fixed-field digestion patterns, six putative rRNA operons were identified in L. lactis subsp. lactis DLll and placed on the map of the chromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced. The gram-positive bacteria Lactococcus lactis subsp. lac- tis and L. lactis subsp. cremoris (formerly Streptococcus lactis and S. cremoris, respectively) are used extensively as starter organisms for the manufacture of cheese. Interest in the molecular biology of these organisms is heightened by their industrial importance and because they and their met- abolic products are generally regarded as safe for human consumption. Analysis of the L. lactis genome has been restricted to measurements of its base composition (36 to 38% G+C [16]) and the use of renaturation kinetics to determine its size (2.75 to 3.1 megabase pairs [Mbp] [13]). Preliminary results from pulsed-field gel electrophoresis (PFGE) indicate some- what smaller genome sizes of 2.0 to 2.7 Mbp for a number of strains of L. lactis (20, 33). Most strains of L. lactis contain four or more different plasmids. The properties of a number of these plasmids have been reported, and a number of plasmid-borne genetic determinants have been characterized at the molecular level (4, 18, 21, 29). In contrast, there are only a few reports of the cloning of chromosomal DNA (26, 35) and a genetic map of the lactococcal chromosome has not been developed. Be- cause of the paucity of chromosomal markers and the unavailability of suitable techniques for transferring chromo- somal genes within the lactococci, PFGE offers the most promise for analyzing and mapping the lactococcal chromo- some. In this article we describe the use of two-dimensional separations with PFGE for the construction of the first map of the chromosome of a member of the genus Lactococcus. The locations on the map of putative rRNA operons have been determined. This physical map will provide the basis for the construction of a genetic map of the lactococcal chromosome and for studying chromosomal evolution in the many strains of this species. * Corresponding author. MATERIALS AND METHODS Strains and plasmids. L. lactis subsp. lactis DL11, a proteinase-negative (Prt-) derivative of L. lactis ATCC 11454 (19), was provided by D. LeBlanc, University of Texas Health Science Center, San Antonio, Tex. It produces the bacteriocin nisin (Nis' [19]), ferments sucrose (Suc+ [19]), and has reduced sensitivity to bacteriophage infection (Rbs+ [22]). It is not plasmid free, but the number and sizes of its plasmids are not known. The plasmid pMC5, with a 4.8-kbp insert encoding most of the rRNA operon of Mycoplasma capricolum (2), was from S. Razin, The Hebrew University-Hadassah Medical School, Jerusalem, Israel. Preparation and digestion of high-molecular-weight DNA. L. lactis subsp. lactis DL11 was grown to an A600 of 0.6 in M17 medium (34) containing 0.5% (wt/vol) glucose at 30°C. Chloramphenicol was added to a final concentration of 100 pug/ml, and incubation was continued for 1 h. Cells were harvested and incorporated into low-melting-temperature agarose (FMC Corporation, Rockland, Maine), and high- molecular-weight DNA was prepared in situ as described previously (33). Unless indicated otherwise, the DNA was digested by incubation for 16 h with 5 to 20 U of restriction endonuclease (Boehringer-Mannheim Pty. Ltd., Mannheim, Federal Republic of Germany [FRG], or New England BioLabs, Beverly, Mass.), using the conditions specified by the supplier. Partial digestion of the DNA was carried out by a modification of the method of Albertsen et al. (1). The restriction enzyme (5 to 20 U) was allowed to diffuse into the agarose block for 1 to 2 h at 25°C in the absence of Mg2", and then digestion was performed for 1 h in the presence of limiting (0.5 mM) Mg2". PFGE. PFGE in the contour-clamped homogeneous elec- tric field (CHEF) mode (7) was done through a 1.0 to 1.2% agarose gel in 0.5 x TBE buffer (TBE is 89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA [pH 8.0]) in a CHEF-DRII apparatus (Bio-Rad Laboratories, Richmond, Calif.) operat- ing at 6 V/cm for 22 h at 15°C. Various pulse-time ramps were used as described in the text. The chromosomes of 2768 JOURNAL OF BACTERIOLOGY, May 1991, p. 2768-2775 0021-9193/91/092768-08$02.00/0 Copyright C 1991, American Society for Microbiology on March 12, 2021 by guest http://jb.asm.org/ Downloaded from
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Vol. 173, No. 9
Physical Map of the Chromosome of Lactococcus lactis subsp.lactis DL11 and Localization of Six Putative rRNA OperonsDEBRA L. TULLOCH,1 LLOYD R. FINCH,' ALAN J. HILLIER,2 AND BARRIE E. DAVIDSON'*
Russell Grimwade School ofBiochemistry, University of Melbourne, Parkville, Victoria 3052,1 andCommonwealth Scientific and Industrial Research Organization, Division ofFood Processing,
Dairy Research Laboratory, Highett, Victoria 3190,2 Australia
Received 2 July 1990/Accepted 15 February 1991
A physical map of the chromosome of Lactococcus lactis subsp. lactis DLl1 was constructed by using thecontour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two-dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, whichshows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, iscircular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DLll chromosome. By usingrDNA from Mycoplasma capricolum to probe Southern blots of pulsed- and fixed-field digestion patterns, sixputative rRNA operons were identified in L. lactis subsp. lactis DLll and placed on the map of thechromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. Thepresence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon tobe deduced.
