Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France
Correspondence
Benjamin Candelon
bcandel{at}jouy.inra.fr
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ABSTRACT |
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The GenBank accession numbers for the sequences determined in this work are given in the text.
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INTRODUCTION |
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In several previous studies, Southern hybridization and inverse PCR were used for ribotyping the B. cereus group strains (Johansen et al., 1996; Lechner et al., 1998
; Patra et al., 2002
; Priest et al., 1994
). However, these works led only to approximate determination of the rRNA operon number (Patra et al., 2002
). Recently the entire genomic sequence of the type strain B. cereus ATCC 14579 was established (Ivanova et al., 2003
). In this study we determined the precise number of rRNA operons and compared them in several strains of the B. cereus group distinguished by different phenotypes such as specific insect pathogenicity for B. thuringiensis (Schnepf et al., 1998
), psychrotolerance for B. weihenstephanensis (Lechner et al., 1998
), or food-related pathogenicity for B. cereus 391-98 (Lund et al., 2000
). Thirteen to fourteen rRNA operons were identified, the highest number ever detected in bacteria. To determine the relationships between these strains, we carried out parallel sequencing of several genes distributed over their chromosomes. The accurate analysis of rRNA operon sequences in the type strain revealed sequence variations, which suggest the existence of two distinct classes of rRNA operons. The eleven rRNA operons of the recently sequenced B. anthracis Ames (Ban Ames) (Read et al., 2003
) exhibit a similar polymorphism, suggesting that the presence of two distinct classes of rRNA operons is a general feature through the B. cereus group.
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METHODS |
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PCR amplification.
PCR using the Expand Long Template PCR System (Roche Diagnostics) was used to obtain templates for sequencing and for preparing DNA hybridization probes. The cycling program was: 94 °C for 5 min; 12 cycles of 94 °C for 10 s and 68 °C for 12 min; 24 cycles of 94 °C for 10 s and 68 °C starting from 12 min and increasing this time by 15 s each cycle. The final extension was done at 72 °C for 10 min.
Sequencing procedures.
PCR products were treated for 1 h at 37 °C with exonuclease I and shrimp alkaline phosphatase (USB). Sequencing reactions were performed using an ABI PRISM sequencing kit (Applied Biosystems). Products of reactions were ethanol precipitated and analysed with an ABI 3700 sequencer (Applied Biosystems).
Separate amplification of each operon was performed using oligonucleotides listed in Table 2. Sequencing was done on each amplification product separately using a set of 32 oligonucleotides with sequences common to all operons (oligonucleotide sequences can be provided on request). PCR amplification and sequencing of the regions between closely located 5S and 16S rRNA genes of operons rrnC and rrnD respectively were done using the following oligonucleotides: FIAH7 (5'GCCAGCTTATTCAACTAGCACTTG3'), FIAH8 (5'GTTTCCCGGAGTTATCCCAGTCTTA3'), FIBH7 (5'GATCCCTGAAAGATGATCAGGTTG3') and FIBH8 (5'GTTCCCATACCGAACACGGAAGTT3'). Assembly was performed manually using Staden's XBAP version 14.0 software (Dear & Staden, 1991
) and consensus sequences of 16S, 23S and 5S rRNA genes as scaffold. The 13 rRNA operon sequences were deposited at NCBI under accession numbers AY224379AY224388.
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Determination of the number of rRNA operons.
Total DNA of each strain was digested with BclI, ClaI or HindIII. An internal fragment of the 16S rRNA gene was used as the probe and the enzymes used had no recognition sites within the 16S rRNA gene but had one or more sites within the 23S rRNA gene. In these conditions, each ribosomal operon was expected to produce only one DNA fragment able to hybridize to the probe. Intensity of each hybridization band was measured using the STORM scanner. After background subtraction, an iterative process was applied to determine the number of rRNA operons corresponding to each hybridized DNA band, assuming that each operon contributed equally to the signal intensity. In iteration the signal corresponding to one rRNA operon was estimated by dividing the intensity of each hybridization band by the postulated number of rRNA operons to which the band corresponded. The relative error of the signals of one rRNA operon, measured by using the different hybridized bands, was then calculated by the formula
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Genetic relationships.
