Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

Youssef G. Abs EL-Osta1,{dagger}, Alan J. Hillier2,3 and Marian Dobos1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2·061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (~0·05–4·3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnAD) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S–23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.


The GenBank/EMBL/DDBJ accession number for the 23S rRNA (rrl) gene reported in this paper is AJ620360.

{dagger}Present address: Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Lactobacillus belongs to the lactic acid bacteria, which comprise a heterogeneous group of micro-organisms. The diversity of the genus is reflected in the range of G+C content (32–55 mol%) of the DNA from different species (Collins et al., 1991; Hammes & Vogel, 1995; Vandamme et al., 1996). A number of species of Lactobacillus have been used for centuries as fermenting agents in the manufacturing of a range of food products. Some species act as food preservatives by virtue of their acidification of the fermented products and/or by the production of bacteriocins, which suppress the growth of food-poisoning bacteria (McKay & Baldwin, 1990; De Vuyst & Vandamme, 1994; Klaenhammer, 1995; Stiles, 1996). Several Lactobacillus species are claimed to have significant beneficial health effects in humans and animals. These effects include anti-carcinogenic and anti-mutagenic activities, control of serum cholesterol levels, enhanced immune responses and improved lactose utilization in individuals with lactose intolerance. The evidence of these beneficial effects has led to the use of a broad range of Lactobacillus species as ‘probiotic’ bacteria (e.g. Gilliland, 1990; Goldin, 1998; Vaughan et al., 1999; Gorbach, 2000; Rolfe, 2000; Mercenier et al., 2003). Also, in recent years, selected Lactobacillus species have been used as vaccine delivery systems, which can be administered orally and can effectively induce protective immune responses (Mercenier et al., 1996; Wells et al., 1996; Pouwels et al., 2001; Seegers, 2002; Scheppler et al., 2002).

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Preparation, digestion and electrophoretic separation of genomic DNA.
L. acidophilus neotype strain ATCC 4356 was grown at 37 °C in MRS medium (De Man et al., 1960). Genomic DNA was extracted in solution as described by Marmur (1961). Also, genomic DNA was prepared and digested in 2 % (w/v) agarose blocks for one- and two-dimensional PFGE analysis in 1 % (w/v) agarose gels, using either a CHEF-DRII or CHEF Mapper apparatus (Bio-Rad), as described previously (Abs EL-Osta et al., 2002). The number and mean sizes of all DNA fragments resolved by PFGE were estimated from >=20 different PFGE runs, using different electrophoretic conditions. These conditions were: 10 h with a pulse time ramped from 0·1 to 10 s (for the separation of 1–50 kbp fragments), 19 h with a pulse time of 3 to 13 s (50–200 kbp fragments), 22 h with a pulse time of 1 to 45 s (200–600 kbp fragments) and 22 h with a pulse time of 20 to 180 s (600–1600 kbp fragments). PFGE was performed in 0·5x TBE (65 mM Tris/HCl, 22·5 mM boric acid, 1·25 mM EDTA, pH 9) buffer at 200 V and 15 °C (constant). Fragment sizes were determined by comparison with Saccharomyces cerevisiae chromosomes, lambda ({lambda}) DNA concatemers and {lambda} DNA digested with HindIII size markers (New England BioLabs). To investigate the presence of DNA fragments <=15 kbp, all digests were resolved in conventional (1 %, w/v) agarose gels and stained with ethidium bromide. To detect the presence of fragments of <=1 kbp, high-molecular-mass chromosomal DNA was digested with each of NotI, I-CeuI, CspI, SmaI, ApaI and SgrAI followed by 5'-end-labelling with [{gamma}-33P]dATP in agarose blocks. Digests were then resolved by conventional agarose gel electrophoresis followed by gel drying and autoradiographic detection (Sambrook et al., 1989).