The gram-positive bacteria Lactococcus lactis subsp. lac-
tis and L. lactis subsp. cremoris (formerly Streptococcuslactis and S. cremoris, respectively) are used extensively as
starter organisms for the manufacture of cheese. Interest inthe molecular biology of these organisms is heightened bytheir industrial importance and because they and their met-abolic products are generally regarded as safe for humanconsumption.
Analysis of the L. lactis genome has been restricted tomeasurements of its base composition (36 to 38% G+C [16])and the use of renaturation kinetics to determine its size(2.75 to 3.1 megabase pairs [Mbp] [13]). Preliminary resultsfrom pulsed-field gel electrophoresis (PFGE) indicate some-what smaller genome sizes of 2.0 to 2.7 Mbp for a number ofstrains of L. lactis (20, 33).Most strains of L. lactis contain four or more different
plasmids. The properties of a number of these plasmids havebeen reported, and a number of plasmid-borne geneticdeterminants have been characterized at the molecular level(4, 18, 21, 29). In contrast, there are only a few reports of thecloning of chromosomal DNA (26, 35) and a genetic map ofthe lactococcal chromosome has not been developed. Be-cause of the paucity of chromosomal markers and theunavailability of suitable techniques for transferring chromo-somal genes within the lactococci, PFGE offers the mostpromise for analyzing and mapping the lactococcal chromo-some.
In this article we describe the use of two-dimensionalseparations with PFGE for the construction of the first mapof the chromosome of a member of the genus Lactococcus.The locations on the map of putative rRNA operons havebeen determined. This physical map will provide the basisfor the construction of a genetic map of the lactococcalchromosome and for studying chromosomal evolution in themany strains of this species.
* Corresponding author.
MATERIALS AND METHODS
Strains and plasmids. L. lactis subsp. lactis DL11, a
proteinase-negative (Prt-) derivative of L. lactis ATCC11454 (19), was provided by D. LeBlanc, University ofTexas Health Science Center, San Antonio, Tex. It producesthe bacteriocin nisin (Nis' [19]), ferments sucrose (Suc+[19]), and has reduced sensitivity to bacteriophage infection(Rbs+ [22]). It is not plasmid free, but the number and sizesof its plasmids are not known.The plasmid pMC5, with a 4.8-kbp insert encoding most of
the rRNA operon of Mycoplasma capricolum (2), was fromS. Razin, The Hebrew University-Hadassah Medical School,Jerusalem, Israel.
