Recently, an unexpectedly high abundance of signals with the bacterial probe EUB338 in samples from a hypersaline crystallizer pond in Alicante (5
) was reported. This result indicated that members of the domain Bacteria
could form an important part of the autochthonous microbiota, in contrast with what had been reported previously (6
). DGGE results, and the subsequent retrieval of the most abundant bacterial 16S rRNA sequences of the hypersaline pond, suggested that most of the EUB338-positive signals in these samples corresponded to hitherto-uncultured members of the domain Bacteria
. These samples had low bacterial diversity, as indicated by DGGE and subsequent 16S rDNA clone library analysis. Sequencing and phylogenetic reconstruction of the two analyzed clone sequences showed that both were affiliated with Rhodothermus
species, forming a deep branch within the CFB phylum (33
). R. marinus
and R. obamensis
are thermophilic (optimum growth temperatures, 65 and 80°C, respectively), moderately halophilic bacteria isolated from marine hydrothermal environments (1
). Thus, to date this deep CFB branch is composed only of extremophilic (e.g., thermophilic and halophilic) Bacteria
. Given the low sequence similarity of EHB-1 and EHB-2 to their closest known relative, EHB can be considered a new genus within the phylum. The highest similarity (86%) between the two bacterial groups is far from the empirical limit of 94% of sequence identity that discriminates genera (12
). Therefore, in accordance with the work of Murray and Schleifer (15
), we propose provisional classification of EHB as “Candidatus
Salinibacter gen. nov.,” with the following short description: phylogenetic position, Cytophaga-Flavobacterium-Bacteroides
phylum; cultivation, noncultivated; gram reaction, negative; morphology, rod; basis of assignment, 16S rDNA sequences (EMBL accession numbers AJ133744
) and oligonucleotide probe EHB412, 5′-TACGCCCCATAGGGGTGT-3′; habitat, hypersaline environments; metabolism and unusual features, extremely halophilic; authors, Antón et al. (this study).
Since the two EHB sequences were closely related, a probe (EHB412) that specifically targeted the candidate genus was designed. This probe was used for quantifying EHB and studying their geographical distribution and growth. In addition, we also determined that these two sequences did not represent the same bacterium, since prokaryotes with different rRNA operons have been described for both the Archaea
and the Bacteria
). For this purpose, probes EHB586 and EHB1451, which specifically targeted EHB-1 and EHB-2, respectively, were designed. The results proved that the two 16S rDNA sequences retrieved by DGGE and cloning analysis corresponded to two different bacterial populations. However, these two populations were closely related (97.6% sequence similarity in the 16S rRNA gene), and therefore the candidate genus was monitored with probe EHB412.
The halophilic nature of these Bacteria
has been shown without having isolated them in pure culture. The first evidence of the strict halophilicity of the EHB was given by their distribution and abundance along the salinity gradient (Table ). EHB appeared only in ponds with at least 20% salinity, and their highest abundance was observed in the crystallizer pond, where they represented nearly the whole bacterial population. The second evidence was given by the enrichment experiments depicted in Fig. . The absence of growth at 15% total salts, together with optimum growth between 20 and 25% total salts, indicated that EHB were strict halophiles (17
). They grew with a generation time of about 0.7 day at these salinities.
To the best of our knowledge (17
), there are currently three known bacterial species which proliferate in saturated salts. One is Halorhodospira halophila
(formerly Ectothiorhodospira halophila
), a halophilic phototrophic bacterium isolated from soda lakes (10
), which grows optimally in an environment around 25% NaCl. The second is Haloanaerobium lacusroseus
), an anerobic bacterium isolated from sediments of a hypersaline lake. There is also one actinomycete, Actinopolyspora halophila
, able to grow in saturated NaCl that was first isolated as a contaminant of culture medium containing 25% NaCl (11
). The ecological relevance of the aforementioned bacteria is unknown, since no studies of their in situ abundance have been carried out.
Moreover, the occurrence of EHB as a significant part of the autochthonous microbiota in hypersaline environments in Alicante is not an isolated case. EHB412-positive bacteria with high rRNA content have been found in salterns of the Canary Islands and the Balearic Islands, and even in the east Mediterranean (Table ). Abundances of these organisms varied with the salinity, but it is worth mentioning that in the salterns of Majorca, the population of a single phylotype represented one-fourth of the total prokaryotic population.
Thus, our results indicate that EHB are part of the autochthonous microbiota in hypersaline environments, which had been repeatedly described as dominated by halophilic Archaea
). However, it is not surprising that the presence of EHB was not detected before if we consider that the most abundant archaeon in these environments has never been cultured (5
). Even for an environment with such a low diversity, culture-dependent techniques have offered a very biased view of the prokaryotic community composition.
Until recently (18
), it had been commonly assumed that Archaea
dwell in extreme habitats (e.g., high temperature, high salt, low oxygen), whereas Bacteria
are restricted to moderate sites. Lately, molecular data have indicated the presence of Archaea
in moderate environments, such as marine waters or soil (18
), while Bacteria
seem to be quite common, for example, at high temperatures that were once considered to be exclusive for Archaea
). The present work is the first report indicating that Bacteria
constitute a significant and important part of the microbiota that inhabit NaCl-saturated water, another classical habitat for Archaea
. Both domains have obviously developed wide ecological competence. Bacteria
seem to be as widespread as Archaea