To investigate the genetic relationships of the different strains, seven genes distributed over the 5·4 Mb chromosome of the entirely sequenced type strain Bce 6A5 (Ivanova et al., 2003) were partially sequenced in all strains. These were: clpC (position on the chromosome 90 kb) encoding the ClpC regulator; purF (300 kb), encoding glutamine phosphoribosylpyrophosphate amidotransferase; gdpD (574 kb), encoding glycerophosphoryl diester phosphodiesterase; yhfL (1068 kb), encoding long-chain fatty acid CoA ligase; panC (1484 kb), encoding pantoate
-alanine ligase; dinB (4103 kb), encoding DNA polymerase IV; plcR (5255 kb), encoding transcriptional regulator PlcR. The corresponding oligonucleotides used for PCR amplification and sequencing were: 5'GTACGCGAAGGTGAAGGAATTGC3' and 5'ATGCATCGATTGCACCTTCTGCTC3' for clpC (the nucleotide sequences used for phylogenetic analyses correspond to positions 151650 from the first primer); 5'CGAAGAATGTGGCGTTTTCGGAA3' and 5'GAAATACTAGAGTCTGGTACACCAG3' for purF (positions 101700); 5'GGTGGTTATGCAAATGCGACAAATG3' and 5'CGCCATAATATAAAATGGCGTCACG3' for gdpD (positions 101600); 5'GCGAAACCTCATCAGATTTTAACC3' and 5'CAGGTACTTCTTCCCCAAGTTCAT3' for yhfL (positions 101600); 5'CGATATCCTCGTGATATTGATAGAG3' and 5'TCCGCATAATCTACAGTGCCTTTC3' for panC (positions 101450); 5'GAGGCGAGAGAATACGGAATACG3' and 5'GCCCATTTGACTCGGATCCACT3' for dinB (positions 101500); and 5'CCCAAGTATGGATATATTGCAAGG3' and 5'CCAATTAATGCCATACTATTAATTCGGC3' for plcR (positions 101500). The resulting sequences were deposited at NCBI under GenBank accession numbers AY224696AY224779. Corresponding sequences of the B. anthracis strain Ames were also used in this study (accession no. AE016879) (Read et al., 2003
). The same source was used for Ban Ames rRNA operon analysis. Five other genes were also tested, but they were eliminated since we did not succeed in amplifying and sequencing them in all strains. The nucleotide sequences from each strain were compared by the neighbour-joining method using the multiple alignment program CLUSTALX 1.8 (Thompson et al., 1997
) and the phylogenetic trees calculated using 1000 bootstrap trials were visualized by the NJplot software (Perriere & Gouy, 1996
).
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RESULTS AND DISCUSSION |
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The quantitative Southern hybridization approach used for strain Bce 6A5 was applied to 11 other strains from the B. cereus group. The selected strains represent the diversity of this group. According to previous studies, strains Bce 6A5, Bce 6A1 and B. thuringiensis subsp. canadensis (Btc) HD224 are closely related (Carlson et al., 1994), B. thuringiensis subsp. israelensis (Bti) and B. thuringiensis subsp. kurstaki (Btk) strains represent the most important insecticidal groups (Hofte & Whiteley, 1989
), B. weihenstephanensis (Bwe) strains represent a large cluster of psychrotolerant strains (Lechner et al., 1998
) and strain Bce 391-98 was characterized as having a high pathogenic potential (Lund et al., 2000
). As in the case of strain Bce 6A5, three enzymes, BclI, ClaI and HindIII, were used. Representative ribotype profiles of these strains, using digestion by HindIII, and the deduced number of rRNA operons are shown in Fig. 2(A, B)
. Most of the studied strains appeared to possess 13 rRNA operons, like the type strain Bce 6A5; 14 rRNA operons were detected for the other strains. If a correct number was used for calculation, the relative statistical error was 34 %. This error increased up to 920 % if the number used for calculation differed by 1 (Fig. 1B
). In all cases, the minimal value of the statistical error correlated with the highest linear regression coefficient. This asserted that the intensity of hybridization signal varies linearly with the number of hybridized fragments, and hence with the number of rRNA operons. Thus, this approach provides an unambiguous estimation of the repeat number of a sequence in a genome even when it is as high as 14.
