1 School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia
2 School of Agriculture and Food Systems, The University of Melbourne, Werribee, Victoria, Australia
3 Food Science Australia, Werribee, Victoria 3030, Australia
Correspondence
Youssef G. Abs EL-Osta
y.osta{at}unimelb.edu.au
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ABSTRACT |
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Present address: Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia.
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INTRODUCTION |
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Despite the significance of some species of Lactobacillus as fermenting agents and as probiotics, there is a paucity of published information on the content, structure and organization of the genome of a number of species representing the genus. Recently, physical and genetic maps of the chromosomes of Lactobacillus sakei (Dudez et al., 2002) and Lactobacillus gasseri ATCC 33323 (Abs EL-Osta et al., 2002
) have been reported. And more recently, the complete genome sequences of Lactobacillus plantarum WCFS1 (Kleerebezem et al., 2003
) and Lactobacillus johnsonii NCC 533 (Pridmore et al., 2004
) have been determined; other projects are under way to sequence the genome of L. acidophilus ATCC 700396 (see http://www.tigr.org).
The availability of genome sequences for key members of the genus Lactobacillus provides the basis for investigating molecular biological processes which could have important implications for the development of genetically modified strains with improved metabolic characteristics, enhanced vaccine delivery properties and/or probiotic effects. It also provides the basis for extrapolating physical and genetic maps of the chromosomes of related species and strains. Furthermore, the construction of a combined physical and genetic map of the chromosome for a well-defined strain of Lactobacillus has important implications for fundamental molecular biological and biochemical studies and for comparative genome analyses.
This paper describes the construction of a combined physical and genetic map of the chromosome of L. acidophilus neotype strain ATCC 4356, and reports the number, composition and orientation of the rrn operons on the chromosome.
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METHODS |
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Fragment nomenclature.
Fragments produced after digestion of L. acidophilus ATCC 4356 genomic DNA with each of the enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI, were designated Ce, Nt, Cs, Sm, Ap and Sg, respectively, followed by a capital letter suffix, A, B, C, etc., in the order of decreasing fragment size (e.g. Tulloch et al., 1991; Ojaimi et al., 1994
; Abs EL-Osta et al., 2002
). Bands consisting of multiple co-migrating fragments of the same or similar sizes were detected based on their relative staining intensity in ethidium-bromide-stained agarose gels; fragments within these bands were numbered 1, 2 or 3 (e.g. SmG1 and SmG2). Fragments produced by digestion of genomic DNA with two different restriction enzymes were designated by the two single-digested fragments from which they were derived (e.g. NtA-CsA is a DNA fragment with a portion being within fragment NtA and the remaining portion within fragment CsA) (Abs EL-Osta et al., 2002
).
Preparation of hybridization probes.
Probes used to analyse the rRNA (rrn) operons and to locate genetic markers on the map were amplified by PCR from genomic DNA of L. acidophilus or from other related species of Lactobacillus or Lactococcus, using specific primers (Tables 1 and 2). Genomic DNA (30100 ng) was amplified in a total reaction volume of 100 µl, as previously described (Abs EL-Osta et al., 2002
). For PCR amplification of DNA template embedded in agarose slices, the weight of each slice was estimated and water added such that the final concentration of agarose was 0·5 % (w/v). The slices were then incubated at 65 °C until dissolved, and an aliquot (410 µl) was used directly in PCR. Amplicons which represented a single band in agarose gels were purified using the QIAquick PCR purification kit (Qiagen), and either labelled with [
-32P]dATP and used as probes, or cloned into Vector pGEM-T Easy (Promega) for transformation into Escherichia coli JM107. The identity of each PCR product was verified by sequencing using the Big Dye Terminator kit (Perkin Elmer Cetus), employing specific primers (Tables 1 and 2
), or primers T7 or SP6 (Promega) for cloned products, followed by comparison with related sequences in the GenBank database.
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RESULTS |
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Visual inspection of the relative intensity of ethidium-bromide-stained bands in PFGE gels suggested that some bands contained two or more co-migrating fragments with the same electrophoretic mobility, and thus the same or similar size (e.g. SmG1 and SmG2; ApB1 and ApB2) (Table 4). The presence and identification of all the co-migrating and closely migrating fragments was subsequently confirmed by hybridization analyses of L. acidophilus chromosomal DNA digests and from the constructed physical map (Fig. 1
).
Based on the total sum of the mean fragment sizes obtained by digesting chromosomal DNA with each of I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI, the chromosomal size of L. acidophilus was estimated at 2·061 Mbp (Table 4).
Map construction
Ordering of the I-CeuI, NotI and CspI chromosomal fragments.
