The divergent movement of the African and Arabian tectonic plates, which is associated with deposition of new oceanic crust, led to the formation of several isolated topographical depressions within the Red Sea basin (4
). Redissolution of buried Miocenic evaporites and/or hydrothermal phase separation, followed by upward migration to the seafloor and later accumulation in enclosed depressions, filled as many as 25 such deeps with highly saline water (13
). In addition to their common extreme salinity (up to 27% [wt/vol] NaCl), all of these brines are anoxic, are enriched in metals and hydrocarbons, and have temperatures higher than 21.7°C, which is the mean deep-seawater temperature of the Red Sea (13
Samples for geochemical and microbiological studies were retrieved from the northernmost brine-filled deeps of the Red Sea during the RV Meteor Cruise 52/3 in 2002. Isolation attempts with a sample of the previously unexplored brine-sediment interface of the Shaban Deep led to the recovery of strain SSD-17BT. This isolate displayed a highly unusual morphology with contractile “tentacle-like” structures and minibodies, and it represents a novel phylogenetic lineage. The phenotypic and phylogenetic data point to the distinctiveness of this organism, which we believe represents a novel taxon at the order level.
The brine-filled deeps of the Red Sea represent unusual environments which remain largely unexplored. During the course of a microbial diversity assessment study of the Shaban Deep, an organism designated SSD-17BT
was recovered from the brine-sediment interface of this deep, which was striking in both its morphology and its dynamic contractility. Phylogenetic studies showed strain SSD-17BT
to represent a new lineage between the phylum Firmicutes
and the phylum Tenericutes
). The Firmicutes
currently comprise the low-G+C gram-positive organisms such as the bacilli and clostridia. The Tenericutes
lineage comprises wall-less species of genera such as Mycoplasma
, as well as cell-walled lineages such as the misnamed taxa Clostridium ramosum
, Clostridium innocuum
, Eubacterium biforme
, and the species of the genera Solobacterium
, and Erysipelothrix
. Even though the branching point of the lineage representing strain SSD-17BT
is equidistant to the lineages of the Firmicutes
and the Tenericutes
), the sequence similarities between the new organism and representatives of the Firmicutes
are higher. This is in part due to the short branch lengths of genera such as Gemella
, and Bacillus
, in contrast to the extended lineages of genera of the phylum Tenericutes
) such as Acholeplasma
, and Coprobacillus
. This new lineage, in addition to strain SSD-17BT
, contains a number of environmental 16S rRNA gene clone sequences at the 91.0 to 94.0% similarity level. These include an environmental clone sequence retrieved during a study of the halophilic and alkalophilic microbial communities from Lake Van in Turkey (23
). There is a significant ecological correlation between the two saline environments, but the other branch within this novel lineage comprises two highly similar environmental clone sequences retrieved from mammalian gastrointestinal tracts, which are not presently known to harbor halophilic taxa.
The species represented by strain SSD-17BT
shares several morphological characteristics with the wall-less species of the class Mollicutes
, including the absence of a cell wall, which was not observed by electron microscopy. Resistance to penicillin and cephalosporin also argues for the absence of a peptidoglycan layer, as in the wall-less mollicutes but unlike the members of the Firmicutes
, namely, the species of the genus Gemella
) and walled mollicutes where penicillin sensitivity has been examined (21
). The ability to be filtered through 450-nm pore membrane filters, typical colonies with a “fried-egg”-like appearance, and the unusual cellular contractility and dynamic morphology of this isolate also indicates the lack of a rigid cell wall and evokes the flagellum-independent movements seen in the strains of the genus Spiroplasma
). However, the new organism possesses a central coccoid body which is not found in the species of the genus Spiroplasma
. The chains of microspheres which appear to derive from the “tentacles” protruding from the central body of strain SSD-17BT
might result from an atypical binary fission process, similar to that observed in isolates of the genus Mycoplasma
). These characteristics of strain SSD-17BT
, like for other wall-less organisms, are probably due to the lack of a rigid cell wall.
