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1 Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan
2 Food Research Laboratories, Mitsui Norin Co. Ltd, 223-1 Miyahara, Fujieda, Shizuoka 426-0133, Japan
Correspondence
Pham Hong Nhung
hongnhung_gifu{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). According to the second edition of Bergey's Manual of Systematic Bacteriology, the genus includes 14 phenospecies that correspond to at least 17 DNA HGs (Martin-Carnahan & Joseph, 2005). Recently, descriptions of novel species, reclassifications and extended descriptions of species with validly published names have resulted in ongoing changes to the taxonomy of the genus Aeromonas.
At present, DNA–DNA hybridization and 16S rRNA gene sequences, along with housekeeping gene sequences, are considered crucial molecular tools for the definition of bacterial species (Stackebrandt et al., 2002). In the genus Aeromonas, however, the high conservation of the 16S rRNA gene sequence (97.8–100 %; Martin-Carnahan & Joseph, 2005) and the existence of several copies of the gene, with intragenomic heterogeneity, in some Aeromonas strains (Morandi et al., 2005) limit the usefulness of the 16S rRNA gene for taxonomic analysis at the species level. DNA–DNA hybridization has not been subjected to extensive analysis of interrelationships for all present species. Moreover, discordances between 16S rRNA gene analysis and DNA–DNA hybridization results (Martínez-Murcia et al., 1992b) or between DNA–DNA hybridization data reported by different authors (Saavedra et al., 2006) have added more fuel to the controversies regarding the sites of Aeromonas species boundaries. It has been reported that housekeeping gene sequences, e.g. gyrB and rpoD, could be used to clarify interspecies phylogenetic relationships within Aeromonas (Yáñez et al., 2003; Soler et al., 2004). In some cases, the different behaviour of gyrB and rpoD sequences has left species delineations (e.g. Aeromonas culicicola and Aeromonas allosaccharophila) in an ambiguous state, so they still need to be resolved (Saavedra et al., 2006).
The dnaJ gene, encoding heat-shock protein 40, which acts as a functional cohort of DnaK in various aspects of protein dynamics (Caplan et al., 1993), has been reported to be a suitable target for phylogenetic study and identification of species of Mycobacterium (Takewaki et al., 1994), Legionella (Liu et al., 2003) and Streptococcus (Itoh et al., 2006). In this study, we used dnaJ gene sequences and extensive DNA–DNA hybridizations in an attempt to clear up the taxonomic confusion regarding interrelationships within the genus Aeromonas.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Strains were grown on tryptic soy agar (Difco) at 28 °C, except for the psychrotolerant Aeromonas salmonicida strains, which were grown at room temperature.
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PCR amplification and sequencing.
DNA was extracted by quick-heat lysis. One colony was suspended in 100 µl distilled water and boiled for 10 min. The supernatant served as a template. Amplification reactions contained 1x PCR buffer, 0.2 mM of each dNTP, 0.1 U Taq polymerase (Takara Shuzo), 0.4 µM of each primer and 1 µl template in a final reaction volume of 20 µl. PCR amplification was carried out in a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems) as follows: a 3 min initial denaturation step at 94 °C, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s and 74 °C for 1 min, with a final extension step of 7 min at 72 °C. Amplified products were examined by agarose gel electrophoresis (1.2 %) and ethidium bromide staining. Purified PCR products were sequenced with the use of a BigDye Terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems) in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions.
Phylogenetic data analysis.
dnaJ sequences were aligned by the CLUSTAL W program (version 1.83) (Thompson et al., 1994). Genetic distances were obtained with the MEGA3 package (Kumar et al., 2004) by the neighbour-joining method (Saitou & Nei, 1987) and Kimura's two-parameter distance model (Kimura, 1980). Phylogenetic trees were constructed by the neighbour-joining and maximum-parsimony methods.
DNA–DNA hybridization.