Preparation and digestion of high-molecular-weight DNA.L. lactis subsp. lactis DL11 was grown to an A600 of 0.6 inM17 medium (34) containing 0.5% (wt/vol) glucose at 30°C.Chloramphenicol was added to a final concentration of 100pug/ml, and incubation was continued for 1 h. Cells wereharvested and incorporated into low-melting-temperatureagarose (FMC Corporation, Rockland, Maine), and high-molecular-weight DNA was prepared in situ as describedpreviously (33). Unless indicated otherwise, the DNA was
digested by incubation for 16 h with 5 to 20 U of restrictionendonuclease (Boehringer-Mannheim Pty. Ltd., Mannheim,Federal Republic of Germany [FRG], or New EnglandBioLabs, Beverly, Mass.), using the conditions specified bythe supplier. Partial digestion of the DNA was carried out bya modification of the method of Albertsen et al. (1). Therestriction enzyme (5 to 20 U) was allowed to diffuse into theagarose block for 1 to 2 h at 25°C in the absence of Mg2", andthen digestion was performed for 1 h in the presence oflimiting (0.5 mM) Mg2".PFGE. PFGE in the contour-clamped homogeneous elec-
tric field (CHEF) mode (7) was done through a 1.0 to 1.2%agarose gel in 0.5 x TBE buffer (TBE is 89 mM Tris-borate,89 mM boric acid, 2 mM EDTA [pH 8.0]) in a CHEF-DRIIapparatus (Bio-Rad Laboratories, Richmond, Calif.) operat-ing at 6 V/cm for 22 h at 15°C. Various pulse-time rampswere used as described in the text. The chromosomes of
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JOURNAL OF BACTERIOLOGY, May 1991, p. 2768-27750021-9193/91/092768-08$02.00/0Copyright C 1991, American Society for Microbiology
a Fragment nomenclature is defined in Materials and Methods.b Numbers in parentheses are the sizes (in kilobase pairs) of fragments produced by the action of both enzymes.Determined from two-dimensional separations by fixed-field gel electrophoresis in the second dimension (see text).
Saccharomyces cerevisiae (Bio-Rad Laboratories), multi-mers of bacteriophage lambda DNA (prepared by themethod of Vollrath and Davis [37] or purchased from Phar-macia LKB Biotechnology, Uppsala, Sweden), and Hindllldigests of lambda DNA were used as molecular size stan-dards.
Two-dimensional PFGE experiments. The method for two-dimensional PFGE was adapted from that described byBautsch (3). Following restriction endonuclease digestion,DNA samples were subjected to PFGE through 1.0% ultra-pure agarose (Bio-Rad Laboratories) in TPE buffer (80 mMTris-phosphate, 8 mM EDTA [pH 8.6]). The lane containingthe separated fragments was excised from the gel and cutinto pieces not exceeding 3 cm in length. Each piece wastransferred to a 2.0-ml microfuge tube for treatment of theDNA in situ with a second restriction endonuclease (80 U in1.0 ml for 16 h). The pieces were then inserted across theorigin of a fresh 1.2% agarose gel for the second-dimensionPFGE. DNA from each of the two single digests and adouble digest was also electrophoresed in the second gel.DNA fragment nomenclature. Fragments produced by di-
gestion with a single restriction endonuclease are designatedNt, Sm, or SI to identify the enzyme (NotI, SmaI, and SalIl,respectively) with a capital letter suffix, A, B, etc., in orderof decreasing size (Table 1). For fragments indistinguishablein size, the suffix is numbered, 1, 2, etc.; e.g., SmCl, SmC2,
and SmC3 in Table 1. New fragments from double digestionsare designated by the two single-digest fragments fromwhich they were derived plus a number in parentheses toindicate the approximate size in kilobase pairs. The suffixhas been omitted when the single-digest fragment was notdefined, e.g., the Sall fragment contributing to the double-digest product SmCl-S1(25) in Table 3.
Synthesis of a 16S rRNA probe. Polymerase chain reaction(PCR) (27) was performed with a GeneAmp DNA Amplifi-cation Reagent Kit (Perkin-Elmer Cetus, Norwalk, Conn.)and an Intelligent Heating Block (Hybaid Limited, Tedding-ton, Middlesex, U.K.). Twenty-mer primers, synthesized ona 381A DNA synthesizer (Applied Biosystems Inc., FosterCity, Calif.), were used to amplify bp 665 to 813 (12) of theM. capricolum 16S rRNA gene in pMC5.DNA hybridizations. 32P-labeled probes were prepared by
random priming (10). Southern blots of PFGE agarose gelswere prepared on GeneScreen Plus nylon membranes (DuPont, Dreieich, FRG) with 0.4 M NaOH as the eluant(24).
32p labeling of DNA in agarose. To facilitate the detectionof small (<10 kbp) DNA fragments, digested DNA waslabeled in the agarose block with [a-32P]dATP as describedby Cocks et al. (8). Following fixed-field electrophoresis ofthe DNA through a 0.8% agarose gel, the gel was dried andthe DNA fragments were detected by autoradiography.