The combined use of one- and two-dimensional PFGE enabled the construction of the physical map of the chromosome for I-CeuI and NotI. These enzymes were selected for the initial construction of the map, because they produced the least number of restriction fragments compared with the other enzymes used (Table 4). Therefore, chromosomal DNA was digested with each of NotI and I-CeuI, and the resultant restriction fragments separated by PFGE. Fragments were then excised from the gel and digested with the second (reciprocal) enzyme before PFGE separation in the second dimension (Fig. 2
). The one-dimensional section of the gel revealed the presence of four I-CeuI fragments (CeA, CeB, CeC and CeD), three NotI fragments (NtA, NtB and NtC) and seven subfragments in the I-CeuI+NotI double digests (Fig. 2
; Table 4
). For ease of interpretation, fragment CeA was placed to the right of the map (Fig. 1
), and the analysis commenced with the digestion of CeA by NotI, which produced three subfragments of 821, 339 (i.e. fragment NtC) and 81 kbp (Table 4
). Since the 821 kbp subfragment was a portion of fragment NtA and the 81 kbp subfragment a portion of fragment NtB, then fragment NtC was positioned within fragment CeA between the 821 and 81 kbp subfragments (Fig. 1
). Furthermore, fragment NtA was positioned on the map with the 821 kbp portion on one end of fragment CeA, and fragment NtB was positioned with 81 kbp on the other end of fragment CeA. Since fragment CeA was placed to the right of the map containing part of fragment NtA (821 kbp), and given the circular nature of the chromosome, the remaining 156 kbp portion of NtA was placed to the left of the map. The 156 kbp subfragment also represented a portion of fragment CeB, and the remaining 288 kbp portion of fragment CeB was contained within fragment NtB (Table 4
). This led to the placement of fragment CeB to the left of the map and confirmed the position of fragment NtB as being adjacent to the 156 kbp portion of fragment NtA (Fig. 1
).
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The remaining fragments (CeC, CeD, CsB and CsE) were located on the map by Southern blot analysis; a 4·1 kbp insert from a clone selected from fragment CeD DNA library was used as a probe in Southern blot analysis of L. acidophilus DNA digested with each of I-CeuI, NotI and CspI. This probe hybridized to fragments CeD, CsA and NtB (Fig. 3), indicating that CeD was adjacent to CeA, and fragment CeC adjacent to CeB, thus allowing the order of the I-CeuI fragments to be determined. Similarly, a probe from the fragment CsE library (3·8 kbp insert) hybridized to fragments CsE, NtB and CeC (Fig. 3
), thus placing fragment CsE adjacent to CsA, and CsB adjacent to CsC, which concluded the ordering of the CspI fragments (Fig. 1
). The order of the I-CeuI and CspI fragments was also confirmed by two-dimensional PFGE analysis of reciprocal double digests of L. acidophilus genomic DNA using I-CeuI and CspI (data not shown).
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The SmaI fragments 40 kbp, excluding the doublets SmG1, G2 and SmK1, K2, were allocated within the larger fragments by two-dimensional PFGE analysis of genomic DNA digested with each of I-CeuI, NotI and CspI (results not shown), and by hybridization analysis using the linking probes (Table 3
). The co-migrating fragments, and those of
40 kbp, were allocated by Southern blot analysis using specific SmaI fragments as probes. Initially the SmaI fragments were excised from either one- or two-dimensional PFGE, radiolabelled and hybridized with genomic DNA digested with each of NotI, CspI and I-CeuI (Table 5
). Subsequently, small (0·35 kbp) probes representing the SmaI fragments (Table 5
) produced by PCR from SmaI bands excised from PFGE gels were radiolabelled and used in Southern blot analysis of PFGE separations of L. acidophilus chromosomal DNA digested with each of I-CeuI, NotI and CspI (Table 5
). Using this approach, the SmaI fragments SmG1, G2, H, K1, K2, M, N1, N2, O, P1, P2, Q, R, S, T, U and V could be located within the larger restriction fragments of NotI, CspI and I-CeuI (Table 5
).
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Analysis of the rRNA (rrn) operons
A typical eubacterial rrn operon consists of a single copy of the 16S (rrs), 23S (rrl) and 5S (rrf) rRNA genes in the transcriptional order 16S23S
5S, with an intergenic spacer between each gene (Wagner, 1994
; Garcia-Martinez et al., 1999
). To assist in the analysis of the rrn operons of L. acidophilus, specific probes were produced by PCR amplification, to particular regions of the operon (Table 2
, Fig. 4
). PCR products PISR2, IU and 23SB were sequenced, leading to the determination of the nucleotide sequence of the rrl gene for L. acidophilus (accession no. AJ620360), which contained a single restriction site for each of ApaI and I-CeuI. Further analysis of the nucleotide sequence of the rrl gene revealed significant identity (up to 88·2 %) to the rrl gene sequences for other lactic acid bacteria.