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 (30–100 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 (4–10 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 [{alpha}-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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Table 1. Oligonucleotide primers used in PCR to amplify specific gene probes used in Southern blot hybridization to position genetic markers on the L. acidophilus physical map

 

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Table 2. Oligonucleotide primers used in PCR to amplify specific rRNA (rrn) probes employed in Southern blot hybridization for the analysis of the rrn operons of L. acidophilus, and to position restriction fragments on the physical map

 
Fourteen linking probes (LC1–LC14) (Table 3) were isolated from a library constructed from L. acidophilus genomic DNA (4–10 kbp) cloned into plasmid pUC19 (Yanisch-Perron et al., 1985, Sambrook et al., 1989). The library was specifically screened for clones carrying plasmids containing inserts with SmaI restriction sites. Based on the white/blue colour selection of transformed E. coli PMC112 (Doherty et al., 1993; Raman et al., 1997), ~60 000 white colonies were obtained, and 10 000 of these were individually examined, by plasmid preparation and restriction digestion, for the presence of inserts containing SmaI sites. Competent E. coli cells were transformed using the method of Hanahan (1983) or by high-voltage electroporation (Dower et al., 1988; Sambrook et al., 1989), and plasmid DNA was isolated using the mini-prep alkaline lysis method (Birnboim & Doly, 1979) or the plasmid Midi Kit (Qiagen). Two partial libraries were also constructed from the DNA of fragments CeD and CsE, respectively (Table 4). Each of these fragments was excised from PFGE agarose gels and partially digested in situ with Sau3AI (Albertsen et al., 1989). The digested DNA was then purified from agarose and used to construct partial libraries using the same approach as used for the construction of genomic libraries.


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Table 3. Hybridization of the linking probes LC1–LC14 to L. acidophilus chromosomal DNA digested with each of SmaI, I-CeuI, NotI and CspI, and double digested with NotI+SmaI and CspI+SmaI

Linkages between the SmaI fragments determined by hybridization analysis are shown.

 

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Table 4. PFGE analysis of single, double and reciprocal double digests of L. acidophilus chromosomal DNA

 
Southern blot hybridization.
DNA resolved in agarose gels by one- or two-dimensional PFGE was transferred to nylon membranes (Hybond-N+, Amersham) by depurination (Reed & Mann, 1985) and hybridized with probes labelled with [{alpha}-32P]dATP by random priming (Feinberg & Vogelstein, 1984). Hybridization and subsequent stringent washings were carried out according to standard procedures (Sambrook et al., 1989).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PFGE analysis of genomic DNA restriction digests, and estimation of chromosomal size
PFGE analysis of undigested genomic DNA from L. acidophilus ATCC 4356 revealed no evidence of extrachromosomal bands on the gels. Furthermore, plasmids were not detected in the cells, by using two different methods for the isolation of large plasmid DNA (Anderson & Mckay, 1983; O'Sullivan & Klaenhammer, 1993), thus providing support for the absence of plasmids in L. acidophilus ATCC 4356. Undigested L. acidophilus DNA in agarose plugs did not migrate in PFGE gels under a number of different pulse conditions (results not shown), thus indicating the circular nature of the chromosome (Ojaimi et al., 1994; Walker et al., 1999), which was subsequently supported by the constructed physical map (Fig. 1).



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Fig. 1. Combined physical and genetic map of the chromosome of L. acidophilus ATCC 4356 incorporating the restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI. (a) The 2·061 Mbp circular chromosome has been linearized at the I-CeuI site in the rrnD operon. Positions of the I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI restriction sites are shown, and fragment names are indicated. (b) Genetic map of the L. acidophilus chromosome constructed based on the physical map shown in (a). The gene loci are positioned on the map to the minimum region localized by hybridization as indicated by boxes and vertical lines. The order of genes given in a particular region is arbitrary and does not necessarily represent the actual order of genes in that region. Arrowheads indicate the direction of transcription of the rrn operons consisting of the gene order rrs-rrl-rrf. The fork heads on the upper section of the genetic map indicate the positions of the rrn genes, with the centre of the fork being the restriction site for I-CeuI. Fragments which were equivocally positioned on the map are shaded.

