1 Service de Bactériologie, Hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, France
2 Laboratoire de Bactériologie, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France
Correspondence
Estelle Jumas-Bilak
bacterio{at}iup.pharma.univ-montp1.fr
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ABSTRACT |
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INTRODUCTION |
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Direct sequencing of 16S rDNA (rrs) has proven to be a stable and specific marker for bacterial identification. While the copy number of 16S rRNA genes may vary from 1 to 15 among eubacterial genomes, it is generally believed that all the copies in an organism are identical or nearly identical in nucleotide sequence. However, scattered nucleotide differences between 16S rDNA copies (so-called micro-heterogeneity) have been described in a few cases. Micro-heterogeneity has been identified in eubacteria, Escherichia coli (Cilia et al., 1996; Martinez-Murcia et al., 1999
), Mycobacterium terrae (Ninet et al., 1996
), Mycobacterium celatum (Reischl et al., 1998
), Paenibacillus polymyxa (Nubel et al., 1996
), members of the Mollicutes (Bascunana et al., 1994
; Heldtander et al., 1998
; Liefting et al., 1996
; Pettersson et al., 1996
) and some actinomycetes (Wang et al., 1997
; Yap et al., 1999
). These cases may represent a fairly common phenomenon, particularly if we consider the limited number of organisms for which the nucleotide sequences are available for each rRNA gene copy. The direct sequencing of the 16S rRNA gene of clinical isolates belonging to the genus Veillonella showed ambiguities suggesting micro-heterogeneity. We investigated the copy number of 16S rRNA genes in the Veillonella genome and each copy was sequenced for a clinical isolate. A relatively high level of heterogeneity in sequences between copies was found. The intra-strain sequence heterogeneity was then tested by PCR-RFLP on 27 clinical strains. The results are discussed in terms of phylogenetic and taxonomic implications.
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METHODS |
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DNA amplification, sequencing and RFLP analysis.
DNAs were rapidly extracted by a boiling/freezing method (Carlier et al., 2002). Two-microlitre samples of extracts were used as the template in the PCR experiments. 16S rDNA was selectively amplified by PCR using 5'-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3' (27f), position 836 (E. coli numbering), as the forward primer and 5'-CACGGATCCTACGGGTACCTTGTTACGACTT-3' (1492r), position 14781508 (E. coli numbering), or 5'-CCCAACATCTCACGACACGA-3' (1090r), position 10831102 (E. coli numbering), as reverse primers in the conditions previously described (Carlier et al., 2002
). Selective amplification of one 16S rDNA copy was performed by cutting SmaI macrorestriction fragments obtained after PFGE migration in low-melting agarose gel (see below). Then, 2 µl melted agarose was used as the PCR template. A 0·7 kb fragment of the heat-shock protein 70 (dnaK) gene was amplified using the following degenerate primers deduced from the alignment of Hsp70 sequences available for the more phylogenetically related species: B1 5'-ATTGAYTTAGGWACAACAAA-3' and B2 5'-GCTTTTTCAGCHGCDTCYTT-3'. The amplification profile was as follows: 35 cycles of 1 min at 94 °C, 1·5 min at 55 °C, and 1 min at 72 °C. The 16S rDNA and dnaK PCR products were directly sequenced on an Applied Biosystems Automatic Sequencer (Genome Express) in both directions by using forward and reverse primers. The RFLP analysis of 16S rDNA PCR products consisted in the digestion of 10 µl amplified samples with 10 U of either Sau3AI or MnlI (Biolabs). The digested samples were analysed by electrophoresis on a 2 % agarose gel.
Phylogenetic analysis.
The 16S rDNA sequences were compared and aligned with sequences deposited in the GenBank database using the programs BLAST (Altschul et al., 1997), LALIGN and DIALIGN (http://www.expasy.ch). The computed alignments were then manually checked and corrected. Pairwise evolutionary distances were computed using the Jukes and Cantor equation implemented in the DNADIST program and a phylogenetic tree was constructed by the neighbour-joining method (PHYLIP programs package available online at http://www.pasteur.fr). A total of 100 bootstrapped trees were sampled to determine a measure of the support for each node on the consensus tree, using the SEQBOOT and CONSENSE programs (PHYLIP package). Prediction of RNA secondary structure by energy minimization was performed by the MFOLD program (Walter et al., 1994
).