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Identification and characterization of the ribosomal intergenic spacers
PCR amplification of L. acidophilus genomic DNA employing a forward primer at the 3'-end of the rrs gene and a reverse primer to a number of different positions within the rrl gene (Fig. 4, Table 2
) always produced two products. Sequence analyses of these products revealed the presence of two types of spacers between the rrs and rrl genes (16S23S spacer), namely a long spacer (451 bp) containing two tRNA genes (tRNAAla and tRNAIle), and a short spacer (201 bp) lacking any tRNA gene, in accordance with previous reports (Nour, 1998
). Sequencing results indicated that the restriction sites for SmaI (within the rrs gene) and I-CeuI (within the rrl gene) were separated by 2513 bp or 2266 bp when the long or short 16S23S rRNA spacers, respectively, were included (Fig. 4
). In Southern blot analysis, probe TR (Table 2
, Fig. 4
) hybridized to fragments CsD, NtA, CeB and SmP1, indicating that operon rrnD contains a long 16S23S spacer and supporting the finding that this operon was inverted with respect to the other three. Interestingly, probe TR also hybridized specifically to fragments SmA, CsA, CeA and NtA, indicating that either a tRNAAla gene or a homologous sequence was present in the 359 kbp SmA-NtA region of the chromosome lacking rrn operons (Fig. 2
).
Mapping of the SmaI fragments to the region containing rrn gene loci
Using a combination of two-dimensional PFGE and Southern blot analyses, the SmaI fragments were assigned to different I-CeuI fragments (results not shown). Also, PFGE analyses of L. acidophilus genomic DNA digested with SmaI+I-CeuI demonstrated that fragments SmF and SmL could be digested with I-CeuI. Fragment SmF was located within CeC and was digested with I-CeuI but not with CspI. Given that all the rrn probes (Table 2), except P16S and TR, hybridized to fragment SmF, and considering that operon rrnA contained a short intergenic spacer, fragment SmF was placed on the map with a portion (2266 bp) being within fragment CeB and the remaining part being within fragment CeC. Similar analyses using I-CeuI digestion and hybridization of rrn probes placed a portion of fragment SmL (2266 bp) within fragment CeD and the remainder within CeA. This confirmed the initial positioning on the map of fragment SmL in the region of operon rrnC via the hybridization with linking probe LC8 (Table 3
). Further hybridization analyses placed fragment SmD on the map in the region of operon rrnB. From all of the rrn probes (Table 2
), only P16S and PISR3 hybridized to fragment SmD. Therefore, it was placed on the map within fragment CeC, at a distance of 2266 bp from the I-CeuI site separating fragments CeC and CeD. Fragment SmS corresponded to part of operon rrnD, and could thus be positioned within CeB, 2513 bp from the junction between fragments CeB and CeA (Fig. 1
).
All of the rrn probes (Table 2) except P16S hybridized to fragment SmP1. Therefore, it could be positioned in the region of operon rrnD, within fragment CeB at a distance of 2513 bp from the I-CeuI site. Hybridization analysis using the rrn probes also enabled the positioning of fragments SmV and SmT in the region of rrnB and rrnC, respectively (Table 2
, Fig. 1
). Similarly, SmN1 was positioned in the vicinity of operon rrnA (Table 2
, Fig. 1
).
Mapping of the remaining SmaI fragments using linking probes
The L. acidophilus genomic library was extensively screened to isolate clones with inserts containing SmaI restriction sites. These inserts were used as probes in Southern blot analyses of L. acidophilus DNA digested with each of SmaI, CspI, NotI and I-CeuI (Table 3) and separated by PFGE. This approach allowed adjacent SmaI fragments to be positioned on the map and the position of restriction fragments produced by the other enzymes to be inferred. Based on the assignment of fragment SmN2 within fragments NtA and CeB, and considering the size of the vacant space on the SmaI map, fragment SmN2 was positioned between fragments SmR and SmS (Fig. 1
). Similarly, SmU was placed between fragments SmG2 and SmQ, thus concluding the arrangement of the SmaI restriction fragments on the map (Fig. 1
).
Construction of the genetic map and extension of the physical map to include ApaI and SgrAI fragments
Based on the physical map, a genetic map was constructed by probing restriction digests of L. acidophilus genomic DNA with specific probes carrying complete or partial genes (Table 1). The genetic loci were allocated to the smallest fragment to which the gene probes hybridized (Fig. 1
). This approach allowed the positioning of 73 gene loci including those of the rrn operons on the map (Fig. 1
).