 
Given the low G+C content of L. acidophilus (32–37 mol%; Kandler & Weiss, 1986), genomic DNA was initially digested with several rare-cutting restriction enzymes which recognize G+C-rich sequences. Based on their ability to digest the genome into a relatively small number of fragments which could be resolved by PFGE (Table 4), the restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI were selected for the construction of the physical map. Chromosomal DNA from L. acidophilus was digested with each of the selected enzymes and the resultant restriction fragments separated by PFGE. Using a range of different electrophoretic conditions in PFGE, it was possible to resolve all restriction fragments >=15 kbp and to determine their sizes accurately compared with size markers (Table 4). By employing conventional agarose gel electrophoresis, fragments <=15 kbp were not detected in the NotI, I-CeuI or CspI digests. However, two fragments of 4 and 8 kbp were detected in the SmaI digest, four fragments of 5, 6 (doublet) and 7 kbp were detected in the ApaI digest and three fragments of 6, 13 and 14 kbp in the SgrAI digest (Table 4). DNA fragments of <=1 kbp are usually found at low molecular concentrations in agarose blocks and are difficult to detect using PFGE or conventional gel electrophoresis. No such fragments were detected in the L. acidophilus chromosomal DNA digests, using the sensitive approach of end-labelling digests with [{gamma}-33P]dATP in agarose blocks, followed by conventional agarose gel electrophoresis and autoradiography.

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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Fig. 2. Two-dimensional PFGE analysis of DNA fragments produced by single, double and reciprocal double digests of L. acidophilus ATCC 4356 chromosomal DNA using the restriction enzymes NotI and I-CeuI. The one-dimensional (1D) section of the gel shows the separations of {lambda} HindIII digest (lane 1), S. cerevisiae chromosomes (lane 2), {lambda} DNA concatemers (lane 3), and L. acidophilus chromosomal DNA digested with I-CeuI (lane 4), NotI (lane 5) and I-CeuI+NotI (lane 6). The two-dimensional (2D) section I of the gel shows the separations of the I-CeuI fragments digested with NotI. The two-dimensional section II of the gel shows the separations of the NotI fragments digested with I-CeuI. Electrophoresis was performed in a 1 % (w/v) agarose gel with pulse times of 1–80 s at 200 V for 21 h in 0·5x TBE buffer at 15 °C in a Bio-Rad CHEF DRII apparatus. Size markers (kbp) are indicated to the left of the gel. The results obtained from this gel are summarized in Table 4.

 
Reciprocal double digests of L. acidophilus genomic DNA with NotI and CspI were also analysed by two-dimensional PFGE (Table 4). Based on the established map for I-CeuI and NotI, CsA was placed on the map spanning 552 kbp at the right end of NtB, the entire size of NtC and 109 kbp of the left end of NtA (Fig. 1). Fragment CsC was located with 130 kbp within NtA and 228 kbp within NtB. Fragment CsD was also located within NtA, between CsC and CsA. Both fragments CsB and CsE were found within NtB, but it was not possible to determine their order from the two-dimensional PFGE results.

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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Fig. 3. Positioning of fragments CsD and CsE on the map. (a) PFGE separations of {lambda} HindIII digest (lane 1), {lambda} DNA concatemers (lane 2), S. cerevisiae chromosomes (lane 3), and L. acidophilus chromosomal DNA digested with I-CeuI (lane 4), CspI (lane 5) and NotI (lane 6). Electrophoresis was performed in a 1 % (w/v) agarose gel with pulse times of 10–180 s at 200 V for 19 h in 0·5x TBE buffer at 15 °C in a Bio-Rad CHEF Mapper apparatus. (b, c), Southern blots of the gel in (a), hybridized with specific probes. Size markers (kbp) are shown to the left.

 
Allocation of the SmaI restriction fragments.
The physical map established for the enzymes I-CeuI, NotI and CspI (Fig. 1) was used as a skeleton to position the SmaI fragments using a combination of the following strategies: (i) assignment of SmaI fragments to those produced by digestion of L. acidophilus DNA with each of NotI, I-CeuI and CspI, (ii) positioning of the SmaI fragments containing restriction sites for I-CeuI, CspI or NotI, (iii) positioning of the SmaI fragments in the region of the rrn operons, and/or (iv) linking of SmaI fragments by hybridization analysis using specific linking probes selected from several L. acidophilus genomic libraries.