PFGE and DNA electrophoresis.
Intact genomic DNA was extracted in agarose plugs as described previously (Marchandin et al., 2001) and submitted to PFGE after restriction by either 40 U of the endonuclease SmaI or 1 U of the intronic endonuclease I-CeuI (Jumas-Bilak et al., 1998
). The macrorestriction fragments were separated with a contour-clamped homogeneous field electrophoresis apparatus, CHEF-DRII (Bio-Rad) in 0·5x TBE at 10 °C. The PFGE parameters for resolution of SmaI fragments were 42 h at 4·5 V cm-1, with switch times ramped from 1 to 15 s in a 1 % agarose gel. I-CeuI fragments were separated using pulse times from 90 to 150 s during 24 h at 5·1 V cm-1 in a 0·8 % agarose gel.
Genomic DNAs digested with 10 U of HindIII, NheI or SpeI were submitted to electrophoresis for 3 h at 80 V in a 0·8 % agarose gel in 0·5x TBE using a SubCell apparatus (Bio-Rad). After electrophoresis, DNA fragments were stained in 0·5x TBE containing 0·5 µg ethidium bromide ml-1 and visualized under UV illumination.
Southern blotting, probes and hybridization.
Electrophoresis gels were transferred onto Nytran N (Scheilcher and Schull) nylon membrane by vacuum blotting in 20x SSC. 16S rDNA digoxigenin-labelled probe was obtained by PCR using primers 27f/1090r as described above with a dNTP mixture containing 0·1 mM digoxigenin-dUTP (Roche). Hybridization of the probe was detected by using chromogenic substrate NBT/BCIP (Roche).
Nucleotide sequence accession numbers.
GenBank accession numbers for 16S rDNA sequences determined in this work are as follows: V. atypica ATCC 17744T, AF439641; V. dispar ATCC 17748T, AF439640; V. parvula CIP 60.1, AF439639; Veillonella sp. strain ADV 360.1 rrsA, rrsB, rrsC and rrsD, AF439642, AF439643, AF439644 and AF439645, respectively. GenBank accession numbers for 70 kDa heat-shock protein (dnaK) gene sequences determined in this work are as follows: V. atypica ATCC 17744T, AF440436; V. dispar ATCC 17748T, AF440435; V. parvula CIP 60.1, AF440437; Veillonella sp. strain ADV 360.1, AY220521.
GenBank accession numbers for previously deposited 16S rDNA sequences used in this work are as follows: V. atypica DSM 20739T (=ATCC 17744T), X84007; V. criceti ATCC 17747T, AF186072; V. dispar DSM 20735T (=ATCC 17748T), X84006; V. parvula DSM 2008, X84005; V. ratti ATCC 17746T, AF186071; Veillonella sp. oral clone X042, AF287781; Veillonella sp. oral clone AA050, AF287782; Anaeroglobus geminatus CIP 106856T, AF338413; Dialister pneumosintes ATCC 33048T, X82500; Megasphaera elsdenii ATCC 25940T, U95027; Acidaminococcus fermentans ATCC 25085T, X59645.
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RESULTS AND DISCUSSION |
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To confirm that strain ADV 360.1 was clonal and thus rule out the hypothesis that the heterogeneity observed could be due to a mix of different strains, we performed the following experiment. The dnaK gene was amplified and sequenced for V. atypica ATCC 17744T, V. dispar ATCC 17748T, V. parvula CIP 60.1 and Veillonella sp. strain ADV 360.1. The sequences showed inter-species variability higher than that observed after comparison of 16S rDNA sequences. For example, V. dispar and V. parvula differed by 7·2 % of nucleotide positions. We also observed the absence of a double sequencing signal corresponding to heterogeneous positions in the dnaK sequence of ADV 360.1. This result was a strong argument in favour of the clonality of this isolate since a mix of strains would have led to a dnaK sequence with several heterogeneous positions.