A total of 21 strains were selected for the DNA–DNA reassociation study. Genomic DNA was prepared using the protocol of Marmur (1961) with minor modifications (Ezaki et al., 1988). DNA–DNA hybridizations were carried out at an optimal temperature of 45 °C in 2x SSC buffer and 50 % formamide, using the fluorometric microplate method (Ezaki et al., 1989).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), was 89.2 %, indicating a divergence of the dnaJ gene that is remarkably greater than that of the 16S rRNA gene (98.7 %), but comparable to that of gyrB (92.2 %) and rpoD (89.3 %) (Soler et al., 2004). Sequence similarity among the Aeromonas strains ranged from 81.2 to 99.9 %, corresponding to between 167 and 1 nucleotide differences. At the intraspecies level, the rate of nucleotide substitution did not exceed 3.3 %, except for Aeromonas hydrophila subsp. dhakensis GTC 2880T, which showed far larger distances (4.7 and 5.1 %, respectively) from A. hydrophila subsp. hydrophila GTC 2793T and A. hydrophila subsp. ranaei GTC 2877T. At the interspecies level, the rate of nucleotide substitutions was greater than 5.2 % for most Aeromonas strain pairs but was only 1.7–3.3 % for a cluster including four species (Aeromonas ichthiosmia, Aeromonas veronii, A. allosaccharophila and A. culicicola) and 1.3 % for Aeromonas encheleia and Aeromonas sp. HG 11. These results brought into question the species delineations of A. culicicola, A. allosaccharophila and Aeromonas sp. HG11.
The phylogeny based on dnaJ sequences of 27 strains constructed by the neighbour-joining method is shown in Fig. 1. The tree generated by the maximum-parsimony method gave the same topology (data not shown). In general, the relationships based on dnaJ sequences were in relatively good agreement with those based on 16S rRNA gene sequences. However, some of the relationships on the 16S rRNA tree were markedly different from those on the dnaJ tree. In terms of the dnaJ gene, all A. hydrophila subspecies were in a monophyletic group, although their substitution rates fell in a rather wide range (1.8–5.1 %), and their allocations were reliable as supported by high bootstrap values. This was in contrast to the 16S rRNA tree, on which A. hydrophila subsp. dhakensis belonged to a separate cluster from the remaining A. hydrophila subspecies (Miñana-Galbis et al., 2004). A. allosaccharophila was closely related to A. veronii on the dnaJ phylogeny but was located in a lineage far from A. veronii on the 16S rRNA tree.
Hybridization results are presented in Table 1. Each value was the mean of triplicate experiments. Repeated experiments exhibited a maximum standard deviation of 4 %. In agreement with the published proposals that A. ichthiosmia and Aeromonas enteropelogenes are respectively considered to be synonyms of A. veronii and Aeromonas trota (Huys et al., 2001, 2002), high genomic relatedness was observed between A. ichthiosmia GTC 2766T and A. veronii strains (83–94 %) as well between A. enteropelogenes GTC 2764T and A. trota GTC 2767T (82–87 %) (Table 1). DNA–DNA relatedness values in reciprocal hybridizations between A. encheleia GTC 2788T and Aeromonas sp. HG11 GTC 2939, between members of the cluster including A. veronii, A. ichthiosmia, A. allosaccharophila and A. culicicola or between Aeromonas bestiarum, Aeromonas popoffii and A. salmonicida strains were also greater than the 70 % threshold recommended for species delineation. These DNA–DNA hybridization results were in agreement with the phylogenetic relationships observed from the dnaJ tree (Fig. 1).
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) and 16S rRNA gene (Fig. 2b) sequence similarities and pairwise DNA–DNA relatedness values show a linear relationship between pairwise dnaJ or 16S rRNA gene sequence similarities and DNA–DNA relatedness values. Regression analysis showed a stronger relationship between DNA–DNA reassociation values and dnaJ sequence similarities (r2=0.706, P<0.001) than between the former and 16S rRNA gene sequence similarities (r2=0.347, P<0.001). As a consequence, phylogenetic relationships between Aeromonas species are in close agreement with the current taxonomic classification of this genus. Additionally, the discriminatory power of the dnaJ sequence is greater than that of the 16S rRNA gene sequence, enabling dnaJ sequences to differentiate clearly any two species. This highlights the fact that the dnaJ gene is a promising phylogenetic marker in the genus Aeromonas.
