Ribosomal multi-operon diversity: an original perspective on the genus Aeromonas

Abstract

16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1-13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker in the genus Aeromonas for both evolutionary and polyphasic taxonomic studies provided that multi-operon fingerprinting approaches are used.

Figure 1. Number of rrn operons and PCR-TTGE patterns for the 195 Aeromonas strains of the study.

Results are presented according to the structure of the population observed in multilocus phylogenetic analysis (MLPA). Unrooted maximum-likelihood tree was based on concatenated sequences of five housekeeping genes (gltA, gyrB, rpoB, tsf and zipA genes), as described in Roger et al. . Only type and references strains are indicated within clades on the tree. The horizontal lines show genetic distance, the scale bar indicating the number of substitutions per nucleotide position. The numbers at the nodes are support values estimated with 100 bootstrap replicates. Only bootstrap values >70 are indicated on the tree. When all members of a clade shared identical features, the common rrn operon number and/or PCR-TTGE pattern was indicated. * The A. caviae clade included the type strain A. caviae CECT 838^T displaying an external position to other members of the A. caviae clade in the tree.

Figure 2. Schematic representation of I-CeuI-restricted DNA fragment sizes for Aeromonas spp. strains with 10 rrn operons.

Data were given for the 142 A. veronii, A. hydrophila, A. aquariorum, A. caviae and A. salmonicida strains with 10 rrn operons with the aim to illustrate interspecific variability between the 5 taxa and the intraspecific variability for the 3 main species. I-CeuI-restricted DNA fragments were numbered in size descending order; the size of the largest fragment 1 was not measured (as discussed in the text). A to F, size distribution for fragments 2 to 7, respectively. Fragment mean sizes (in kilobases) were indicated in the corresponding tables according to the species and by a blue line in the corresponding scheme A to F. Standard deviation values (SD) and coefficient of variation (CV) were indicated in kb and %, respectively, in the corresponding tables according to the species except for species with less than 10 representatives; +/−1 SD were represented by black dotted lines and +/−2 SD by red dotted lines in the corresponding scheme A to F. Schematic representations for the 3 smallest fragments with mean size lower than 97 kb (mean size and SD of 93.7±23 kb, 66.7±9.1 kb and 40.5±7.5 kb, respectively) were not presented here because they were not informative.