FIG. 1. (A) PFGE of restriction endonuclease digests of L. lactissubsp. lactis DL11 DNA. DNA was digested with ApaI (lane 1),BssHII (lane 2), NotI (lane 3), RsrII (lane 4), Sall (lane 5), Sfi1 (lane6), and SmaI (lane 7). The bars at the side show the positions ofbands for markers of the sizes indicated. The pulse time was rampedfrom 1 to 70 s. (B) PFGE of SmaI-digested L. lactis subsp. lactisDL11 DNA (lane 1), lambda DNA concatomers (lane 2), and aHindlIl digest of lambda DNA (lane 3). Numbers on the side are thesizes of the lambda DNA markers at the positions shown by thebars. The pulse time was ramped from 5 to 10 s to optimizeseparation of the SmaI-digested DNA.
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RESULTS
Restriction digestion patterns of L. lactis subsp. lactis DL11genomic DNA. L. lactis subsp. lactis DL11 has been studiedin many laboratories because it produces the bacteriocinnisin (19), it ferments sucrose (19), and it carries at least onegenetic determinant that confers phage resistance (22). In-tact DNA isolated from L. lactis subsp. lactis DL11 wasdigested with restriction endonucleases that were expectedto have a limited number of cleavage sites in the genome(Fig. 1A). Those enzymes which generated the fewest DNAfragments were NotI, Sfil, RsrII, and SmaI. Sfil and RsrIIwere not satisfactory for primary mapping studies becausethey generated fragments which, on the basis of intensity ofstaining with ethidium bromide, were present in variousmolar ratios. This may have been due to preferential cuttingby these enzymes at some sites, as a result of their multiplerecognition sites (30). ApaI, BssHII, and SalI digested thegenome relatively frequently, each producing more than 25fragments. NotI and SmaI were chosen for the constructionof a physical map of the L. lactis subsp. lactis DL11chromosome because they produced a limited number ofDNA fragments: six for NotI (Fig. 1A) and 21 for SmaI (Fig.1A and B; further data described below). The faint band at amobility corresponding to 39 kbp in the NotI digest (lane 3,Fig. 1A) was not always observed (e.g., lane 4, Fig. 4) anddid not appear to arise from chromosomal DNA (see below).The sizes of the DNA fragments generated by NotI and
SmaI are shown in Table 1. The two smallest SmaI frag-ments, SmN and SmO, were observed on PFGE gels onlywhen large amounts of DNA were loaded. Their sizes weredetermined following fixed-field gel electrophoresis of aSmaI digest of L. lactis subsp. lactis DL11 DNA that hadbeen 32P-labeled in the agarose block.
Construction of a physical map of the L. lacds subsp. kctisDL11 chromosome. (i) Two-dimensional separations of recip-rocal NotI and SmaI double digests. Two-dimensional gels ofreciprocal double digests of L. lactis subsp. lactis DL11
FIG. 2. Two-dimensional PFGE separation of the DNA frag-ments generated by (A) NotI-SmaI and (B) SmaI-NotI doubledigests of L. lactis subsp. lactis DL11 DNA. The pulse time wasramped from 1 to 10 s except for the first dimension (left to right) ofthe NotI-SmaI digest, when it was ramped from 30 to 60 s. Thearrows show the locations of the 27-kbp fragment discussed in thetext. (C and D) Southern blots of the gels in panels A and B,respectively, hybridized with the insert from pMC5. Lane 1 of eachpanel shows the two-dimensional separation, with migration of theproducts of the first digest from left to right, and the other lanesshow single-dimension separations of digests of L. lactis subsp.lactis DL11 DNA by SmaI plus Notl (lane 2), SmaI (lane 3), andNotI (lane 4). The bars at the side of each panel show the positionsof bands for markers of the sizes indicated. The letters under eachpanel help to locate fragments (described in Table 1) generated bythe first restriction endonuclease digestion.
