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Applied and Environmental Microbiology, November 2003, p. 6740-6749, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6740-6749.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Atieh Khamis, Philippe Colson, Didier Raoult, and Bernard La Scola^*

Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 13385 Marseille Cedex 05, France

Received 23 June 2003/ Accepted 18 August 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability hot spots in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aquatic bacteria such as Legionella, Pseudomonas, Stenotrophomonas, Burkholderia spp., and Acinetobacter spp. may colonize hospital water supplies and have previously been shown to be causally associated with cases of nosocomial infections (23). Free-living amoebae have been shown to be a reservoir of pathogens, such as Legionella spp., Burkholderia pickettii, and Cryptococcus neoformans (2, 26). The most studied of amoeba-resisting bacteria (ARB) is Legionella pneumophila, the agent of Legionnaires' disease (27), which frequently results from exposure to contaminated aerosols. There are growing hints that additional ARB might be implicated in community-acquired pneumonia, including Legionella-like amoebal pathogens and members of the genus Parachlamydia (19). As part of the research into the diversity of bacterial agents associated with amoebae in hospital water supplies, we previously identified new -proteobacteria belonging to the Bradyrhizobiaceae (13). Moreover, we demonstrated that patients with nosocomial pneumonia who were hospitalized in a public hospital where contaminated water was found had elevated titers of antibodies against these bacteria (14) and that patient seroconversion to Bosea massiliensis was frequent in patients hospitalized in intensive care units and was associated with the occurrence of ventilator-acquired pneumonia (17). Among the Bradyrhizobiaceae, bacteria of the genera Bosea and Afipia were the most frequently isolated. Due to the fastidiousness of these bacteria (3, 15, 16), identification is mostly based on 16S rRNA gene sequence (15, 16, 21). However, the 16S rRNA genes of these bacteria show very low variability: bacteria with only 1 base difference may belong to different species, as evidenced by DNA-DNA hybridization studies (15, 16, 25). To develop a molecular tool for both identification of cultured bacteria and detection from human samples, we decided to develop a sequence-based identification assay. Among the universal genes that can be used for this purpose, the RNA polymerase ß-subunit-encoding gene (rpoB) was extensively used by our team for Bartonella spp. (22), Staphylococcus spp. (5), and Enterobacteriaceae (20), as well as for Mycobacterium (11) and Legionella spp. (12). The RNA polymerase ß' subunit is encoded by the rpoC gene. This gene has a low level of homology with rpoB and has been less studied for sequence-based identification. Herein we investigate the usefulness of rpoB sequencing for differentiation and identification of Afipia and Bosea. As rpoB is large (>4,000 bp), we also determined regions of variability in the sequence that are bordered by conserved sequences with the objective of designing universal primers for amplification of a small but discriminative sequence for routine Afipia and Bosea identification.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains.
The bacterial stains used in this study are listed in Table 1. These strains were routinely grown on buffered charcoal-yeast extract agar plates (bioMérieux, Marcy l'Étoile, France) as previously described (13).

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TABLE 1. List of the species for which rpoB partial or complete sequences were determined

rpoB gene amplification and sequencing.
The sequences of rpoB from the most closely related species of the studied bacteria were aligned in order to produce a consensus sequence. The chosen bacteria were Sinorhizobium meliloti, Mesorhizobium loti, Bartonella henselae, and Bartonella quintana (GenBank accession numbers SME591787, AP002994, AF171070, and AF165994, respectively). The consensus sequence was used to generate primers that were used in PCRs, for genome walking (24), and for sequencing. Additional primers were selected from ongoing base sequence determinations. All primers used in this study are summarized in Table 2. Bacterial DNA was extracted from a heavy suspension of strains with the QIAamp blood kit (Qiagen, Hilden, Germany) according to manufacturer's recommendations. All PCR mixtures contained 2.5 x 10^-2 U of Taq polymerase per µl; 1x Taq buffer; 1.8 mM MgCl₂ (Gibco BRL, Life Technologies, Cergy Pontoise, France); 200 µM concentrations of dATP, dCTP, dTTP, and dGTP (Boehringer Mannheim GmbH, Hilden, Germany); and 0.2 µM concentrations of all primers (Eurogentec, Seraing, Belgium). PCR mixtures were subjected to 35 cycles of denaturation at 94°C for 30 s, primer annealing for 30 s (at a temperature 5°C below the melting temperature [T_m] of the primer with the lowest T_m), and extension at 72°C for 2 min. Every amplification program began with a denaturation step of 95°C for 2 min and ended with a final elongation step of 72°C for 10 min. Complete determination of the rpoB sequence ends was achieved by using the sequences of both 3' and 5' ends of the gene and amplifying by PCR using the Universal GenomeWalker kit (Clontech Laboratories, Palo Alto, Calif.). Briefly, genomic DNA was digested with EcoRV, DraI, PvuII, StuI, and ScaI. DNA fragments were ligated with a GenomeWalker adaptor, which had one blunt end and one end with a 5' overhang. The ligation mixture of the adaptor and the genomic DNA fragments were used as a template for PCR. This PCR was performed with an adaptor primer supplied by the manufacturer and specific primers to walk downstream the DNA sequence. For the amplification, 1.5 U of ELONGASE (Boehringer Mannheim) was used with a mixture containing 10 pmol of each primer, 20 mM (each) deoxynucleoside triphosphate, 10 mM Tris-HCl, 50 mM KCl, 1.6 mM MgCl₂, and 5 µl of template in a final volume of 50 µl. Genome walking was performed with the Universal GenomeWalker kit according to the manufacturer's recommendations. Amplicons were purified for sequencing with a QIAquick spin PCR purification kit (Qiagen) by following the protocol of the supplier. Sequencing reactions were carried out with the reagents of the ABI Prism 3100 DNA sequencer (dRhod.Terminator RR Mix; Perkin-Elmer Applied Biosystems) by following the standard automated-sequencer protocol.