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 (0·3–5 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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Table 5. Allocation of the co-migrating and small L. acidophilus SmaI restriction fragments to the restriction fragments produced from the digestion of L. acidophilus chromosomal DNA with each of I-CeuI, NotI and CspI

 
Mapping of the SmaI fragments containing restriction sites for NotI or CspI.
A single NotI restriction site was detected within each of the fragments SmA and SmB (Table 3), which resulted in their positioning on the map in the region of two NotI restriction sites (Fig. 1). The third NotI site was detected within fragment SmK1 by hybridization with linking probe LC8 (Table 3), which allowed its positioning on the map adjacent to fragment SmL and localization of the boundary between the two fragments within fragment NtB (Fig. 1). Two-dimensional PFGE of reciprocal double digests of genomic DNA with SmaI+CspI identified a CspI restriction site within fragments SmC and SmD, enabling these fragments to be mapped (Fig. 1). Based on the two-dimensional PFGE and hybridization results, six of the 26 SmaI fragments could be accurately positioned on the map. Mapping of the remaining SmaI fragments was achieved by hybridization analyses using probes targeting the ribosomal operons as well as linking and gene specific probes.

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 16S->23S->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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Fig. 4. Schematic representation of the two types of rRNA operons found in L. acidophilus ATCC 4356. (a) Operon containing the short 16S–23S rRNA intergenic spacer region. The rRNA genes (rrs, rrl and rrf) are represented by boxes and the locations of the restriction sites for the restriction enzymes SmaI, ApaI and I-CeuI are shown. (b) Section of the rRNA operon showing the long 16S–23S rRNA intergenic spacer region containing the genes tRNAIle and tRNAAla. The positions of the probes used to analyse the rRNA operons are indicated with black bars (see also Table 2). Amplicon 23SB was not used as a probe.

 
The enzyme I-CeuI recognizes a highly conserved 26 bp sequence within the rrl genes of most eubacteria (Liu et al., 1993). Therefore, the digestion of L. acidophilus genomic DNA with I-CeuI into four fragments was indicative of the presence of four rrn operons on the chromosome (Fig. 1). This was verified by Southern blot analysis in which each of three ribosomal probes P16S, P23S and P5S (Table 2) hybridized to four bands on a Southern blot of L. acidophilus chromosomal DNA digested with each of BamHI, HindIII and BglII (results not shown). Considering that none of the three probes contained restriction sites for BamHI, HindIII and BglII, and that the hybridizing fragments were small enough to rule out the possibility of tandem repeated operons, the hybridization results suggested the presence of a single copy of each of the rrs, rrl and rrf genes in each rrn operon, assuming that none of these genes was present external to any rrn operon. Analyses of genomic DNA digested with I-CeuI and hybridized with each of the probes P16S, P23S and P5S (Table 2) indicated that operon rrnD (which is located at the junction between fragments CeA and CeB) was oriented in the opposite direction relative to the other three operons (Fig. 1).

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 (16S–23S 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 16S–23S 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 16S–23S 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).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PFGE combined with Southern blot analyses allowed the construction of a physical and genetic map of the L. acidophilus ATCC 4356 chromosome. The size of the circular chromosome was determined to be 2·061 Mbp, which is similar to that of L. acidophilus IP7613 (1·85 Mbp), L. gasseri IP102991 (2·02 Mbp) (Roussel et al., 1993), L. gasseri ATCC 33323 (1·96 Mbp) (Abs EL-Osta et al., 2002), L. delbrueckii subsp. bulgaricus (2·30 Mbp) (Leong-Morgenthaler et al., 1990), L. sakei (1·845 Mbp) (Dudez et al., 2002) and L. johnsonii NCC533 (1·99 Mbp) (Pridmore et al., 2004) but smaller compared with L. plantarum (3·3–4 Mbp) (Chevallier et al., 1994; Kleerebezem et al., 2003). The size difference between the former species and L. plantarum may relate to insertion/deletion events and/or different evolutionary rates within the genus Lactobacillus. Interestingly, the chromosomes of Lactobacillus species are smaller than those of a number of other prokaryotes, such as E. coli K-12 (4·7 Mbp) (Smith et al., 1997), Pseudomonas aeruginosa (5·9 Mbp) (Römling et al., 1989), Streptomyces griseus (7·8 Mbp) (Lezhava et al., 1995) and Bacillus cereus (5·7 Mbp) (Kolsto et al., 1990). This may relate to the evolution of the lactobacilli in nutritionally rich environments, during which some of the genes encoding enzymes involved in non-essential amino acid biosynthetic pathways may have been lost (Klaenhammer, 1995).

