M. Carmen Fusté

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA-DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85(T) and CDC 2555-87 were low (0.6-1.1%). The DNA G+C content of the type strain was 62.2mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85(T) (=CECT 4254(T)=ATCC 43946(T)=LMG 17321(T)).

Bacterial lipoxygenases, a new subfamily of enzymes? A phylogenetic approach

Lipoxygenases (EC. 1.13.11.12) are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. They catalyze the dioxygenation of 1Z,4Z-pentadiene moieties of polyunsaturated fatty acids obtaining hydroperoxy fatty acids. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates, and fungi, but now, some lipoxygenase sequences have been detected on prokaryotic organisms, changing the idea that lipoxygenases are exclusively a eukaryotic affair. Lipoxygenases are involved in different types of reactions on eukaryote organisms where the biological role and the structural characteristics of these enzymes are well studied. However, these aspects of the bacterial lipoxygenases have not yet been elucidated and are unknown. This revision discusses biochemical aspects, biological applications, and some characteristics of these enzymes and tries to determine the existence of a subfamily of bacterial lipoxygenases in the context of the phylogeny of prokaryotic lipoxygenases, supporting the results of phylogenetic analyzes with the comparison and discussion of structural information of the first prokaryotic lipoxygenase crystallized and other eukaryotic lipoxygenases structures.

Molecular Phylogenetics and Temporal Diversification in the Genus Aeromonas Based on the Sequences of Five Housekeeping Genes

Several approaches have been developed to estimate both the relative and absolute rates of speciation and extinction within clades based on molecular phylogenetic reconstructions of evolutionary relationships, according to an underlying model of diversification. However, the macroevolutionary models established for eukaryotes have scarcely been used with prokaryotes. We have investigated the rate and pattern of cladogenesis in the genus Aeromonas (γ-Proteobacteria, Proteobacteria, Bacteria) using the sequences of five housekeeping genes and an uncorrelated relaxed-clock approach. To our knowledge, until now this analysis has never been applied to all the species described in a bacterial genus and thus opens up the possibility of establishing models of speciation from sequence data commonly used in phylogenetic studies of prokaryotes. Our results suggest that the genus Aeromonas began to diverge between 248 and 266 million years ago, exhibiting a constant divergence rate through the Phanerozoic, which could be described as a pure birth process.

Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas.

The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.

Reclassification of Aeromonas hydrophila subspecies anaerogenes

Technological advances together with the continuous description of new taxa have led to frequent reclassifications in bacterial taxonomy. In this study, an extensive bibliographic revision, as well as a sequence analysis of nine housekeeping genes (cpn60, dnaJ, dnaX, gyrA, gyrB, mdh, recA, rpoB and rpoD) and a phenotypic identification of Aeromonas hydrophila subspecies anaerogenes were performed. All data obtained from previous physiological, phylogenetic, and DNA-DNA hybridization studies together with those presented in this study suggested that A. hydrophila subspecies anaerogenes belonged to the species Aeromonas caviae rather than A. hydrophila. Therefore, the inclusion of A. hydrophila subsp. anaerogenes in the species A. caviae is proposed.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BackgroundThe bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species.ResultsWe sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an ω ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence.ConclusionThe models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection.ReviewersThis article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

Draft Genome Sequence of Aeromonas molluscorum Strain 848TT, Isolated from Bivalve Molluscs

ABSTRACT We report here the draft genome sequence of Aeromonas molluscorum 848T, the type strain of this Aeromonas species, which was isolated from wedge shells (Donax trunculus) obtained from a retail market in Barcelona, Spain, in 1997.

Prediction of whole-genome DNA G + C content within the genus Aeromonas based on housekeeping gene sequences ☆

Different methods are available to determine the G+C content (e.g. thermal denaturation temperature or high performance liquid chromatography, HPLC), but obtained values may differ significantly between strains, as well as between laboratories. Recently, several authors have demonstrated that the genomic DNA G+C content of prokaryotes can be reliably estimated from one or several protein coding gene nucleotide sequences. Few G+C content values have been published for the Aeromonas species described and the data, when available, are often incomplete or provide only a range of values. Our aim in this current work was twofold. First, the genomic G+C content of the type or reference strains of all species and subspecies of the genus Aeromonas was determined with a traditional experimental method in the same laboratory. Second, we wanted to see if the sequence-based method to estimate the G+C content described by Fournier et al. [7] could be applied to determine the G+C content of the different species of Aeromonas from the sequences of the genes used in taxonomy or phylogeny for this genus.

Direct evidence of recombination in the recA gene of Aeromonas bestiarum.

Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.

Potential pathogenicity of Aeromonas hydrophila complex strains isolated from clinical, food, and environmental sources

Aeromonas are autochthonous inhabitants of aquatic environments, including chlorinated and polluted waters, although they can also be isolated from a wide variety of environmental and clinical sources. They cause infections in vertebrates and invertebrates and are considered to be an emerging pathogen in humans, producing intestinal and extra-intestinal diseases. Most of the clinical isolates correspond to A. hydrophila, A. caviae, and A. veronii bv. Sobria, which are described as the causative agents of wound infections, septicaemia, and meningitis in immunocompromised people, and diarrhoea and dysenteric infections in the elderly and children. The pathogenic factors associated with Aeromonas are multifactorial and involve structural components, siderophores, quorum-sensing mechanisms, secretion systems, extracellular enzymes, and exotoxins. In this study, we analysed a representative number of clinical and environmental strains belonging to the A. hydrophila species complex to evaluate their potential pathogenicity. We thereby detected their enzymatic activities and antibiotic susceptibility pattern and the presence of virulence genes (aer, alt, ast, and ascV). The notably high prevalence of these virulence factors, even in environmental strains, indicated a potential pathogenic capacity. Additionally, we determined the adhesion capacity and cytopathic effects of this group of strains in Caco-2 cells. Most of the strains exhibited adherence and caused complete lysis.