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TABLE 2. Primers used for amplification and sequencing of the entire rpoB gene in this study

rpoB sequence analysis.
The nucleotide sequences of the rpoB gene fragments obtained were processed into sequence data with Sequence Analysis software (Applied Biosystems), and partial sequences were combined into a single consensus sequence with Sequence Assembler software (Applied Biosystems). Multiple sequence alignments were made, and percentages of similarity among the different species with rpoB and the 16S rRNA gene were obtained, with CLUSTALW (28) on the EMBL-EBI World Wide Web server (http://www.ebi.ac.uk/clustalw/). Phylogenetic trees were obtained from DNA sequences by three different methods: neighbor joining, maximum parsimony, and maximum likelihood (6). Bootstrap replicates were performed in order to estimate the node reliability of the phylogenetic trees obtained. Bootstrap values were obtained from 1,000 trees generated randomly with SEQBOOT in the PHYLIP software package.

Strategy for determination of discriminative partial sequences.
To search for parts of sequences with high variability bordered by conserved regions, we created a simple analysis tool on Microsoft Excel 97 software that analyzes, reveals, and graphically represents variability along nucleotide sequences. This program (SVARAP, for sequence variability analysis program) can analyze and simultaneously process sets of up to 100 sequences of a maximal length of 4,000 nucleotides (hypertext link Téléchargement at the URL http://ifr48.free.fr/recherche/jeu_cadre/jeu_rickettsie.html). All sequences of our set of sequences (including the sequence used as an outgroup) were aligned with ClustalX, version 1.8 (29). The program calculates the consensus nucleotide (defined as the most frequent nucleotide at a site in the studied set of sequences), the absolute number of each of four nucleotides (G, A, C, and T) or the number of deletions or insertions, and their frequencies (percentages). The variability is considered the proportion of sequences for which the nucleotide at a position is different from the nucleotide found in the consensus sequence generated from the set of studied sequences. It is generated by the following formula: 100 - the maximum frequency for each of the four nucleotides at a given position. The program also calculates the number of nucleotides of different nature that are present at a given site. All these data are available in different sheets in tables or plotted in graphical windows. The data are then processed to calculate for a window of 60 nucleotides median, mean, and highest and lowest variability, with standard deviations.

After this analysis was done, the most variable area in rpoB was identified, and a primer pair designed from the border conserved area was used for PCR amplification of this area. PCR conditions that incorporated this consensus primer pair (Br3200F-Br3950R; Table 2) were those described above. These primers were used for amplification of the hypervariable region for all the strains for which complete rpoB sequences were previously determined and 27 additional strains (Table 1). Amplified fragments were then sequenced with the same primers under conditions described above.