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 16S–23S 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.


   ACKNOWLEDGEMENTS
 
Many thanks to the late Professor Barrie E. Davidson for initiating this project and for all of his help and inspiration, especially during the PhD candidature of Youssef G. Abs EL-Osta. We would also like to thank: Todd Klaenhammer for originally providing L. acidophilus ATCC 4356; Peter Pouwels for plasmid pBK1 carrying the S-layer protein gene SlpA of L. acidophilus; Takao Igarashi for plasmid pMN115 carrying the manganese catalase gene catM of L. plantarum; and Jean Delcour for plasmid pGIN012 carrying the D-LDH gene of L. delbrueckii subsp. bulgaricus.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Abs EL-Osta, Y. G., Hillier, A. J., Davidson, B. E. & Dobos, M. (2002). Pulsed field gel electrophoretic analysis of the chromosome of Lactobacillus gasseri ATCC33323. Electrophoresis 23, 3321–3331.[CrossRef][Medline]

Albertsen, H. M., Le Paslier, D., Abderrahim, H., Dausset, J., Cann, H. & Cohen, D. (1989). Improved control of partial DNA restriction enzyme digest in agarose using limiting concentrations of Mg++. Nucleic Acids Res 174, 808.

Anderson, D. G. & Mckay, L. L. (1983). Simple and rapid method for isolating plasmid DNA from lactic streptococci. Appl Environ Microbiol 46, 549–552.[Medline]

Bentley, R. W., Leigh, J. A. & Collins, M. D. (1991). Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Int J Syst Bacteriol 41, 487–494.[Abstract]

Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513–1523.[Abstract]

Boot, H. J., Kolen, C. P., Andreadaki, F. J., Leer, R. J. & Pouwels, P. H. (1996). The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J Bacteriol 178, 5388–5394.[Abstract/Free Full Text]

Branny, P., De La Torre, F. & Garel, J. R. (1998). An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase. Microbiology 144, 905–914.[Medline]

Canard, B. & Cole, S. T. (1989). Genome organization of the anaerobic pathogen Clostridium perfringens. Proc Natl Acad Sci U S A 86, 6676–6680.[Abstract]

Chevallier, B., Hubert, J. C. & Kammerer, B. (1994). Determination of chromosome size and number of rrn loci in Lactobacillus plantarum by pulsed-field gel electrophoresis. FEMS Microbiol Lett 120, 51–56.[CrossRef][Medline]

Cole, S. T. & Saint Girons, I. (1994). Bacterial genomics. FEMS Microbiol Rev 14, 139–160.[CrossRef][Medline]

Collins, M. D., Rodrigues, U., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez-Murica, A., Phillips, B. A., Williams, A. M. & Wallbanks, S. (1991). Phylogenetic analysis of the genes Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77, 5–12.[CrossRef]

Davidson, B. E., Kordias, N., Dobos, M. & Hillier, A. J. (1996). Genomic organisation of lactic acid bacteria. Antonie Van Leeuwenhoek 70, 161–183.[Medline]

De Man, J. C., Rogosa, M. & Sharpe, E. (1960). A medium for the cultivation of lactobacilli. J Appl Bacteriol 23, 130–135.

De Vuyst, L. & Vandamme, E. J. (1994). Bacteriocins of Lactic Acid Bacteria, 1st edn. Glasgow, UK: Chapman & Hall.