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Firstly, on the basis of the high DNA–DNA relatedness obtained in a previous study (75.6 %) (and corroborated in this study; 68–72 %) and the impossibility of separation by either biochemical tests or 16S rRNA gene sequence, it was suggested previously that A. salmonicida and A. bestiarum might represent a single taxon (Martínez-Murcia et al., 2005). However, housekeeping gene sequence analysis revealed genetic divergence between these two species. gyrB showed nucleotide substitutions (2.2–3.3 %) on the borderline between the intra- and interspecies ranges (Yáñez et al., 2003), but rpoD, with 6.8–8.7 % divergence (Soler et al., 2004), and dnaJ, with 5.2–6.2 % divergence in this study, allowed easy differentiation of A. salmonicida and A. bestiarum as two separate taxa.
To date, one of the most debatable issues within the genus concerns A. encheleia and Aeromonas sp. HG11. Comparison of 16S rRNA gene sequences of A. encheleia and Aeromonas sp. HG11 strains showed that they were genetically different and phylogenetically separate (Martínez-Murcia, 1999). In a study on the polymorphism of tRNA intergenic spacers, these two species also presented different tDNA-PCR patterns (
aganowska & Kaznowski, 2005). On the other hand, they were found to share similar combined intergenic spacer region (ISR)-RFLP patterns and to constitute one group (aganowska & Kaznowski, 2004). Divergences determined for sequences of gyrB (Yáñez et al., 2003), rpoD (Soler et al., 2004) and dnaJ (in the present study) were respectively 2.1–2.2, 1.4–1.7 and 1.3 %. Compared with the intra- and interspecies ranges of sequence divergence observed in the genus Aeromonas, these values fall within limits that suggest that these taxa might represent a single species. DNA–DNA reassociation, the determinate criterion, gave different results. Esteve et al. (1995) showed that DNA–DNA relatedness between A. encheleia and Aeromonas sp. HG11 strains was only 12 %, whereas Huys et al. (1997a) reported a value of 84 %, considerably exceeding the threshold value of 70 % for membership of the same species. Our hybridization data (82–85 %) were comparable to those reported by Huys et al. (1997a). Thus, we suggest that A. encheleia and Aeromonas sp. HG11 belong to the same genetic group.
Heated discussion is also taking place with respect to A. culicicola and A. allosaccharophila. They were described as two novel Aeromonas species by Pidiyar et al. (2002) and Martínez-Murcia et al. (1992a). In later studies, from evidence deduced from phenotypic characteristics, amplified fragment length polymorphism and DNA–DNA hybridization results, Huys et al. (1996, 2001, 2005) cast doubt on the status of these species as separate from A. veronii. However, contradictions between hybridization data from Huys et al. (2001, 2005) and from the original descriptions of the three species have resulted in the exact positions of these species in the genus Aeromonas remaining unclear. Study of gyrB and rpoD housekeeping gene sequences (Soler et al., 2004) showed that the nucleotide substitution rates of these species were slightly greater than the intraspecies substitution rate (approx. 2 %), but it is worth noting that the collection of Aeromonas strains used by Soler et al. (2004) did not include members of the A. hydrophila subspecies. When A. hydrophila subspecies were added, the highest intrasubspecies substitution rates were increased to 4.4 % for gyrB and 5.1 % for rpoD. In our study, strains of A. culicicola and A. allosaccharophila, together with two A. veronii biovars and A. ichthiosmia (a later synonym of A. veronii), clustered in a very close genetic group. Sequence divergences among them (1.7–3.3 %) were the lowest in comparison with interspecies divergence in the entire genus and were comparable to the intraspecies divergence. The high genomic relatedness (67–97 %; Table 1) observed between the five strains A. ichthiosmia GTC 2766T, A. culicicola GTC 2882T, A. allosaccharophila GTC 2879T, A. veronii bv. Veronii GTC 2800T and A. veronii bv. Sobria GTC 2938 corroborated the phylogenetic relationships obtained from housekeeping gene analysis, indicating a highly close genetic relationship between them. In terms of the status of A. culicicola and A. allosaccharophila in the genus Aeromonas, Huys et al. (2001, 2005) have endeavoured to prove the affiliation of A. culicicola and A. allosaccharophila to A. veronii by strong phenotypic and genotypic evidence, but the positions of these species remain debatable because of contradictions between sets of DNA–DNA hybridization data and a disagreement between recently published phylogenetic data (Soler et al., 2004; Saavedra et al., 2006). Our DNA–DNA hybridization results and phylogenetic analysis support the results given by Huys et al. (2001, 2005) to justify the recognition of A. allosaccharophila and A. culicicola as later heterotypic synonyms of A. veronii.
