Abstract
An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2+/-0.9%. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were <or=3.5%, while interspecific divergence rates fell between 3.7 and 16.9%, excluding A. sharmana. In this study, cpn60 UT sequencing was shown to be a universal, useful, simple and rapid method for the identification and phylogenetic affiliation of Aeromonas strains.
Highlights
Classification of the genus Aeromonas, which belongs to the Gammaproteobacteria, remains complex from a taxonomic point of view because of the continuous description of novel species, the rearrangement of strains and species described so far and the discrepancies observed in different DNA–DNA hybridization studies (Huys et al, 1997, 2001, 2005; Martınez-Murcia, 1999; Esteve et al, 2003; MinanaGalbis et al, 2007)
Sequence analysis of different housekeeping genes has been recommended for species delineation in addition to DNA–DNA hybridization in order to increase discriminatory power and the robustness of phylogenetic relationships with regard to 16S rRNA gene sequence analysis (Stackebrandt et al, 2002)
We sequenced the universal target (UT) region of the cpn60 gene, a 555 bp fragment corresponding to positions 274–828 of the complete gene, in 35 strains representing all species and subspecies of the genus Aeromonas (Table 1)
Summary
Classification of the genus Aeromonas, which belongs to the Gammaproteobacteria, remains complex from a taxonomic point of view because of the continuous description of novel species, the rearrangement of strains and species described so far and the discrepancies observed in different DNA–DNA hybridization studies (Huys et al, 1997, 2001, 2005; Martınez-Murcia, 1999; Esteve et al, 2003; MinanaGalbis et al, 2007). Hill et al (2004) have analysed sequences of this gene from a wide variety of bacterial, archaeal and eukaryotic species They concluded that a 549–567 bp region of the cpn gene (the universal target or UT), amplified by universal PCR primers, is representative of the complete gene We have sequenced and analysed a 555 nt sequence, corresponding to the UT region of the cpn gene, in type and reference strains of all Aeromonas described to date. This should allow us to evaluate its applicability for species delineation and identification within Aeromonas. Sequencing of the cpn gene increases the number of housekeeping genes sequenced from Aeromonas species, but may facilitate simple and rapid Aeromonas species identification
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