Nucleotide sequence accession numbers.
GenBank accession numbers for 16S rRNA and rpoB sequences obtained in this study are listed in Table 1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Determination of rpoB sequences in Afipia, Bosea, and Bradyrhizobium species.
The rpoB sequences varied in length, the longest being that of Afipia felis, with 4,137 bp, and the shortest being that of B. massiliensis, with 4,113 bp (Table 1). The percentage of homology between different species ranged from 83 to 97% (Table 3). It was always lower for rpoB than for 16S ribosomal DNA (rDNA), even for species not well discriminated by 16S rRNA gene sequences (Table 3). In the Afipia genus, A. birgiae and A. massiliensis, which have 99% homology with 16S rRNA gene sequences, have only 96% homology in rpoB. Nearly all members of the genus Bosea that have homologies above 98% for the 16S rRNA gene have homologies that range from 90 to 92% in rpoB. The exceptions in the genus are B. eneae and B. vestrisii, which have only 97% homology in rpoB but whose 16S rRNA gene sequences differ by only 1 position. The phylogenetic trees constructed with the different methods have the same topology except for the relations between Bradyrhizobium spp. and the group of the three Afipia genospecies. Bacteria of the genus Bosea form a group independent from Afipia (Fig. 1). B. vestrisii and B. eneae are separated from other Bosea species, whereas the recently described B. minatitlanensis is closely related with B. thiooxidans. Bosea sp. strain 7F appears as a well-separated species. In the group of Afipia, a cluster that contains A. massiliensis, A. birgiae, A. broomeae, A. clevelandensis, A. felis, and A. felis genospecies A is well separated from other species with high bootstrap values. The two Bradyrhizobium spp. cluster together as Afipia genospecies 1 and 2. The positions of Afipia genospecies 3 strains vary with the technique used to construct the tree and are never supported by high bootstrap values.

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TABLE 3. Percent homology observed between Afipia and Bosea species according to the gene analyzed

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FIG. 1. Dendrogram representing phylogenetic relationships of Afipia and Bosea by the neighbor-joining method. The tree was derived from alignment of complete rpoB sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent). Sinorhizobium meliloti was used as an outgroup.

Strain identification with discriminative partial sequences.
Study of sequence variability allowed detection of four highly variable sequences bordered by conserved regions (Fig. 2). These regions were between positions 481 and 1141, 1741 and 2041, 2881 and 3241, and 3361 and 3841. As the last region was the most variable (no. 4 in Fig. 2), especially the central part of 408 to 420 bp from position 3380 to position 3800 of the sequence of A. felis (AY242824), taken as reference, we designed a consensus primer pair (Br3200F-Br3950R; Table 2) that allowed amplification of a 740- to 752-bp fragment that contains the 408- to 420-bp hypervariable region in all species. Sizes of the amplified fragment and hypervariable region vary according to the species. The hypervariable region was determined for all the strains for which a complete rpoB sequence was determined and for 27 additional strains, 3 of A. felis, 2 of A. broomeae, 1 of B. eneae, 2 of B. vestrisii, 11 of B. massiliensis, and 8 of Bradyrhizobium liaoningense (Table 1). With the exception of those for Afipia genospecies 1 and 2, the percentages of homology observed with the 408- to 420-bp partial rpoB were always lower than those observed with the complete sequence (Table 4). Interestingly, homology between B. eneae and B. vestrisii was lowered to 96%. Among strains belonging to the same species the homology for this fragment ranged from 98 to 100%. The homologies between strains 34614 and 34617^T of B. eneae, between strains 34620 and 34635^T of B. vestrisii, between strain 34649 and isolates 18 and 21 of B. massiliensis, and between all strains of A. felis were 100%. The homology was 98% between strain 34649, strain 63287^T, and isolates 40 to 286 of B. massiliensis, which had the same sequence; between strain B-91-007286^T of A. broomeae and the two other strains of this species that had the same sequence; and between strains 34635^T and 63286 of B. vestrisii. The homology between strain ESG2281^T and isolate 93 of B. japonicum was 99%, and that between ESG2281^T and all other isolates of B. japonicum that shared the same partial sequence was 98%. The trees constructed by using the hypervariable region have the same topologies as those obtained with the complete sequence, but bootstrap values are lower and the distribution of some Bosea spp. is modified (Fig. 3). However, different species remain clearly differentiated.

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FIG. 2. Graphical representation of range site variability (y axis) in rpoB sequences of species studied per window of 60 nucleotides (x axis: position). Hypervariable regions bordered by conserved regions are numbered from 1 to 4.

