Andrea Legat

Evaluation of the LIVE/DEAD BacLight Kit for Detection of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples

ABSTRACT Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.

Halococcus dombrowskii sp. nov., an archaeal isolate from a Permian alpine salt deposit.

Several extremely halophilic coccoid archaeal strains were isolated from pieces of dry rock salt that were obtained three days after blasting operations in an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225-280 million years ago). On the basis of their polar-lipid composition, 16S rRNA gene sequences, cell shape and growth characteristics, the isolates were assigned to the genus Halococcus. The DNA-DNA reassociation values of one isolate, strain H4T, were 35 and 38% with Halococcus salifodinae and Halococcus saccharolyticus, respectively, and 65.8-67.8% with Halococcus morrhuae. The polar lipids of strain H4T were C20-C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate. Whole-cell protein patterns, menaquinone content, enzyme composition, arrangements of cells, usage of carbon and energy sources, and antibiotic susceptibility were sufficiently different between strain H4T and H. morrhuae to warrant designation of strain H4T as a new species within the genus Halococcus. It is proposed that the isolate be named Halococcus dombrowskii, and the type strain is H4T (= DSM 14522T = NCIMB 13803T = ATCC BAA-364T).

Very similar strains of Halococcus salifodinae are found in geographically separated permo-triassic salt deposits.

The authors have previously isolated a novel extremely halophilic archaeon, Halococcus salifodinae Blp, from Austrian rock salt deposited about 250 million years ago. In this study they compared strain Blp with two other halococci isolated independently from geographically distant salt deposits of similar age, and with two recent isolates (N1 and H2) from the same site as strain Blp. Strain BG2/2 was from a salt mine in Germany and strain Br3 from a halite deposit in England; both resembled Hc. salifodinae Blp in cellular and colonial morphology. Strains Blp, BG2/2 and Br3 had identical 16S rRNA sequences, very similar whole-cell protein patterns, which were different from those of other halococci, similar G+C contents and identical sequences in a 108-base insertion in their 5S rRNA gene. Other similarities included composition and relative abundances of polar lipids, antibiotic susceptibility, enzymic activities and Fourier-transform infrared spectra. Strains N1 and H2 showed similar morphology, whole-cell protein patterns and biochemical characteristics as strains Blp, Br3 and BG2/2. Their partial 16S rRNA sequences (682 and 641 bases, respectively) were indistinguishable from those of strains Blp, Br3 and BG2/2. Therefore strains N1 and H2 can be considered as reisolates of Hc. salifodinae which were obtained 8 years after the first samples were taken from that mine. The results presented suggest that viable halophilic archaea, which belong to the same species, occur in widely separated evaporite locations of similar geological age, and support the notion that these halophilic isolates from subterranean salt deposits may be the remnants of populations which inhabited ancient hypersaline seas.

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.

Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece

Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2-97.1%. The DNA-DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75%, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2%, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5-66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T=NCIMB 14426T=ATCC BAA-1602T).

Astrobiology with haloarchaea from Permo-Triassic rock salt

Several viable halophilic archaebacteria were isolated previously from rock salt of Permo-Triassic age in an Austrian salt mine; one of these strains was the first to be recognized as a novel species from subterranean halite and was designated Halococcus salifodinae . The halophilic microorganisms have apparently survived in the salt sediments over extremely long periods of time. Halobacteria could therefore be suitable model organisms for exploring the possibility of long-term survival of microbes on other planets, in particular, since extraterrestrial halite has been detected in meteorites and is assumed to be present in the subsurface ocean on Europa. Our efforts are directed at the identification of the microbial content of ancient rock salt and the development of procedures for the investigation of the halobacterial response to extreme environmental conditions. Using modified culture media, further halophilic strains were isolated from freshly blasted rock salt and bore cores; in addition, growth of several haloarchaea was substantially improved. Molecular methods indicated the presence of at least 12 different 16S rRNA gene species in a sample of Alpine rock salt, but these strains have not been cultured yet. The exploration of Mars is a target of space missions in the 21st century; therefore, testing the survival of haloarchaea under conditions comparable to present-day Mars, using a simulation chamber, was begun. Preliminary results with Halococcus and Halobacterium species suggested at least tenfold higher survival rates when cells were kept in liquid brines than under dry conditions; staining of cells with the LIVE–DEAD kit, which discriminates between damaged and intact membranes, corroborated these data.

Responses of haloarchaea to simulated microgravity.

Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterraneis resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites).

From Intraterrestrials to Extraterrestrials — Viable Haloarchaea in Ancient Salt Deposits

During several periods in the Earth’s history, immense sedimentation of halite and some other minerals from hypersaline seas took place. An estimated 1.3 million cubic kilometers of salt were deposited in the late Permian and early Triassic periods alone (ca. 240 to 280 million years ago; Zharkov 1981). The continental land masses were concentrated around the paleoequator and formed the supercontinent Pangaea (Fig. 5.1). Salt sediments developed in large basins, which were connected to the open oceans by narrow channels. The paleoclimate was warm and arid in a wide belt around the equator, causing large-scale evaporation. About 100 million years ago, fragmentation of Pangaea was beginning; the continents were displaced to the north, and folding of new mountain ranges such as the Alps and Carpathians was underway (Einsele 1992). As a result of these movements driven by plate tectonics, huge salt deposits are found today predominantly in the northern regions of the continents, e.g. in Siberia, northern and central Europe (Zechstein series), south-eastern Europe (Alps and Carpathian mountains), and the midcontinent basin in North America (Zharkov 1981). Growing interest is emerging in the exploration of microbial life in subterranean environments, such as deep sub-seafloor sediments, crustal rocks, sedimentary rocks, and also ancient salt deposits (for a review, see Pedersen 2000). It has been estimated that the total amount of carbon in the “intraterrestrial” prokaryotic mass on Earth may be as large or even exceed that of plants and prokaryotes growing on the surface of the Earth (Pedersen 2000).

24 The Assessment of the Viability of Halophilic Microorganisms in Natural Communities

Publisher Summary This chapter describes protocols for staining of haloarchaea with fluorescent dyes and correlation with CFUs, including improved media for growth of cells from environmental samples. Staining of micro-organisms with fluorescent dyes in the presence of high ionic strength (up to 4.2 M NaCl) is possible. Morphology, size and the presence of nucleoids (which is considered to be indicative of active cells) can be detected in the epifluorescence microscope; an assessment of the intactness or damage of membranes, whether of bacterial or archaeal composition can be made. Extremes of pH do not interfere with the application of the LIVE/DEAD ® kit. Staining with DAPI does not distinguish between viable and dead bacterial cells, which are also true for haloarchaea. The LIVE/DEAD ® kit is thought to permit a differentiation between active and dead cells. For information about the true status of microbial cells, determination of CFUs is still the most valuable approach, though not always feasible. A useful property of the dyes of the LIVE/DEAD ® kit is their noninterference, when used at low concentrations, as for staining with subsequent growth experiments.

Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters.

Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing hypotheses on prokaryotic evolution, such as the molecular clock concept, or the net-like history of genome evolution. A comparison of available taxonomic characteristics from strains of Hcc. salifodinae and other Halococcus species, most of them originating from surface waters, is presented. The cell wall polymer of Hcc. salifodinae was examined and found to be a heteropolysaccharide, similar to that of Hcc. morrhuae. Polyhydroxyalkanoate granules were present in Hcc. salifodinae, suggesting a possible lateral gene transfer before Permian times.