DNA with NotI and SmaI (lanes 1, Fig. 2A and B) were usedas the first step in constructing a map. The double-digestproducts, separated in a single dimension, are shown in lanes2. In lane 1 of Fig. 2B, the undigested SmaI fragmentsconstituted the strongly fluorescing bands aligned along anapproximate diagonal, with the products of the SmaI frag-ments digested by NotI detectable as the fainter bands belowthe diagonal. Interpretation of these gels by the approach ofBautsch (3) allowed definition of most of the products ofNod-SmaI digestion listed in Table 1. As an example, thearrowed 27-kbp DNA fragment was generated when NtCwas cut by SmaI (Fig. 2A) and also by the digestion of SmH1with NotI (Fig. 2B). Thus, it is listed in Table 1 as NtC-SmH1(27).The internal consistency of the data in Table 1 was
demonstrated by the fact that in all cases the sum of the sizesof the fragments generated by digestion of a NotI (or SmaI)fragment with SmaI (or NotI) was, within experimentalerror, the same as the size of the intact NotI (or SmaI)fragment. The presence of two 60-kbp fragments [NtA-
FIG. 3. (A) Physical map of the chromosome of L. lactis subsp. lactis DL11. Radiating out from the center, the five annuli show the scale(Mbp) and digestion sites for NotI (Nt), SmaI (Sm), Sall (SI), and all three enzymes, respectively. Only the Sall sites located between mapcoordinates 0.345 and 0.990 Mbp are shown. The NotI site between NtA and NtB has been arbitrarily defined as map coordinate 0.0 Mbp.(B) Map of the putative rRNA operons of L. lactis subsp. lactis DL11. Fragments that hybridized with both the pMC5 probe and a 16S rDNAprobe (PCR product) are stippled, and those that hybridized only with the pMC5 probe are hatched. In each case, only the minimum-sizedfragment that hybridized is marked. The locations of putative rRNA operons (rrnA to rrnF) are shown by arrows pointing in the direction oftranscription of the operon. Sm, SmaI; S1, Sall; Nt, NotI.
SmC2(60) and NtE-SmH1(60)] in the NotI-SmaI doubledigest complicated the interpretation of the data. However,these fragments were distinguished from each other becauseonly NtA-SmC2(60) hybridized with a probe containing therRNA operon from M. capricolum (see below).To map small fragments (<10 kbp) which could not be
detected by ethidium bromide staining of separations such asthose shown in Fig. 2, it was necessary to use fixed-field gelelectrophoresis for the second dimension. In this way (datanot shown), SmN and SmO were located within NtE andNtA, respectively, while NtB-SmL(3.8) was observed fromboth NtB and SmL, and NtD-SmJ2(10) was confirmed asarising from NtD and SmJ2.
In addition to the products from SmaI digestion of the sixNotI fragments (NtA to NtF), lane 1 of Fig. 2A shows thefaint 39-kbp NotI fragment (NtG) undigested by SmaI. Acorresponding product could not be identified in the recip-rocal NotI digest of the SmaI fragments in lane 1 of Fig. 2B,since the 39-kbp fragment from SmJ was accounted for asNtC-SmJ2(39), which was derived from NtC in lane 1 of Fig.2A. However, the gel shown in Fig. 2B did not include theNotI products from the well of the first-dimension SmaIdigest. In another equivalent two-dimensional gel, a 39-kbpfragment was observed after NotI digestion of DNA in ornear the well of the first-dimension SmaI digest, consistent
with the fragment's deriving from NotI digestion of closedcircular DNA which had shown little mobility in the first-dimension PFGE. Another possibility, that there was afurther SmJ fragment cut by NotI to give a second 39-kbpfragment forming a doublet with NtC-SmJ2(39), could not beaccommodated with the data for other NotI-SmaI fragments.The data in Table 1 allowed the mapping of all the Notl
sites in the L. lactis subsp. lactis DL11 chromosome and theSmaI sites at map coordinates 0.023, 0.708, 0.843, 0.978,1.069, 1.464, 1.513, 1.668, 2.498, and 2.574 Mbp (Fig. 3A).These data also established that the chromosome is circular.The internal consistency of the data for NotI and SmaIsingle- and double-digestion products allowed no scope forplacement of a further 39-kbp NotI fragment in the chromo-some, so we concluded that the anomalous NotI fragmentwas of extrachromosomal origin. The SmaI sites definingfragments internal to NtA, -C, -D, and -E could not bemapped from the data in Table 1.