The linking probes used to complete the SmaI map consisted of relatively large L. acidophilus DNA fragments (ranging in size from 1·5 to 9·85 kbp) (Table 3). Approximately 800 bp of both the 3'- and 5'-ends of the linking probes were sequenced, and the data used to search for similar sequences in the GenBank database. The similarity searches revealed that the 3' end of probe LC4 had 57 % identity (over 213 bp) to the RNA methyltransferase gene yfi of Bacillus subtilis (accession no. F69806). Hybridization analysis using probe LC4 (Table 3
) allowed the positioning of the putative yfi gene on the map in the region spanning fragments SmC and SmK2 (Fig. 1
). However, further hybridization analyses of ApaI and SgrAI digests using probe LC4 (Table 3
) narrowed the region within which the putative yfi gene was contained (Fig. 1
). Similarly, probe LC2 (Table 3
) had 79·3 % identity (over 800 bp) to the tpi gene of L. delbrueckii (Branny et al., 1998
). Hybridization results achieved using this probe (Table 3
) confirmed the previous positioning of the tpi gene locus using the homologous gene probe (Table 1
). Comparison of the partial sequences of the other linking probes (Table 3
) did not reveal any significant matches to any gene found in current sequence databases.
Based on the positions of gene loci and the physical map constructed for I-CeuI, NotI, CspI and SmaI, an extended physical map for ApaI and SgrAI was constructed by employing similar strategies to those used to position the SmaI fragments (data not shown). Southern blot analyses of PFGE separations of L. acidophilus DNA digested with ApaI and SgrAI using the linking, rrn (Table 2) and gene probes (Table 1
) allowed the physical map to be extended to include the enzymes ApaI and SgrAI (Fig. 1
).
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DISCUSSION |
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The constructed physical and genetic map of the L. acidophilus ATCC 4356 chromosome contained 86 restriction sites for six endonucleases (I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI) and 73 genes, including the rrn genes. An interesting finding was the clustering of restriction sites on the L. acidophilus physical map for some endonucleases. Of the 26 SmaI restriction sites, 15 (61·5 %) clustered in a 725 kbp region (35·2 %) of the chromosome, between coordinates 373 kbp and 1098 kbp (Fig. 1). Similarly, of the 27 ApaI sites 17 (63 %) were clustered in a 703 kbp region (34·1 %) of the chromosome, between coordinates 362 kbp and 1065 kbp (Fig. 1
). Also in the latter region were three of the five CspI and three of the four I-CeuI restriction sites. Clustering of the rrn operons was also detected in similar regions of the chromosome. In contrast, no such clustering was observed for SgrAI restriction sites (Fig. 1
). Clustering of restriction sites for some endonucleases indicates differences in the G+C content between different parts of the genome. It seems that the regions with restriction enzyme clustering are richer in G+C content compared with other regions of the chromosome, which is supported by the finding that in L. plantarum the G+C content is not evenly distributed on the chromosome (Kleerebezem et al., 2003
). This aspect could be investigated once the entire genome sequence for L. acidophilus has been determined. PFGE-hybridization analyses of I-CeuI digests revealed that the chromosome of L. acidophilus ATCC 4356 contains four rrn operons. The number of rrn operons has been reported as five in L. plantarum (Kleerebezem et al., 2003
), six in L. gasseri ATCC 33323 (Abs El-Osta et al., 2002
), six in L. johnsonii (Pridmore et al., 2004
) and seven in L. sakei (Dudez et al., 2002
). There appears to be an inverse relationship between genome size and the number of rrn operons. For example, L. acidophilus, whose genome is 2·061 Mbp, has four rrn operons, whereas the smaller genomes of L. gasseri (1·96 Mbp), L. johnsonii (1·99 Mbp) and L. sakei (1·845 Mbp), contain six, six and seven rrn operons, respectively. This observation is reinforced by other examples, such as Pseudomonas aeruginosa, which possesses a 5·9 Mbp genome (Römling et al., 1989
) with four rrn operons (Hartmann et al., 1986
), contrasting with Clostridium perfringens, which has a 3·6 Mbp genome containing nine rrn operons (Canard & Cole, 1989
). An interesting feature was the clustering of the rrn operons on the chromosome of L. acidophilus. While the four operons clustered in a region of 820 kbp (representing 40 % of the chromosome), three of them clustered in a region representing only 18 % of the chromosome (Fig. 1
). Chromosomal clustering of the rrn loci has not been found in L. plantarum (Kleerebezem et al., 2003
), but it has been described for L. gasseri (Abs EL-Osta et al., 2002
), L. sakei (Dudez et al., 2002
) and Lactococcus lactis (Tulloch et al., 1991
; Le Bourgeois et al., 1992