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TABLE 4. Comparison of the percent homology observed between Afipia and Bosea species according to the size of the rpoB gene studied

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FIG. 3. Dendrogram representing phylogenetic relationships of Afipia and Bosea by the neighbor-joining method. The tree was derived from alignment of partial rpoB sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent). Sinorhizobium meliloti was used as an outgroup.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The description of new bacterial species is currently based on results of DNA-DNA hybridization and phenotypic characters, so-called polyphasic classification data (8, 32). This method of classification has two major drawbacks: the difficulty of performing DNA-DNA hybridization, which is an expensive, technically complex, and labor-intensive procedure, and the scarcity of reproducible and distinguishable phenotypic characteristics for several bacterial species. The development of gene amplification and sequencing, especially that of the 16S rRNA gene sequences, has simplified the identification and the detection of fastidious bacteria, especially those lacking distinguishable phenotypic characteristics. However, as previously described for several species, including Bacillus spp. (1, 7), the 16S rDNA gene alone is not variable enough to allow confident discrimination between different species in some genera. This is the case for bacteria that belong to the genus Afipia and Bosea which we recently described (15, 16). For example, A. felis and A. felis genospecies A represent two distinct genospecies on the basis of DNA-DNA hybridizations and phenotypic data such as susceptibility to antibiotics, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, and whole-cell fatty acid composition (8, 32), but they exhibit levels of 16S rRNA gene sequence similarity of 99.9%. As the comparison of 16S rDNA gene sequences is not sensitive enough for the reliable delineation of several species, comparison of sequences from a more divergent part of the genome, such as the rrs-rrl intergenic spacer, is more suitable, and this approach has been used for other members of the -proteobacterium subgroup, including Nitrobacter spp. (9) and Bradyrhizobium (30). Our data, based on rpoB sequences of these bacteria, confirm that this gene is probably polymorphic enough to replace or supplement the 16S rRNA gene for definitive identification of Afipia and Bosea bacteria, as the two closest bacteria by 16S rRNA gene comparisons, with 1 different position (<0.1%), differ by at least 3% with rpoB. The results of rpoB sequencing support our proposal for removing Afipia genospecies 1 and 2 from the genus Afipia but still do not allow definition of the positions of Afipia genospecies 3 and related strains (15). The rpoB sequences of A. felis and A. felis genospecies A that have homology of only 94% are in agreement with results of DNA-DNA hybridization and clearly confirm that these are different species. Sequencing rpoB could also help classify, without the use of DNA-DNA hybridization, some isolates that are misidentified as Afipia based on 16S rRNA gene sequencing in the GenBank database. The 16S rRNA gene sequences given for Afipia genospecies 8 and 9 are in fact those of Bosea spp. (16).

The major drawback of rpoB sequencing is that the length of the gene (>4,000 bp) does not allow routine molecular identification or detection in clinical samples. For this purpose, we developed a simple tool that allowed determination of regions with high variability flanked by conserved areas. This tool allowed the design of universal primers for amplification and sequencing of a 740- to 752-bp fragment containing a hypervariable region of 408 to 420 bp for identification of all species tested in the phylum. Moreover, the percentages of homology observed in this partial sequence analysis correlate well with results of DNA-DNA hybridization (Table 5). With this partial sequence, a percentage of homology 98% ensures that two bacterial isolates belong to the same species whereas a percentage 96% indicates that they belong to two different species. A. felis and A. felis genospecies A, which are two genospecies on the basis of DNA-DNA hybridization results (45%), appear also as two genospecies by partial rpoB sequence comparison (94%). The development of partial rpoB sequencing allows the quick and accurate identification of bacteria in the genera Afipia and Bosea and detection of potential new species that will be used for surveys of hospital water system colonization and detection of human infection. Last, the procedure for designing PCR primers for amplification of hypervariable areas may be used in primer design for multilocus sequence typing (MLST). MLST is a typing method based on sequence comparisons of multiple loci (18). In this technique, partial sequences of housekeeping genes are determined and used to construct matrices that allow analysis of genetic relationships among isolates of a single species (18, 31). The number of alleles observed by using a given sequence is almost directly proportional to the number of polymorphic sites in the sequence (31). Actually, partial sequences are chosen randomly. Thus, in order to increase the number of alleles without increasing the sizes of determined sequences, it seems important to determine the most-variable regions in a given set of sequences. The SVARAP tool we propose herein could be useful for this purpose.

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TABLE 5. Comparison between DNA-DNA relatedness and percent homology in the rpoB hypervariable region for Bosea spp. and some Afipia spp.

 
We are indebted to J. S. Dumler for reviewing the manuscript and S. Ouattara for providing the B. minatitlanensis strain.

 
* Corresponding author. Mailing address: Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 05, France. Phone: 33.04.91.38.55.17. Fax: 33.04.91.83.03.90. E-mail: bernard.lascola{at}medecine.univ-mrs.fr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2003, p. 6740-6749, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6740-6749.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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