(ii) Two-dimensional gels of NotI and partial SmaI digests.Determination of the sizes of fragments in two-dimensionalseparations of NotI and partial SmaI digests (Fig. 4 andTable 2) allowed the remaining SmaI sites in both NtC andNtD to be mapped. For NtD, which has 10- and 150-kbpNotI-Smal fragments at either end (Fig. 3A), the sizes of theSmaI partial digestion products were consistent with the
FIG. 4. Two-dimensional PFGE separation of the DNA frag-ments generated by NotI and partial SmaI double digestion of L.lactis subsp. lactis DL11 DNA (lane 1). A NotI digest was separatedin the first dimension with a pulse time ramped from 30 to 70 s.Agarose blocks containing NtA to NtC and NtD to NtF wereexcised and subjected to partial SmaI digestion, and then theproducts were separated with a pulse time ramped from 30 to 60 s.The letters under the figure show the positions of the NotI frag-ments. The right-hand lanes of the gel show single-dimensionseparations of digests of L. lactis subsp. lactis DL11 DNA withSmaI plus NotI (lane 2), SmaI (lane 3), and NotI (lane 4). The barsat the side show the positions of bands for markers of the sizesindicated.
10-kbp fragment NtD-SmJ2(10) being linked to SmF and the150-kbp fragment NtD-SmA(150) being linked to SmJ3. ForNtC, the sizes of the SmaI partial digestion products werecompared with the sizes of the partial digestion productsexpected for all the possible arrangements of SmB, SmJl,and SmH2 within NtC. The only arrangement that fitted thedata accurately was that shown in Fig. 3A.
TABLE 2. Fragments produced by partial SmaI digestion of NtCand NtD from L. lactis subsp. lactis DL11a
Size from gels of partialNotI SmaI digestion Deduced component
65 SmJ3a Fragment nomenclature is defined in Materials and Methods.
(iii) Mapping of the SmaI sites in NtA. PFGE separationsinvolving digestion with a third enzyme (Sall) were used tolocate the remaining unmapped SmaI sites in NtA. In thefirst step, a NotI digest was electrophoresed to allow theexcision of agarose blocks containing pure NtA. Theseblocks were then used for reciprocal two-dimensional PFGEexperiments in which the NtA was digested first with SmaI(or SailI) and then with SailI (or SmaI). The data obtained bythis approach are summarized in Table 3.The majority of fragments in these two separations were
identified by their size. The two 60-kbp fragments, NtA-SmC2(60) and SmCl-SlC(60), were distinguishable becauseonly NtA-SmC2(60) (map coordinates 0.708 to 0.768 Mbp)hybridized with a probe made from pMC5 (see below andFig. 3B). SmM, which also hybridized with pMC5, wasdistinguished from the other 22-kbp fragments in a similarfashion. Analysis of the data in Table 3 allowed the mappingof SmaI sites at map coordinates 0.148, 0.273, 0.408, 0.508,0.530, and 0.660 Mbp and the SaIlI sites at 0.348, 0.478,0.567, and 0.575 Mbp (Fig. 3A). The location of the SalI siteat map coordinate 0.818 Mbp was ascertained by measuringthe sizes of SlA and its SmaI digestion products.
(iv) Mapping of SmN and SmO. As noted above, the twosmallest SmaI fragments, SmN and SmO, were found to belocated in NtE and NtA, respectively. To map them pre-cisely, a Sall digest of L. lactis subsp. lactis DL11 DNA wassubjected to PFGE, digested with SmaI, and then subjectedto fixed-field electrophoresis in the second dimension (re-sults not shown). SmN was produced from the 87.5-kbp SailIfragment SIF2. The map location of SlF2 (and SlK2 andSlM2) was determined by the two-dimensional techniquedescribed above on reciprocal double digests of purified NtEwith SmaI and Sail. Since SIF2 was found to overlap oneend of SmC3, the location of SmN was unequivocallyestablished at that end (Fig. 3A). SmO was produced from a242-kbp Sall fragment, SlA, indicating that it must bealongside SmK. On the basis of hybridization data and theobservation that SmO contains a 16S rRNA gene (seebelow), the location of SmO was established as shown inFig. 3A.