The fatty acid composition indicates that the organism from the Shaban Deep, like several wall-less members of the class Mollicutes
, a few walled relatives (namely, Erysipelothrix
and the most closely related members of the Firmicutes
, such as Gemella
), the lactic acid bacteria, and Aerococcus
, possesses straight-chain saturated and unsaturated fatty acids (21
). MK-4 has been rarely detected in bacteria but has been found in Mycoplasma arthriditis
and Acholeplasma axanthum
), although most authors have not found respiratory quinones in wall-less members of the class Mollicutes
). On the other hand, trace amounts of MK-7 have been found in Erysipelothrix tonsilarum
), although respiratory quinones have not been detected or sought in other walled mollicutes or the most closely related genera of the Firmicutes
Despite many similarities to the wall-less members of the Mollicutes
, the ability of isolate SSD-17BT
to grow at between 1.5 and 18% NaCl clearly clashes with the characteristic osmotic fragility and the obligate association with plant or animal hosts for growth, which are hallmarks of members of the class Mollicutes
). The relatively simple medium for growth (which does not contain complex organic components such as serum), the extreme environment from which the organism was isolated, and the lack of host dependency of strain SSD-17BT
, in addition to the phylogenetic position, strongly support the proposal of a novel genus and species which we name Haloplasma contractile
gen. nov., sp. nov. Moreover, the distinct phylogenetic lineage of strain SSD-17BT
argues for the proposal of a higher-level taxon to accommodate this bacterium, perhaps at the phylum level. However, only one strain of this lineage has been isolated, and the phylogenetic data available indicate that other organisms of the same lineage inhabit not only saline environments, such as deep sea brines and Lake Van (AY642583), but also mammalian intestines (AJ400262), clearly indicating that the organisms of this distinct lineage have characteristics which could be very different from those of strain SSD-17BT
. The absence of other isolates precludes the description of a novel class or phylum at this point. Nevertheless, the organism from the Shaban Deep has distinct physiological and biochemical characteristics and phylogenetic position to justify the proposal of a new order. We are therefore of the opinion that strain SSD-17BT
belongs to a novel family, for which we propose the name Haloplasmataceae
fam. nov., within a novel order, for which we propose the name Haloplasmatales
Description of Haloplasmatales ord. nov. (Rainey, da Costa, Antunes, and Huber).
Haloplasmatales (Ha.lo.plas.ma.ta′les. N.L. n. Haloplasma, type genus of the order; suff. -ales, ending denoting an order; N.L. fem. pl. n. Haloplasmatales, the Haloplasma order). The description of this order is the same as that for the genus Haloplasma. The order contains the family Haloplasmataceae. The type genus of the order is Haloplasma.
Description of Haloplasmataceae fam. nov. (Rainey, da Costa, Antunes, and Huber).
Haloplasmataceae (Ha.lo.plas. ma.ta′ce.ae. N.L. n. Haloplasma, type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Haloplasmataceae, the Haloplasma family). The description is the same as that for the genus Haloplasma. The family contains the type genus Haloplasma.
Description of Haloplasma gen. nov. (Antunes. Rainey, da Costa, and Huber).
Haloplasma gen. nov. (Ha.lo.plas′ma. Gr. n. hals, salt; Gr. neut. n. plasma, something formed or molded, a form; N.L. neut. n. Haloplasma, a salt-loving form). Gram-negative, non-spore-forming, pleomorphic cells sometimes with “tentacle-like” protrusions. Moderately halophilic. Mesophilic. Grows in medium with neutral pH. Strictly organotrophic. Strictly anaerobic. Denitrifying and fermentative. Oxidase and catalase negative. Cell wall is not detected. Straight chain fatty acids are present. The major polar lipids are phosphatidylglycerol and bisphosphatidylglycerol (DPG). MK-4 is the major respiratory quinone. The G+C content of the DNA of the type species is 33.6 mol%. The type species of this genus is Haloplasma contractile.
Description of Haloplasma contractile sp. nov. (Antunes. Rainey, da Costa, and Huber).
Haloplasma contractile sp. nov. (con.trac.ti′le. N.L. neut. adj. contractile, contractile). Haloplasma contractile possesses, in addition to the characteristics in the description of the genus, pleomorphic cells, usually consisting of a central coccoid body (0.5 to 1.8 μm) with one or two “tentacle-like” protrusions (up to 20 μm in length and 0.1 to 0.25 μm in diameter). Motility may occur by cellular contraction of the protrusions. Colonies in MM-X agar are very small (0.03 to 0.05 mm in diameter), gold-yellow, and round, often with a “fried-egg”-like appearance. The NaCl range for growth is 1.5 to 18% (wt/vol); optimum growth is at about 8% (wt/vol) NaCl. Growth occurs at between 10 and 44°C (optimal temperature, 30 to 37°C), and the pH range is between about 6.0 and 8.0 (optimal pH, 7.0). Cells are able to be filtered through 450-nm pore membrane filters. Resistant to penicillin G, ampicillin, and cephalosporin but susceptible to bacitracin and chloramphenicol. Nitrate and nitrite are reduced; lactate was detected from the fermentation of the medium components. Urea and arginine were not hydrolyzed. Phosphatase was not detected. l-Arabinose, d-psicose, α-ketobutyric acid, α-ketoglutaric acid, and α-ketovaleric acid were metabolized with the Biolog GN2. Peptone and yeast extract are required for growth. The predominant cellular fatty acids are 16:0, 18:0, and 18:1 ω9c. The G+C content of the DNA of the type species is 33.6 mol%. Isolated from the anoxic brine-sediment interface of the Shaban Deep, Red Sea. The type strain, SSD-17BT, has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, as strain DSM 18853 and in the Japan Collection of Microorganisms (JCM), Saitama, Japan, as strain JCM 14575.