The differences in nucleotides between the three Veillonella species, and between the four 16SrRNA gene copies of strain ADV 360.1, are listed in Table 2. Nine positions have been determined to be variable between V. dispar and V. parvula. Among them, eight were proved to be polymorphic between rrs copies of the strain ADV 360.1. In fine, only one of these variable nucleotides (position 193) could be considered as valuable for differentiation between V. parvula and V. dispar. However, comparison of the two currently available 16S rDNA sequences for V. dispar ATCC 17748T (accession numbers X84006 and AF439640) showed that position 193 is variable. Consequently, none of the variable positions between V. dispar and V. parvula could be retained as species-specific. In contrast, 11 nucleotides differentiated V. atypica from V. dispar and V. parvula. Among them, eight were not polymorphic between operons and can be retained for species specification (Table 2
). Thus, V. atypica could be separated from the two other human species on the basis of 16S rDNA sequences.
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As described for members of a multigene family, rrn operons are subject to homogenization processes. Thus, rRNA sequences generally show low variability between copies in a genome and within species or subspecies (Liao, 2000). However, a total of 15 positions variable between copies have been identified in the same strain in this work (Table 2
). Despite a few descriptions of micro-heterogeneity, the general extent of 16S rRNA heterogeneity within a bacterial species has received little attention, except in few works (Cilia et al., 1996
; Clayton et al., 1995
; Martinez-Murcia et al., 1999
; Ueda et al., 1999
). Clayton et al. (1995)
observed that slightly different 16S rRNA sequences were deposited in the databases for strains belonging to the same species. They thought most of the differences were not caused by sequencing errors but by real intra-specific variations. Part of these variations could be due to inter-operon heterogeneity. Thus, the authors showed that 18 % of rrs sequence pairs deposited for the same strain exhibited 15 % heterogeneity. We compared sequences of rrs copies obtained from 32 completely sequenced genomes of Gram-positive bacteria and Proteobacteria using the BLAST program. With respect to the 16S rDNA region analysed in this work (from 82 to 1109 following E. coli numbering), the percentage of variation in sequence between copies of the same organism ranges between 0 and 1·2 %. The distribution of the strains according to the percentage of variation was: <0·1 %, 19 strains; 0·11 %, 12 strains; and >1 %, one strain. The same distribution was obtained when complete sequences were compared. Thus, the level of variations in the sequences of rrs copies of Veillonella sp. strain ADV 360.1 (1·43 %) could be considered high and somewhat atypical.
16S rDNA PCR-RFLP of 27 clinical isolates of Veillonella sp.
The nucleotides variable between rrs copies of the strain ADV 360.1 occurring in positions 591 and 648 involved polymorphism of two Sau3AI restriction sites. In the same way, the variable position 184 involved MnlI polymorphism (Table 2). These enzymes have been previously described as effective in the identification of Veillonella species by 16S rDNA PCR-RFLP (Sato et al., 1997a
, b
). Particularly, Sau3AI digestion clearly distinguished V. dispar from V. parvula. We performed a PCR-RFLP assay by Sau3AI digestion of the PCR products obtained with primers 27f and 1090r. V. parvula gave a profile formed by five bands of 59, 116, 169, 308 and 491 bp. A profile consisting of three bands of 116, 169 and 910 bp was obtained for V. dispar (data not shown). These results were in accordance with the number and size of Sau3AI restriction fragments calculated on the sequences. Sau3AI digestion of PCR products obtained from each rrs copy of strain ADV 360.1 is shown in Fig. 2
. The profiles obtained for rrsA and rrsD were similar to the V. dispar profile, whereas the rrsB and rrsC profiles corresponded to the V. parvula one. The RFLP profiles were in accordance with the polymorphism in rrs sequences described above. The PCR product obtained from total genomic DNA of strain ADV 360.1 gave a complex RFLP profile consisting of a mix of the bands obtained in lanes 14 (Fig. 2
). Using this method, we rapidly searched for the occurrence of sequence heterogeneity between rrs copies in a panel of 27 human isolates of Veillonella sp. Among these strains, four (14·8 %) gave the profile described for V. parvula CIP 60.1, three (11·1 %) gave the profile characteristic of V. dispar type strain, and 20 (74·1 %) gave mixed profiles. These 20 isolates were shown to be unrelated after comparison of SmaI macrorestriction patterns (data not shown). The general aspect of the PFGE profiles suggested that each DNA was extracted from a single strain and not from a mixture of strains. Thus, the clonality of each isolate proved for the strain ADV 360.1 was highly probable for the other isolates. These results suggested that inter-operon heterogeneity was a common phenomenon that occurred widely in the genus Veillonella.