Location of rRNA genes. In eubacteria, genes encoding 5S,16S, and 23S rRNAs are normally grouped in multicistronicoperons, present in multiple copies in the chromosome (11).To map the rRNA genes in L. lactis subsp. lactis DL11, weused the plasmid pMC5 as a probe. This plasmid has a4.8-kbp insert that encodes the entire rRNA operon of M.capricolum except for 40% of the 16S rRNA at its 5' end (2)(Fig. 5). Southern blots of PFGE separations of L. lactissubsp. Iactis DL11 DNA that had been digested with ApaI,BglI, BssHII, SacII, SalI, and XbaI yielded, respectively,six, five, four, six, three, and five DNA fragment bands thathybridized with the insert from pMC5. Double digests byApaI and Sall, BglI and Sall, and Bgil and BssHII all yieldedsix bands that hybridized with the probe. Figures 2C and Dshow the results when the insert was used to probe Southernblots of the gels shown in Fig. 2A and B, respectively. Theinsert was also used to probe Southern blots of a gel from atwo-dimensional PFGE separation of SmaI-Sall and Sall-SmaI digests of NtA (see above, section iii) and a gel fromfixed-field electrophoresis of SmaI-digested DNA (resultsnot shown). Hybridization was observed with SmM, SmD,SmK, SmC2, SmN, SmI, SmL, SmG, SmO, and SmC3.Hybridization was significantly more intense with the firstsix of these SmaI fragments than with the remaining four.From all these data, it seemed likely that there were sixrRNA operons in the L. lactis subsp. lactis DL11 chromo-
First digestion with SalIcNtA-SlA(200) 200 SmD-SlA(95), NtA-SmC2(60), SmK(45)sic 130 Smg-SIC(70), SmCl-SlC(60)SID 110 SmEl-SlD(85), SmE2-SlD(29)SF1 95 SmD-SlF1(37), SmG-SlF1(37), SmM(22)SIKM 50 SlK1(50)SlM1 33 SIM1(33)SlNld 25 SlN1(25)SlN2d 25 SIN2(25)SlOld 22 S101(22)S102d 22 S102(22)a Fragment nomenclature is defined in Materials and Methods.b Numbers in parentheses indicate the size (in kilobase pairs) of DNA fragments.c Fragments smaller than 20 kbp seen after Sall-SmaI digestion were not detected after SmaI-Sall digestion.dOn the basis of intensity of bands, SIN and SIO were doublets.
some and that there was a SmaI site within the region oflactococcal DNA that hybridized with the pMC5 probe.To examine this possibility further, another rDNA probe
was made by PCR amplification of the region betweennucleotides 665 and 813 (12) of the M. capricolum 16S rRNAgene (Fig. 5). This region corresponds to one end of theinsert in pMC5, is highly conserved in eubacteria (9), anddoes not contain a SmaI site. The 32P-labeled probe washybridized to Southern blots of the gels probed with pMC5or equivalent gels (data not shown). Six different DNAfragments, those at map coordinates 0.478 to 0.508 Mbp(SIF1-SmG), 0.508 to 0.530 Mbp (SmM), 0.660 to 0.664 Mbp(SmO), 0.664 to 0.709 Mbp (SmK), 0.844 to 0.979 Mbp(SmC3), and 2.574 to 2.580 Mbp (SmL-NtB), hybridized toapproximately equivalent extents with this probe (Fig. 3B).Each of these fragments had also hybridized with the pMC5probe. Southern blots of EcoRI restriction fragments, sepa-rated by fixed-field gel electrophoresis, also showed sixbands hybridizing with the PCR probe (data not shown).These observations establish that six regions of the L. lactissubsp. lactis DL11 chromosome have sequence homologywith 16S rRNA and are consistent with the suggestion thatthere are six rRNA operdns in the chromosome.
p 16S
Probe Size
23S 5S t
4.8 kbp
148 bp
FIG. 5. Details of the probes used to locate rRNA operons in L.lactis. Shown is the typical organization of a eubacterial rRNAoperon, with the promoter (p) and terminator (t). The insert in pMC5is a 4.8-kbp fragment of M. capricolum DNA encoding most of an
rRNA operon from this species. PCR was used to amplify a 148-bpDNA fragment located near the 5' end of the 4.8-kbp insert.
