IV. RESULTS AND DISCUSSION
3.1. Background
3.5.2. Multilocus sequence analysis (MLSA)
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3.5.2. Multilocus sequence analysis (MLSA)
In order to complement the intraspecific diversity studies of Halococcus strains, two coding proteins hypervariable positions using Gblocks program (http://molevol.ibmb.csic.es/Gblocks_-‐server.html). The total number of substitutions independently of whether they were synonymous or nonsynonymous, as well as the number of insertions-‐deletions, were calculated for each gene. After the translation of the each alignment, number of informative positions, and accession numbers are specified.
Results and Discussion: Chapter 3
Results and Discussion: Chapter 3 calculations. The bar indicates 2% sequence divergence.
Results and Discussion: Chapter 3 this observation, two different phylogenetic reconstructions were performed. The first was done using sequences from the three genes used for MLSA analysis (Fig. 34), and the second, additionating the 16S contribution of atpB and ITS genes, whose phylogenetic reconstructions also showed that these strains were not closely related (see Fig.33).
Results and Discussion: Chapter 3
Figure 34: PHYML-‐based phylogenetic reconstructions of a concatenated of the three genes studied, showing also the inclusion of 16S rRNA gene sequences. The bootstrap values are obtained after 100 calculations. The bar indicates 2% sequence divergence.
Results and Discussion: Chapter 3
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3.6. Conclusions
In this chapter, the suitability of the salt glands of the seabird C. diomedea as a potential habitat for cultivable extremely halophilic aerobic prokaryotes has been analyzed. All the microorganisms isolated using the methods described above were members of the Archaea domain, whereas members of the Bacteria domain were not detected. The sequencing of the 16S rRNA gene in fourteen selected colonies affiliated the new isolates with Halococcus spp.
Members of this genus have been found in environments as distinct as solar saltern brines (Javor, 1984), stromatolites (Goh et al., 2006), fish sauce (Namwong et al., 2007), alpine salt deposits from the Permian age (Stan-‐Lotter et al., 2002), and salted codfish (Farlow, 1880). All such findings indicate that Halococcus spp. may be very versatile as revealed by the enormous range of environments in where they thrive, or highly resistant to hostile environments as low-‐salinity systems (Rodríguez-‐Valera et al., 1979). Seven of the fourteen strains that affiliated with the genus Halococcus were further studied. The phylogenetic reconstruction based on the nearly complete sequence of the 16S rRNA gene of the isolates affiliated them with the three species Hcc. morrhuae, Hcc. dombrowskii (Stan-‐Lotter et al., 2002), and Hcc. quingdaonensis (Wang et al., 2007). In particular, for this group of species, the sequence divergence is so low (27 changes in homologous positions in the whole alignment) that no clear branching order could be revealed. Actually, all of them form a clade without resolution at the species level. The lack of resolution power at the species category level of the small subunit of the ribosome is well known, and thus DDH is appealed to solve the discrimination (Stackebrandt et al., 2002). In our case, the identification of the new isolates to the existing species was clear by using DDH. The results indicated that six of them could be identified as members of Hcc. morrhuae, and one to Hcc.
dombrowskii. However, the phenotypic traits studied showed that the strain collection was endorsed with a certain degree of diversity. The problem is that, due to the diverse metabolism shown by the strains in study, no clear phenotypic property is found to assure a distinct species status (Stackebrandt et al., 2002).
The fact that genospecies cannot be phenotypically discriminated is not unusual for species that account with a large set of isolates, and this is especially evident in widely spread species with versatile metabolism (Rosselló-‐Móra & Amann, 2001). In such cases, when difficulties to discriminate genomic groups by means of phenotypic traits arise, the plausible classification may be as genomovars (Rosselló-‐
Móra & Amann, 2001). The difficulties in discriminating both species are even reinforced by our studies on MLSA. The taxonomic problem raised here has no trivial solution.
Results and Discussion: Chapter 3
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As Hcc. dombrowskii, numerous species are yearly classified basing their studies on a single strain, a fact that hampers the recognition of the taxon diversity, and endorses the species description with a high degree of rigidity (Christensen et al., 2001). The problems arise when new closely related strains are isolated and their genetic and phenotypic profiles do not match that of the type strain, leading to the species description emendation, or even to reclassification. Some microbiologists consider the current definition too coarse to circumscribe what may be a unit assumed to be species (Konstantinidis & Tiedje, 2007). However, others appeal to the use of a pragmatic species circumscription for prokaryotes (Rosselló-‐Móra & Amann, 2001), in where a certain degree of intraspecific variation is allowed. In this case, applying a relaxed definition would simplify the discussion on the species colonization of bird nostrils, as it would recognize just one colonizing species.
In any case, and independently of the fact that we may recognize one or two species colonizing nostrils, it is clear that this environment constitutes an ideal habitat for the colonization of haloarchaea due to the presence of the salt crystals produced by the salt gland. The isolation of closely related members from all sampled nostrils, indicates that Halococcus are part of their natural culturable epizootic microbial community. Even if we consider that two species of the same genus coexist on the nostril mucosa, the observation is not surprising. As much as nine different species of Deinococcus have been demonstrated to coexist in the same environment (Rainey et al., 2005). In any case, all the data analyzed here showed both species to be very closely related although phenotypically indistinguishable, and most probably sharing an ecological niche.
Finally, the demonstration that the salt-‐excreting glands of seabirds constitute an appropriate habitat for extremely halophilic prokaryotes may have as well implications in their dispersion mechanism. The migratory habits of C. diodomea allows them to reach very distant environments in a constant yearly basis (González-‐Solís et al., 2007), and may help in the dispersion of Halococcus spp. through Earth. This may constitute one of the most rapid and effective mechanisms to blurry allopatric speciation for members of this archaeal species.
V. CONCLUSIONS
Conclusions
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1. The intraspecific metabolic geographical patterns of Salinibacter ruber strains isolated from different geographical areas can be readily revealed by High Resolution Mass Spectrometry (ICR-‐FT/MS) approach, whereas standard genetic methods as Multilocus Sequence Analysis (MLSA) and other molecular fingerprinting failed to resolve the corresponding genetic-‐
geographic patterns.
2. Mediterranean, Atlantic and Peruvian strains can be distinguished by some membrane-‐
associated compounds, which could be the result of the influence of the different environmental conditions of the sites in where they had been isolated.
3. The metabolic response of the two closest relative S. ruber strains M8 and M31 under stress or at the stationary phase of the growth curve revealed a gradual loss of culturability. However, the abrupt drop in the culturability was not accompanied by a decrease in FISH counts, pointing to a transition to viable but non-‐cultivable state rather than cell death especially under adverse conditions.
4. The most remarkable metabolomic changes during the transition from exponential to stationary phase and during stress conditions were attributed to modifications in the composition of the cell envelope, and especially in the cell membrane. These changes were related to the length or saturation ratio of the fatty acids involved in the glycerolipid and glycerophospholipid metabolisms.
5. High Resolution Mass Spectrometry (ICR-‐FT/MS) approach showed to have a higher resolution power for revealing biogeographical patterns and metabolic changes under adverse conditions, demonstrating that the pathways related to the synthesis and metabolism of cell envelope compounds in the extremely halophilic bacteria Salinibacter ruber could be the first line of defence guarding the organism from environmental conditions.
6. The isolation of closely related Halococcus strains from salt-‐excreting glands of seabirds demonstrate that they constitute an appropriate habitat for extremely halophilic prokaryotes and may constitute a rapid and effective mechanism of dispersal the members of this archaeal and other halophilic species.
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