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Genetic relationships between strains of the B. cereus group
To verify the relationships between strains presented above, we sequenced seven genes distributed over the chromosome of Bce 6A5 in the 12 studied strains. We also added the corresponding sequences of B. anthracis strain Ames (Ban Ames) (Read et al., 2003) to get a wider view of the B. cereus group. Trees were constructed using the nucleotide sequences obtained for all genes. Examples of representative dendrograms are given in Fig. 3
(gdpD, panC and plcR genes). The resulting trees were almost fully congruent, for all the genes tested, except plcR, and they confirmed the separate clustering of the five Bwe psychrotolerant strains from the type strain Bce 6A5 and its relatives. For all tested genes, no difference was found between the two Bti strains, confirming their high clonality as mentioned above. In most cases, the two pathogenic strains Bce 391-98 and Ban Ames appeared to be relatively isolated but slightly closer to the psychrotolerant strains than to others. The genes dinB (not shown), panC and plcR of strain Bce 391-98 appeared to be very close to those of strain Bwe 10204 (less than 2 bp differences in 500 bp sequenced: Fig. 3B, C
), suggesting that those strains belong to the same group. However, the difference was greater for the gdpD gene (Fig. 3A
) and strain Bce 391-98 lacked the ability to grow at 8 °C. In comparison, the Ban Ames strain appeared to be much more isolated. On the whole, the trees based on parallel sequencing and on the riboprofiles exhibited similar topologies.
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Comparison of the rRNA operons in Bce 6A5
Bce 6A5 appeared to possess 13 rRNA operons. As expected, all are located close to the replication origin. Physical separation of these 13 rRNA operons, amplified using LR PCR, allowed us to sequence them separately by primer walking and to study their variability in the conserved 16S, 23S and 5S regions and in the less conserved regions flanking these genes.
Sequences of the 16S, 23S and 5S genes were different in few single base positions (Fig. 4). We detected seven, seven and two alleles for the 16S, 23S and 5S rRNA genes, respectively. Seven copies of the 16S rRNA gene (A, E, G, H, I, J and M) exactly corresponded to the consensus sequence determined previously (Ticknor et al., 2001
). None of the other alleles reported in this paper for different strains were detected during the present study. The 16S gene alleles corresponding to the rRNA operons B, D, F, K and L were all unique and contained one nucleotide difference compared to the consensus. The 16S gene in the rrnC operon contained two differences.
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The intergenic 16S23S area, differing considerably between strains, has often been used to classify strains (Bourque et al., 1995; Daffonchio et al., 1998
, 2000
; Harrell et al., 1995
). Our consensus sequence for this region corresponds exactly to the sequence reported earlier for the strain Bce 6A5 (Bourque et al., 1995
; Harrell et al., 1995
) (not shown). The alleles presented by operons A, B, C and D are different. We detected an insertion of tRNA-Ile and tRNA-Ala genes in operons A and B. This feature explains the existence of homoduplex and heteroduplex polymorphisms in this region, which have been used for classifying the strains (Daffonchio et al., 2000
). Separate amplification of different operons by LR PCR, using the oligonucleotides reported here (Table 2
), and subsequent sequencing of the intergenic 16S23S and 23S5S areas, can be suggested as a more precise approach for characterizing and classifying strains.
The consensus sequences of 23S and 5S genes have not been reported yet. From our data they are the same as the alleles corresponding to operons rrnD, G, H, I and J. Concerning the 23S gene, it is remarkable that the operon rrnB contains multiple single-nucleotide differences clustered in two regions, 310335 and 24282485. This might indicate that this allele originated from two recombinational events. Alleles of operons rrnC, E and L have several differences clustered close to position 1560, suggesting their common recombinational origin. Presence of identical 5S alleles that differ from the consensus in these three operons is consistent with this hypothesis. However, the same 5S allele was also found in operons rrnK and M, having the 23S alleles closer to the consensus. It is therefore probable that independent recombination events took place in the 23S and 5S genes. Concerning the 5S genes, only two alleles were detected, differing at four nucleotide sites, indicating that they probably diverged due to a recombination event.
