Sergiu Fendrihan

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.

Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria

The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522(T) was exposed to UV doses over a wavelength range of 200-400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m(2), and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m(2). The estimated D(37) (dose of 37 % survival) for Hcc. dombrowskii was > or = 400 kJ/m(2). However, exposure of cells to UV flux while in liquid culture reduced D(37) by 2 orders of magnitude (to about 1 kJ/m(2)); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.

Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite

Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of aw ≤ 0.75, but exposure to buffered 4 m LiCl (aw ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.

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).

Adaption of Microbial Life to Environmental Extremes

In this chapter, we will explore the different adaptations of extremophiles to life in the extreme cold. We generally forget that the Earth is mostly cold and that most ecosystems are exposed to temperatures that are permanently below 5 C. Such low mean temperatures mainly arise from the fact that 70% of the Earth’s surface is covered by oceans that have a constant temperature of 4–5 C (below a depth of 1000m), irrespective of the latitude. The polar regions account for another 15% of the surface, to which the glacier and alpine regions must also be added. Here, we will take an illustrated look in particular at the Antarctic environment, as it is by far the coldest environment on Earth – the lowest temperature on the surface of the Earth ( 89.2 C) was recorded at the Russian Vostok Station, at the centre of the East Antarctic ice sheet. Antarctica is a place where organisms are often subjected to combined stresses including desiccation, limited nutrients, high salinity, adverse solar radiation and low biochemical activity. The incredibly harsh environment of the Antarctic continent precludes life in most of its forms, and the microorganisms are therefore dominant. Generally, conditions include air temperatures that average well below freezing all year round, strong winds that increase the effects of the cold, light which varies from months of total darkness to total sunlight and little free available water, with all but 2% of the continent covered with ice. Given this combination of extremes, it is surprising that anything lives on the continent at all, let alone thrives there. For the organisms that do manage to adapt, however, the benefits from a lack of competition in the extreme cold are enormous, and this is often seen in very large population sizes. So how do some organisms survive extreme cold, and others exploit and take advantage of it? As organisms have been subjected to these stresses over extremely long periods of time, a range of adaptations have evolved; some species have adapted to live at the limits, some produce specific compounds such as antifreeze, some remain viable but frozen in a state of suspended animation whilst higher organisms can adapt their life cycles in such a way that when conditions are harsh they die, leaving the next generation to recover as conditions improve. By looking at a range of different strategies in a wide variety of organisms, it is hoped to bring together general mechanisms of adaptation to life in the extreme cold.

Halophilic Archaea: Life with Desiccation, Radiation and Oligotrophy over Geological Times

Halophilic archaebacteria (Haloarchaea) can survive extreme desiccation, starvation and radiation, sometimes apparently for millions of years. Several of the strategies that are involved appear specific for Haloarchaea (for example, the formation of halomucin, survival in fluid inclusions of halite), and some are known from other prokaryotes (dwarfing of cells, reduction of ATP). Several newly-discovered haloarchaeal strategies that were inferred to possibly promote long-term survival—halomucin, polyploidy, usage of DNA as a phosphate storage polymer, production of spherical dormant stages—remain to be characterized in detail. More information on potential strategies is desirable, since evidence for the presence of halite on Mars and on several moons in the solar system increased interest in halophiles with respect to the search for extraterrestrial life. This review deals in particular with novel findings and hypotheses on haloarchaeal long-term survival.

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.

Survival Strategies of Halophilic Oligotrophic and Desiccation Resistant Prokaryotes

Viable halophilic and halotolerant Archaea and Bacteria have been found in ancient salt deposits around the world. The first cultivations of halophilic microorganisms from Permian salt sediments (about 250 million years old) were reported in the 1960s (Reiser and Tasch, 1960; Dombrowski, 1963) and met with considerable skepticism. Some 30 years later, detailed taxonomic descriptions of halophilic Bacteria and Archaea (haloarchaea) obtained from ancient evaporites began to be published (Norton et al., 1993; Denner et al., 1994; Stan-Lotter et al., 2002; Mormile et al., 2003; Gruber et al., 2004; Vreeland et al., 2007). Sequences of small ribosomal RNA (16S rRNA) genes and other molecules allowed more meaningful comparisons of isolates with known strains than was possible before. In many cases, no exact matches of sequences from subsurface isolates with those of known strains from surface waters were found (McGenity et al., 2000). This does not necessarily mean that they do not exist in surface environments, merely that they have not been isolated yet from there. In one other case, three strains of Halococcus salifodinae with identical 16S rRNA sequences were found in three geographically separated subsurface regions, all of Permo-Triassic age: strain BIp from Permian Zechstein rock salt, mined at Bad Ischl, Austria; strain Br3 from solution-mined Triassic Northwich halite; and strain BG2/2 from a core of Permian Zechstein salt, Berchtesgaden, Germany (Stan-Lotter et al., 1999; McGenity et al., 2000). A detailed characterization of the three independently isolated halococci revealed that they were very similar and should be considered as strains of the same species (Stan-Lotter et al., 1999). During the Permian period, the large hypersaline Zechstein Sea covered an area of about 250,000 km2 over much of northern Europe. This sea would have provided a connection between the areas from which the three strains of Hcc. salifodinae were isolated (Stan-Lotter et al., 1999; McGenity et al., 2000; Radax et al., 2001). It is conceivable that Hcc. salifodinae was present in the Zechstein Sea and became trapped in the evaporating salts.

The Likelihood of Halophilic Life in the Universe

The search for extraterrestrial life has been declared as a goal for the twenty-first century by several space agencies (Foing, 2002). Potential candidates are microorganisms on or in the surfaces of moons and planets. Extremely halophilic archaea (haloarchaea) are of astrobiological interest since viable strains have been isolated from million-year-old deposits of halite (McGenity et al., 2000; Stan-Lotter et al., 1999, 2002; Fendrihan et al., 2006), suggesting the possibility of long-term survival under desiccation. Extraterrestrial halite has been identified, for example, in Martian meteorites (Treiman et al., 2000), in chloride-containing surface pools on Mars (Osterloo et al., 2008), and in the presumed salty ocean beneath the ice cover of Jupiter’s moon Europa (McCord et al., 1998). These discoveries make a consideration of the potential habitats for halophilic life in space intriguing. Recent data on the physical occurrence of liquid saline water on Mars (Smith et al., 2009; Renno et al., 2009) have added another novel aspect to this notion, since such “cryobrines” would provide liquid phases on the Martian surface, allowing perhaps metabolic activity of halophilic microorganisms.