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It is striking that differences between rrs sequences did not appear at random. The variable positions occurred in stems and loops belonging to hypervariable and variable regions of the 16S rRNA secondary structure model. None of them coincided with invariable positions or positions that were conserved in a large majority of bacteria. Moreover, genes A and D have identical sequences but differed from genes B and C by 13 and 14 nucleotides, respectively, whereas rrsB and rrsC differed by only three nucleotides. This fact suggested that the gene duplication followed an independent acquisition of the two parental copies, probably by recombination events after lateral transfer.
Phylogenetic analysis and taxonomy of the genus Veillonella
The 1048 bp nucleotide sequences of rrsA, rrsB, rrsC and rrsD of strain ADV 360.1 were aligned together with seven deposited sequences obtained from the three human species of Veillonella, two human Veillonella strains unidentified at the species level and two species of Veillonella isolated from rodents (V. criceti and V. ratti). Four related species belonging to the Sporomusa sub-branch, Anaeroglobus geminatus, Dialister pneumosintes, Megasphaera elsdenii and Acidaminococcus fermentans, were also included and aligned. The last one was added as outgroup organism. A phylogenetic tree obtained by the neighbour-joining method after computing a distance matrix using the DNADIST program implemented with the Jukes and Cantor algorithm is presented in Fig. 3. The tree topology confirmed that the rrsA and rrsD sequences were closely related to the sequence of V. dispar whereas rrsB and rrsC were related to that of V. parvula. These branches were associated with bootstrap scores of 96 % and 94 %, respectively. Thus, the sequence variation among rrs copies of strain ADV 360.1 affected the phylogenetic position of this strain. The level of heterogeneity observed in this work did not affect the phylogenetic results obtained with organisms exhibiting distant relationship but has to be considered for studies at the level of the species or the subspecies.
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Previous studies have shown that comparison of 16S rRNA gene sequences gives results in accordance with the existence of the seven species previously described on the basis of serological data (Sato et al., 1997a, b
; Tanner et al., 2000
). The topology of the phylogenetic tree constructed in this work suggests that V. dispar type strain and V. parvula CIP 60.1 associated with strain ADV 360.1 and the two unidentified Veillonella sp. strains form a homogeneous group. No cluster representative of two species could be highlighted in this group, mainly for three reasons: (i) similarity in sequence of about 99 % between 16S rDNAs; (ii) heterogeneous distribution of the rrs copies in the same strain; (iii) branching of the two unidentified strains, which did not allow their affiliation to one of the two defined species. The Veillonella species have been described by serotyping (Rogosa, 1965
, 1984
) and then confirmed by DNA/DNA hybridization (Mays et al., 1982
). However, the DNA/DNA hybridization data were hardly interpretable and they seem to be insufficient to support any taxonomic conclusions. Our results based on 16S rDNA analysis did not suggest the separation of V. parvula and V. dispar in two independent clusters. However, the value of this marker in splitting species is limited since an intra-operon variability higher than an inter-species variability was observed. The sequencing of other housekeeping genes in a multi-locus sequencing typing (MLST) approach as recommended by Stackebrandt et al. (2002)
could clarify the inter-species relations in the genus Veillonella. The sequencing of dnaK and rpoB genes is on-going in our laboratory in order to obtain additional arguments to discuss the unification of V. parvula and V. dispar. However, our study has already revealed that 16S rDNA-based methods are not suitable for the identification of V. dispar and V. parvula.
A polyphasic approach which includes phenotypic and sequencing data is recommended for bacterial systematics (Murray et al., 1990; Vandamme et al., 1996
; Stackebrandt et al., 2002
). As a result, and in relation to the development of molecular methods, a dramatic increase of the number of 16S rDNA sequences deposited in databases has been seen. However, analyses at the intra-species and/or at the intra-chromosomal level have been completely ignored in most sequencing analyses. Our results, together with previously published data (Martinez-Murcia et al., 1999
; Ninet et al., 1996
), suggest that it is necessary to determine rrs copy number and to sequence each copy in a large panel of bacterial species. Indeed, evaluating the general extent of rrs heterogeneity in the bacterial world could clarify its implications for 16S rDNA-based phylogeny, taxonomy and identification.
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ACKNOWLEDGEMENTS |
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Received 19 November 2002;
revised 23 January 2003;
accepted 3 March 2003.
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