) and for prokaryotes other then the lactic acid bacteria, such as E. coli, Bacillus subtilis, Haemophilus influenzae, Pasteurella multocida and Streptococcus thermophilus (Lee et al., 1989
; Bentley et al., 1991
; Roussel et al., 1994
; Itaya, 1997
; Hunt et al., 1998
). In these bacteria, the rrn loci are often located within one-third to one-half of the chromosome. Each rrn operon in L. acidophilus ATCC 4356 contained a single copy of each rrn genes in the usual gene order (rrs
rrl
rrf) which is found in most bacteria (Wagner, 1994
; Gürtler & Stanisich, 1996
). If these genes are indeed transcribed in the same order as in most other bacteria (Davidson et al., 1996
), then one of the four operons of L. acidophilus (rrnD) would be transcribed in the reverse direction compared with the other three, relative to a common region within fragment CeB (Fig. 1
). The location and orientation of the rrn operons are of relevance because they indicate the position of oriC, the origin of replication (Tulloch et al., 1991
). In eubacteria, such as E. coli, replication and transcription of the rrn operons are collinear (Cole & Saint Girons, 1994
). If this were the case also in Lactobacillus, then the results obtained in this study indicate that oriC is located within fragment CeB. This conclusion is supported by the finding that the genes gyrB and dnaA, which are usually found near the origin of replication for a wide range of bacteria (Ogasawara & Yoshikawa, 1992
), are located within fragment CeB on the L. acidophilus map (Fig. 1
). Hence, fragment CeB should be a useful source of DNA for attempts at shotgun cloning and functional analyses of the genes found at the origin of replication in the genome of Lactobacillus. Other important ribosomal regions characterized were the 16S23S rRNA intergenic spacers. Interestingly, operon rrnD (Fig. 4
) had a long spacer (451 bp) containing two tRNA genes (tRNAIle and tRNAAla), whereas the remaining three operons each contained a short spacer (201 bp) without any tRNA genes. Southern blot analyses also suggested that a sequence homologous to at least part of tRNAAla was located in a region of the chromosome (fragment ApB2-SgF2) (Fig. 1
) lacking rrn operons. This is in agreement with the findings that a number of tRNA gene sequences are found outside the rrn operons of L. plantarum WCFS1 (Kleerebezem et al., 2003
) and L. johnsonii NCC533 (Pridmore et al., 2004
).
After having established the physical map of the chromosome of L. acidophilus ATCC 4356 for the restriction enzymes I-CeuI, NotI, CspI and SmaI, 61 markers representing a wide range of genes were located to the genetic map. These included partial genes for enzymes involved in the proteolytic system, the glycolytic pathway, carbohydrate metabolism, and amino acid and purine/pyrimidine biosynthesis (Table 1). The genetic map of L. acidophilus also included a number of genes proposed to be associated with important probiotic properties, such as those relating to the bacterial adaptation to heat or cold shocks imposed during the production, storage and/or distribution of probiotic food products (Vaughan et al., 1999
; Walker et al., 1999
). Specifically, the genes groES and groEL (induced under a heat shock) were mapped to fragment SmB (214 kbp), and cspL (induced under a cold shock) was mapped to fragment SmA (500 kbp) of the chromosome (Fig. 1
). This should provide the starting point for isolating these genes, in order to investigate the molecular biological processes triggered under heat- or cold-shock responses in L. acidophilus. Moreover, the positioning on the map of the slpA and dltA genes (Fig. 1
), both of which are associated with the adhesion/colonization of probiotic lactobacilli in the host intestine (Boot et al., 1996
; Vaughan et al., 1999
), could have important implications for detailed investigations into probiotic processes and mechanisms.
In conclusion, the availability of a physical and genetic map of the chromosome of L. acidophilus ATCC 4356 should have important implications for fundamental and applied research on Lactobacillus. The map constructed provides a platform for comparative genome analyses and opportunities to develop methods for the accurate identification of Lactobacillus species and strains. For instance, the rrn genes or the intergenic spacers could be utilized in PCR-based assays for the quality control of starter cultures prior to, during and after fermentation processes, and during the storage of fermented products containing live bacterial cultures. Such tools would also be applicable to investigating the systematics, ecology and population genetics of Lactobacillus and other lactic acid bacteria.
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ACKNOWLEDGEMENTS |
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Received 4 March 2004;
revised 15 October 2004;
accepted 2 November 2004.
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