Doherty, J. P., Lindeman, R., Trent, R. J., Graham, M. W. & Woodcock, D. M. (1993). Escherichia coli host strains SURETM and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains. Gene 124, 29–35.[CrossRef][Medline]

Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 6127–6145.[Abstract]

Dudez, A. M., Chaillou, S., Hissler, L., Stentz, R., Champomier-Verges, M. C., Alpert, C. A. & Zagorec, M. (2002). Physical and genetic map of the Lactobacillus sakei 23K chromosome. Microbiology 148, 421–431.[Medline]

Feinberg, A. P. & Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137, 266–267.[Medline]

Garcia-Martinez, J., Acinas, S. G., Anton, A. I. & Rodriguez-Valera, F. (1999). Use of the 16S–23S ribosomal genes spacer region in studies of prokaryotic diversity. J Microbiol Methods 36, 55–64.[CrossRef][Medline]

Gilliland, S. E. (1990). Health and nutritional benefits from lactic acid bacteria. FEMS Microbiol Rev 87, 175–188.[CrossRef]

Goldin, B. R. (1998). Health benefits of probiotics. Br J Nutr 80, S203–S207.[Medline]

Gorbach, S. L. (2000). Probiotics and gastrointestinal health. Am J Gastroenterol 95 (Suppl 1), S2–S4.[CrossRef]

Gürtler, V. & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16–23 rDNA spacer. Microbiology 142, 3–16.[Medline]

Hammes, W. P. & Vogel, R. F. (1995). The genus Lactobacillus. In The Genera of Lactic Acid Bacteria, vol. 1, pp. 18–54. Edited by B. J. B. Wood & W. H. Holzapfel. Glasgow, UK: Blackie.

Hanahan, D. (1983). Studies of transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[Medline]

Hartmann, R. K., Toschka, H. Y., Ulbrich, N. & Erdmann, V. A. (1986). Genomic organization of rDNA in Pseudomonas aeruginosa. FEBS Lett 195, 187–193.[CrossRef][Medline]

Hunt, M. L., Ruffolo, C. G., Rajakumar, K. & Adler, B. (1998). A physical and genetic map of the Pasteurella multocida A : 1 chromosome. J Bacteriol 180, 6054–6058.[Abstract/Free Full Text]

Itaya, M. (1997). Physical map of the Bacillus subtilis 166 genome: evidence for the inversion of an approximately 1900 kb continuous DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment. Microbiology 143, 3723–3732.[Medline]

Kandler, O. & Weiss, H. (1986). Regular, nonsporing gram-positive rods. In Bergey's Manual of Systematic Bacteriology, vol. 2. Edited by P. H. A. Sneath and others. Baltimore: William & Wilkins.

Klaenhammer, T. R. (1995). Genetics of intestinal lactobacilli. Int Dairy J 5, 1019–1058.[CrossRef]

Kleerebezem, M., Boekhorst, J., van Kranenburg, R. & 17 other authors (2003). Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 100, 1990–1995.[Abstract/Free Full Text]

Kolsto, A. B., Gronstad, A. & Oppegaard, H. (1990). Physical map of the Bacillus cereus chromosome. J Bacteriol 172, 3821–3825.[Medline]

Le Bourgeois, P., Lautier, M., Mata, M. & Ritzenthaler, P. (1992). Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1402. J Bacteriol 174, 6752–6762.[Abstract]

Lee, J. J., Smith, H. O. & Redfield, R. J. (1989). Organisation of the Haemophilus influenzae Rd genome. J Bacteriol 171, 3016–3024.[Medline]

Leong-Morgenthaler, P., Ruettener, C., Mollet, B. & Hottinger, H. (1990). Construction of the physical map of Lactobacillus bulgaricus. Proc Third Symp Lactic Acid Bact A28.

Lezhava, A., Mizukami, T., Kajitani, T., Kameoka, D., Redenbach, M., Shinkawa, H., Nimi, O. & Kinashi, H. (1995). Physical map of the linear chromosome of Streptomyces griseus. J Bacteriol 177, 6492–6498.[Abstract/Free Full Text]

Liu, S. L., Hessel, A. & Sanderson, K. E. (1993). Genomic mapping with I-CeuI, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria. Proc Natl Acad Sci U S A 90, 6874–6878.[Abstract/Free Full Text]

Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218.

McKay, L. L. & Baldwin, K. A. (1990). Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol Rev 87, 3–14.[CrossRef]

Mercenier, A., Dutot, P., Kleinpeter, P., Aguirre, M., Paris, P., Reymund, J. & Slos, P. (1996). Development of lactic acid bacteria as live vectors for oral or local vaccines. Adv Food Sci 18, 73–77.