It should also be noted that, in this study, A. bestiarum GTC 2790T and A. popoffii GTC 2881T showed high DNA–DNA relatedness (71–81 %), which is rather in disagreement with the result given in the original description of A. popoffii (53 %; Huys et al., 1997b). Despite the tight relationship, these species were clearly distinguishable on the basis of housekeeping gene sequence divergence (3.0, 4.4 and 5.5 %, respectively, for gyrB, rpoD and dnaJ). A likely explanation for the discrepancy observed between sequencing and hybridization data in the case of A. bestiarum and A. salmonicida or A. bestiarum and A. popoffii is that the measure of DNA sequence relatedness by DNA–DNA hybridization may be too crude, or at least not adequately fine to separate highly related species (Martínez-Murcia et al., 2005).
In conclusion, our findings indicate that analysis of dnaJ sequences offers advantages over the 16S rRNA gene in defining phylogenetic relationships within the genus Aeromonas. Results obtained from analyses of dnaJ sequences and DNA–DNA hybridization in this study provide additional information on the interrelationships of the genus Aeromonas, allowing greater accuracy and reliability to be obtained. However, additional reference strains for each species are required for validation of the cut-off point of dnaJ sequence divergence for species delineation.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Esteve, C., Gutierréz, M. C. & Ventosa, A. (1995). Aeromonas encheleia sp. nov., isolated from European eels. Int J Syst Bacteriol 45, 462–466.
Ezaki, T., Takeuchi, N., Liu, S. L., Kai, A., Yamamoto, H. & Yabuuchi, E. (1988). Small-scale DNA preparation for rapid genetic identification of Campylobacter species without radioisotope. Microbiol Immunol 32, 141–150.[Medline]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Huys, G., Coopman, R., Janssen, P. & Kersters, K. (1996). High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting. Int J Syst Bacteriol 46, 572–580.
Huys, G., Kämpfer, P., Altwegg, M., Coopman, R., Janssen, P., Gillis, M. & Kersters, K. (1997a). Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended descriptions of the species Aeromonas eucrenophila and A. encheleia. Int J Syst Bacteriol 47, 1157–1164.
Huys, G., Kämpfer, P., Altwegg, M., Kersters, I., Lamb, A., Coopman, R., Lüthy-Hottenstein, J., Vancanneyt, M., Janssen, P. & Kersters, K. (1997b). Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs. Int J Syst Bacteriol 47, 1165–1171.