Two distinct types of rRNA operons in Bce 6A5 and Ban Ames
An important difference in the overall organization of rRNA operons in Bce 6A5 is the presence of multiple tRNA genes downstream of operons rrnC, E, K, L and M (Fig. 5A). Operons K, L and M have respectively 15, 20 and 22 tRNA genes downstream, with a partially similar order of codon specificity. The closely related operons C and E both have nine tRNA genes downstream, although the codon specificities are different. All the five operons having multiple tRNA genes downstream also differ from the others by their 5S rRNA allele (Fig. 4
) and 23S5S region (Fig. 5B
). These observations suggest the existence of two distinct types of rRNA operons in Bce 6A5. A phylogenetic tree constructed using the sequences of the 23S5S region presented in Fig. 5(B)
confirms the existence of two types of rRNA operons (Fig. 6
). The 13 rRNA operons in Bce 6A5 could therefore be divided into two classes. The first (class I) includes operons rrnA, B, D, F, G, H, I and J, all devoid of multiple tRNA genes downstream, and the second (class II) includes operons rrnC, E, K, L and M, all having many (75 in total) tRNA genes downstream (Fig. 5A
), and distinct 5S gene (Fig. 4
) and 23S5S intergenic region (Figs 5B and 6
). It is worth noting that the 23S5S intergenic region of all class II operons contains sequences (TTGACT as -35 box and TA(C,A or T)AAT as -10 box) which are very similar to the Gram-positive and Gram-negative bacteria
A promoter consensus sequence (TTGACA as -35 box and TATAAT as -10 box). Moreover, class II rRNA operons do not have any obvious transcription terminator structure downstream of the 5S gene (Fig. 5B
). Thus, the 5S rRNA gene and the downstream tRNA genes might be transcribed independently from the 16S and 23S genes. In contrast, all class I operons, except rrnF, contain a clearly identifiable stemloop sequence downstream of the 5S gene, which should act as a transcription terminator (Fig. 5B
). The operon rrnF appears to be intermediate between the two classes. This operon has two tRNA genes downstream of the 5S rRNA gene and 12 tRNA genes upstream of the 16S gene. Its 5S allele is identical to those of class I rRNA operons and no promoter or terminator close to the 5S rRNA gene was detected for this operon. We assigned rrnF to class I, although it may have a particular significance compared to the two classes.
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Evidence for the diversity of rRNA sequences in a single organism has been recently accumulating. Some authors have already evoked the existence of distinct types of rRNA operons based on the heterogeneity of the small-subunit rRNA genes in different domains of life, indicating the wide occurrence of this phenomenon (Carranza et al., 1996; Liefting et al., 1996
; Maden et al., 1987
; Mylvaganam & Dennis, 1992
; Reischl et al., 1998
; Yap et al., 1999
). Concerning the B. cereus group, a potential psychrotolerant signature has already been found in the 16S sequence (Pruss et al., 1999
). Recently the existence of different types of rRNA operons based on the whole organization and sequence of the operons was reported in Escherichia coli (Ohnishi et al., 2000
) and Clostridium perfringens (Shimizu et al., 2001
). However, none of these studies revealed divergence as significant as the presence of a potential alternative promoter within the 23S5S intergenic region. The putative promoter highlighted in this study within this region in Bce 6A5 as well as in Ban Ames may enable the expression of tRNA genes independently of the global regulation of expression of the whole rRNA operon. The expression of tRNA genes could thus be regulated according to the amount of amino acids available for protein synthesis. Such a mechanism would be useful for B. cereus and B. anthracis for adaptation to the environment and the colonization of new environmental niches.
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ACKNOWLEDGEMENTS |
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Received 28 October 2003;
revised 28 November 2003;
accepted 2 December 2003.
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