DISCUSSION
The restriction map presented for the L. lactis subsp.lactis DL11 chromosome was obtained by the analysis ofpulsed- and fixed-field gel electrophoresis patterns of single,double, and partial restriction endonuclease digests of high-molecular-weight DNA. It is the first map of a lactococcalchromosome. The derivation of this map emphasizes thefeasibility of using totally physical means to characterize thechromosome of organisms for which a genetic map or clonedchromosomal markers are unavailable (25). The use ofrDNA from another bacterial genus (Mycoplasma) as aprobe helped significantly in constructing the map. This is anapproach which may be applicable to chromosome mappingfor other bacteria.The map of the L. lactis subsp. lactis DL11 chromosome
is circular, and the sum of the sizes of the mapped Notl andSmaI fragments indicates that the size of the chromosome is2.58 Mbp. This value is in reasonable agreement withprevious determinations of the total genome size of three L.lactis strains (2.75, 2.8, and 3.1 Mbp) by renaturation kinet-ics (13). Estimates of total genome size obtained by summingthe sizes of all the fragments seen after PFGE of SmaI andApaI digests, while less accurate, also yield values rangingfrom 2.0 to 2.7 Mbp, depending on the strain of L. lactis (20,33). The chromosome of L. lactis is therefore considerablysmaller than those of Pseudomonas aeruginosa (5.9 Mbp[25]), Bacillus subtilis (4.7 Mbp [36]), Escherichia coli (4.55Mbp [31]), and Clostridium perfringens (3.6 Mbp [6]), butlarger than those of Haemophilus influenzae (1.98 Mbp [15])and M. mycoides (1.2 Mbp [23]).
L. lactis is moderately demanding in its nutritional re-quirements, since it must obtain most of its amino acids,purines, pyrimidines, and vitamins from the growth medium.By comparison with enterobacteria, Bacillus spp., andpseudomonads, it is able to utilize only a narrow range of
carbon sources, a further indication of limited biochemicalcomplexity. The relatively small size of the lactococcalchromosome is consistent with the idea that its limitednutritional flexibility is a consequence of the absence ofgenes that would be needed to encode a wide range ofmetabolic functions.The data obtained by probing restriction fragments of L.
lactis DNA with the two probes derived from M. capricolumrDNA indicated the presence in the lactococcal chromosomeof six regions that are homologous to the M. capricolumrRNA operon. From this we suggest that L. lactis containssix rRNA operons. By comparison, E. coli has 7 rRNAoperons (17), B. subtilis has 10 (32), C. perfringens has 9 (6),P. aeruginosa has 4 (11), and M. capricolum has 2 (28). TherRNA hybridization data indicate that each of the putativerRNA operons in L. lactis subsp. lactis DL11 contains aSmaI site. Hence, the precise location of each operon on thephysical map could be determined (Fig. 3B). Five of the sixrRNA operons are clustered in a region representing 20% ofthe chromosome, and all six are located in 40% of thechromosome. Similarly, rRNA operons are clustered in thechromosomes of E. coli (5), B. subtilis (14), and C. perfrin-gens (6).
If it is assumed that the operon organization of lactococcalrRNA genes in L. lactis subsp. lactis DL11 is the same asthat in other eubacteria (promoter, 16S, 23S, 5S, termina-tor), it is possible to deduce the 'orientation of the rRNAoperons in the chromosome (Fig. 3B), since the larger probeused in the hybridization studies spans the SmaI site and theshorter probe, comprising only 16S rDNA, does not. Thisanalysis reveals that the five putative operons between mapcoordinates 0.5 and 1.0 Mbp are in one orientation and thatthe sixth, near the zero map coordinate, is in the otherorientation (Fig. 3B). If replication of the chromosomeproceeded bidirectionally from an origin located betweenmap coordinates 0 and 0.5 Mbp, DNA replication througheach of these operons would be codirectional with transcrip-tion, as it is in E. coli (5).
ACKNOWLEDGMENTS
Sincere thanks to Jane Whitley, Eva Tanskanen, Nina Baseggio,Pam Miller, Rima Youil, and Tristan Orpin for helpful advice and toPhillip Arnold for synthesizing the oligonucleotides.
This work was supported by a grant from the Australian ResearchCouncil.
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