Haloarchaea From Salt Sediments

The huge salt sediments on Earth, which were deposited during evaporation of sea water or continental brines, are the origin of evaporitic rocks. Similarly as for other natural environments, progress in methodology has increased greatly the knowledge of microbial diversity. As recently as 1981, Larsen (1981) described mined rock salts as free from bacteria, although isolations of halophilic microorganisms from ancient salt sediments had occasionally been reported since the early decades of the twentieth century (for references, see Grant et al., 1998; McGenity et al., 2000).

Particularly large salt sediments stem from the Permian and Triassic ages (280 to 192 million years ago); the discovery of viable microorganisms, which have likely survived in fluid inclusions in the halite, is of special interest to astro-biology. Grant (2004) thoroughly discussed the problems associated with the isolation of culturable microorganisms from ancient halite, verification of their presumed antiquity, similarities of their genes to those of extant microbes and their mechanisms of longevity in the absence of resting stages, such as spores.

Our group isolated from alpine Permian rock salt, which was collected from the salt mine in Bad Ischl, Austria, a haloarchaeon, which, based upon polyphasic taxonomic data, was recognized as a novel species and named Halococcus salifodinae (Denner et al., 1994). This was the first isolate from ancient rock salt, which was formally classified and deposited in several international culture collections. Two independently isolated strains, Br3 (from solution-mined brine in Cheshire, England) and BG2/2 (from a bore core from the mine of Berchtesgaden, Germany) resembled Hc. salifodinae BIp in numerous properties, including the characteristic morphology of tetrads arranged in large clusters (Fig. 1); in addition, rock salt samples were obtained eight years later from the same site and several halococci were recovered from these samples, which proved to be identical to strain BIp (Stan-Lotter et al., 1999). The data suggested that viable halophilic archaea, which belong to the same species, occur in geographically separated evaporites of similar geological age.

Another halococcal isolate from the Bad Ischl salt formation, which differed from the previously described strains, was subsequently identified as a novel species and named Halococcus dombrowskii (Stan-Lotter et al., 2002). Hc. salifodinae and Hc. dombrowskii have so far not been found in any hypersaline surface waters, or any location other than salt mines. Recently, a series of non-coccoid strains was obtained from a freshly drilled bore core at the salt mine in Altaussee, Austria (about 40 km distance from Bad Ischl), which were similar in their 16S rRNA sequence to Halobacterium salinarum NRC-1, from which the whole genome sequence is known; however, other properties were different and consequently, a novel species was created, Halobacterium noricense (Gruber et al., 2004). Figure 2 shows the two species of the genus Halobacterium. A single rod-shaped Halobacterium isolate from 97,000 year old rock salt in the US was described by Mormile et al. (2003) and deemed to resemble Hb. salinarum NRC-1. From the Permian-aged Salado formation in New Mexico, a strain of a novel genus, Halosimplex carlsbadense (Vreeland et al., 2002) was isolated. Table 1 contains a list of the formally classified isolates from alpine rock salt, the Salado formation and a strain from a British salt mine.

Enveloped Virus Electron Micrograph
Figure 1. Scanning electron micrograph of Halococcus salifodinae Br3 DSM 13046. Bar, 0.5 |m. (Photograph taken by G. Wanner.)
Halococcus Dombrowskii
Figure 2. Scanning electron micrograph of Halobacterium salinarum NRC-1 (left panel) and Halobacterium noricense DSM15987T (right panel). Bars, 1 |im. (Photographs taken by C. Frethem.)

Although the microbial content of ancient rock salt is generally low - estimates range from 1-2 cells/kg of salt from a British mine (Norton et al., 1993) to 1.3 x 105 colony forming units (CFUs) per kg of alpine rock salt (Stan-Lotter et al., 2000), and even up to 104 CFUs per g of Permian salt of the Salado formation in the USA (Vreeland et al., 1998) - the reports showed that viable haloarchaeal isolates were obtained reproducibly by several groups around the world. Taken

Table 1. Validly described haloarchaeal isolates from Permo-Triassic rock salt and salt mine brine.

Organism, strain

Type strain (T); catalogue numbers

Origin

Reference

Halococcus

DSM8989T

Rock salt (lumps)

Denner et al., 1994

salifodinae BIp

ATCC51437T

Bad Ischl, Austria

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