Huys, G., Kämpfer, P. & Swings, J. (2001). New DNA-DNA hybridization and phenotypic data on the species Aeromonas ichthiosmia and Aeromonas allosaccharophila: A. ichthiosmia Schubert et al. 1990 is a later synonym of A. veronii Hickman-Brenner et al. 1987. Syst Appl Microbiol 24, 177–182.[CrossRef][Medline]
Huys, G., Denys, R. & Swings, J. (2002). DNA–DNA reassociation and phenotypic data indicate synonymy between Aeromonas enteropelogenes Schubert et al. 1990 and Aeromonas trota Carnahan et al. 1991. Int J Syst Evol Microbiol 52, 1969–1972.[Abstract]
Huys, G., Cnockaert, M. & Swings, J. (2005). Aeromonas culicicola Pidiyar et al. 2002 is a later subjective synonym of Aeromonas veronii Hickman-Brenner et al. 1987. Syst Appl Microbiol 28, 604–609.[CrossRef][Medline]
Itoh, Y., Kawamura, Y., Kasai, H., Shah, M. M., Nhung, P. H., Yamada, M., Sun, X., Koyana, T., Hayashi, M. & other authors (2006). dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. Syst Appl Microbiol 29, 368–374.[CrossRef][Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
aganowska, M. & Kaznowski, A. (2004). Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp. Syst Appl Microbiol 27, 549–557.[CrossRef][Medline]
aganowska, M. & Kaznowski, A. (2005). Polymorphism of Aeromonas spp. tRNA intergenic spacers. Syst Appl Microbiol 28, 222–229.[CrossRef][Medline]
La Scola, B., Zeaiter, Z., Khamis, A. & Raoult, A. (2003). Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm. Trends Microbiol 11, 318–321.[CrossRef][Medline]
Liu, H., Li, Y., Huang, X., Kawamura, Y. & Ezaki, T. (2003). Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella. Microbiol Immunol 47, 859–869.[Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Martin-Carnahan, A. & Joseph, S. W. (2005). Genus I. Aeromonas Stanier 1943, 213AL. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part B, pp. 557–578. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
Martínez-Murcia, A. J. (1999). Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501. Int J Syst Bacteriol 49, 1403–1408.
Martínez-Murcia, A. J., Esteve, C. & Collins, M. D. (1992a). Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. FEMS Microbiol Lett 91, 199–206.[CrossRef]
Martínez-Murcia, A. J., Menlloch, S. & Collins, A. D. (1992b). Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations. Int J Syst Bacteriol 42, 412–421.
Martínez-Murcia, A. J., Soler, L., Saavedra, M. J., Chacón, M. R., Guarro, J., Stackebrandt, E. & Figueras, M. J. (2005). Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from Aeromonas bestiarum. Int Microbiol 8, 259–269.[Medline]
Miñana-Galbis, D., Farfán, M., Fusté, M. C. & Lorén, J. G. (2004). Aeromonas molluscorum sp. nov., isolated from bivalve molluscs. Int J Syst Evol Microbiol 54, 2073–2078.
Morandi, A., Zhaxybayeva, O., Gogarten, J. P. & Graf, J. (2005). Evolutionary and diagnostic implications of intragenomic heterogeneity in the 16S rRNA gene in Aeromonas strains. J Bacteriol 187, 6561–6564.
Pidiyar, V., Kaznowski, A., Narayan, N. B., Patole, M. & Shouche, Y. S. (2002). Aeromonas culicicola sp. nov., from the midgut of Culex quinquefasciatus. Int J Syst Evol Microbiol 52, 1723–1728.[Abstract]
Saavedra, M. J., Figuera, M. J. & Martínez-Murcia, A. J. (2006). Updated phylogeny of the genus Aeromonas. Int J Syst Evol Microbiol 56, 2481–2487.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Soler, L., Yáñez, M. A., Chacon, M. R., Aguilera-Arreola, M. G., Catalán, V., Figueras, M. J. & Martínez-Murcia, A. J. (2004). Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes. Int J Syst Evol Microbiol 54, 1511–1519.
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C., Nesme, X., Rosselló-Mora, R., Swing, J. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
Takewaki, S., Okuzumi, K., Manabe, I., Tanimura, M., Miyamura, K., Nakahara, K., Yazaki, Y., Ohkubo, A. & Nagai, R. (1994). Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species. Int J Syst Bacteriol 44, 159–166.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Yáñez, M. A., Catalán, V., Apráiz, D., Figueras, M. J. & Martínez-Murcia, A. J. (2003). Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences. Int J Syst Evol Microbiol 53, 875–883.
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