Mercenier, A., Pavan, S. & Pot, B. (2003). Probiotics as biotherapeutic agents: present knowledge and future prospects. Curr Pharm Des 9, 175–191.[Medline]

Nour, M. (1998). 16S–23S and 23S–5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis. Res Microbiol 149, 433–448.[CrossRef][Medline]

Ogasawara, N. & Yoshikawa, H. (1992). Genes and their organisation in the replication origin region of the bacterial chromosome. Mol Microbiol 6, 629–634.[Medline]

Ojaimi, C., Davidson, B. E., Saint Girons, I. & Old, I. G. (1994). Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosome of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii. Microbiology 140, 2931–2940.[Medline]

O'Sullivan, D. J. & Klaenhammer, T. R. (1993). Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59, 2730–2733.[Abstract]

Pouwels, P. H., Vriesema, A., Martinez, B., Tielen, F. J., Seegers, J. F., Leer, R. J., Jore, J. & Smit, E. (2001). Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens. Methods Enzymol 336, 369–389.[Medline]

Pridmore, R. D., Berger, B., Desiere, F. & 12 other authors (2004). The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci U S A 101, 2512–2527.[Abstract/Free Full Text]

Raman, V., Woodcock, D. & Hill, R. J. (1997). Typical unstable long tandem repeats in Escherichia coli show increased stability in strain PMC 107 and are stable when incorporated into the Drosophilia melanogaster genome. Anal Biochem 245, 242–245.[CrossRef][Medline]

Reed, K. C. & Mann, D. A. (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13, 7207–7221.[Abstract]

Rolfe, R. D. (2000). The role of probiotic cultures in the control of gastrointestinal health. J Nutr 130, 396S–402S.[Medline]

Römling, U., Grothues, D., Bautsch, W. & Tummler, B. (1989). A physical genome map of Pseudomonas aeruginosa PAO. EMBO J 8, 4081–4089.[Abstract]

Roussel, Y., Colmin, C., Simonet, J. M. & Decaris, B. (1993). Strain characterization, genome size and plasmid content in the Lactobacillus acidophilus group (Hansen and Mocquot). J Appl Bacteriol 74, 549–556.[Medline]

Roussel, Y., Pebay, M., Guedon, G., Simonet, J. M. & Decaris, B. (1994). Physical and genetic map of Streptococcus thermophilus A054. J Bacteriol 176, 7413–7422.[Abstract]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Scheppler, L., Vogel, M., Zuercher, A. W., Zuercher, M., Germond, J. E., Miescher, S. M. & Stadler, B. M. (2002). Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle. Vaccine 20, 2913–2920.[CrossRef][Medline]

Seegers, J. F. (2002). Lactobacilli as live vaccine delivery vectors: progress and prospects. Trends Biotechnol 20, 508–515.[CrossRef][Medline]

Smith, C. L., Econome, J. G., Schutt, A., Klco, S. & Cantor, C. R. (1997). A physical map of the Escherichia coli K12 genome. Nature 236, 1448–1453.

Stiles, M. E. (1996). Biopreservation by lactic acid bacteria. Antonie Van Leeuwenhoek 70, 331–345.[CrossRef][Medline]

Tulloch, D. L., Finch, L. R., Hillier, A. J. & Davidson, B. E. (1991). Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons. J Bacteriol 173, 2768–2775.[Medline]

Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, P. & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60, 407–438.[Medline]

Vaughan, E. E., Mollet, B. & de Vos, W. M. (1999). Functionality of probiotics and intestinal lactobacilli: light in the intestinal tract tunnel. Curr Opin Biotechnol 10, 505–510.[CrossRef][Medline]

Wagner, R. (1994). The regulation of ribosomal RNA synthesis and bacterial cell growth. Microbiology 161, 100–109.

Walker, D. C., Girgis, H. S. & Klaenhammer, T. R. (1999). The groESL chaperone operon of Lactobacillus johnsonii. Appl Environ Microbiol 65, 3033–3041.[Abstract/Free Full Text]

Wells, J. M., Robinson, K., Chamberlain, L. M., Schofield, K. M. & Le Page, R. W. F. (1996). Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek 70, 317–330.[Medline]

Yanisch-Perron, C., Vieire, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]

Received 4 March 2004; revised 15 October 2004; accepted 2 November 2004.



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