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mayA–junem 2007 ericthe anmagazine of sigmaS xic, thei scientificen researchtis societyt Volume 95 Number 3 May–June 2007 A reprint from American Scientist the magazine of Sigma Xi, The Scientific Research Society

This reprint is provided for personal and noncommercial use. For any other use, please send a request to Permissions, American Scientist, P.O. Box 13975, Research Triangle Park, NC, 27709, U.S.A., or by electronic mail to [email protected]. ©Sigma Xi, The Scientific Research Society and other rightsholders Extreme Microbes

Salt-loving microorganisms are helping biologists understand the unifying features of life and molecular secrets of survival under extreme conditions

Shiladitya DasSarma

een from the air, the irregular grid the microscopic world in a drop of near- Sof evaporation ponds at the south saturated brine contains a menagerie of end of San Francisco Bay in Califor- bizarre life forms that defy these con- nia is a kaleidoscopic quilt of reds and ventions. Unlike the extremophiles that purples. These ponds take their col- have adapted to a single extreme condi- ors from the single-celled microorgan- tion, haloarchaea are metabolically ver- isms that live there, strange beings that satile. They can grow aerobically (with thrive in concentrated salt solutions. oxygen), anaerobically (in the absence Such extreme conditions kill almost of oxygen) or phototrophically (using every form of life on the planet, but not light energy), and can adapt to fluctua- the salt-loving archaea. They are mem- tions in temperature, pH and metal-ion bers of an ancient kingdom that exist- concentration. They are resistant to des- ed even before Earth had an oxygen at- iccation, sunlight and ionizing radiation. mosphere. The fact that many archaea As a result, they can be found in anoxic live under impossible circumstances— salt marshes, hydrothermal vents, pe- boiling temperatures, lethal radia- rennially frozen Antarctica and pockets tion, near-complete desiccation—has of brine deep underground and under led scientists to dub them “extremo- the seafloor. philes.” These organisms may even be The biggest hurdle to studying most capable of hitchhiking through space. species of archaea is recreating in the As human beings, our intuitive con- laboratory the extreme conditions cept of life is influenced by the visible they need. By contrast, the salt-lov- biosphere—temperate terrestrial, fresh- ing haloarchaea are easily cultivated, water and marine environments. But and microbiologists have used them in molecular, genetic and physiological Shiladitya DasSarma received his Ph.D. in biochem- experiments. Haloarchaea grow best istry in 1984 from the Massachusetts Institute of under hypersaline conditions, from Technology and then took up a two-year postdoctoral slightly concentrated seawater to near- fellowship at Massachusetts General Hospital and saturated brine. These attributes, plus Harvard Medical School. He served on the faculty the availability of genomic data and Figure 1. In places where salty brine is evap- of the University of Massachusetts-Amherst from orated to harvest its dissolved minerals, such tools for molecular manipulation, have 1986 to 2001. In 2001, he moved his laboratory to as Lac Rose (“Pink Lake”) in Senegal, the the Center of Marine Biotechnology at the Univer- elevated them to the status of “model” evaporation ponds or salterns take on oth- sity of Maryland Biotechnology Institute, where he organisms that shed light on other ex- is a full professor. He also holds a teaching appoint- tremophiles, including other archaea bacteria and those evolutionary relics ment in the Graduate Program in Life Sciences at and even higher organisms. called archaea. Although the two look the University of Maryland-Baltimore. His labora- the same under the microscope, archaea tory group includes his wife, Priya DasSarma, who Diversity and Unity have molecular characteristics that are earned her M.S. in microbiology from the University Until the 1970s, scientists believed that more similar to nucleus-containing of Massachusetts with a certification to teach biol- all prokaryotes—those single-celled mi- eukaryotes—organisms such as yeasts, ogy. She currently specializes in biotechnology edu- croorganisms that lack a nucleus—were plants and animals. These traits confirm cation and science outreach for students of all ages. Writing on page 230, she discusses the advantages “bacteria.” The pioneering work of Carl that archaea are fundamentally distinct of Halobacterium NRC-1 as a classroom tool. Ad- Woese at the University of Illinois at from bacteria. dress: University of Maryland Biotechnology Insti- Urbana-Champaign, and his colleagues Such distinctions notwithstand- tute, 701 East Pratt Street, Baltimore, MD 21202. proved otherwise. Today, prokaryotes ing, scientists using haloarchaea have Internet: [email protected] are divided into two groups: the “true” made several landmark findings

© 2007 Sigma Xi, The Scientific Research Society. Reproduction 224 American Scientist, Volume 95 with permission only. Contact [email protected]. Ron Giling/Peter Arnold, Inc. erworldly hues. The colors—oranges, reds, pinks and purples—come from halophilic microorganisms, which thrive under such forbidding conditions. As a result of an evolutionary history filled with genetic swaps with other microbes, haloarchaea can withstand not only high concentrations of salt but also extremes of temperature, pH and solar radiation. Such traits make them candidates for interplanetary travel, locked away in salty capsules within meteoric chunks of rock. that have wider implications for mi- Another early discovery first made a mill wheel harnesses the current of crobial and multicellular life forms. in haloarchaea was the protein bac- a river to do useful work. In archaea, For example, evidence from haloar- teriorhodopsin. First identified by the purple-tinted bacteriorhodop- chaea helped H. Gobind Khorana at Walther Stoeckenius of the University sin proteins cluster in a specialized MIT (with whom I began my studies of California, San Francisco, bacterio- region of the cell surface called the of haloarchaea) to establish the stan- rhodopsin uses photons of sunlight purple membrane, where they enable dard genetic code—the Rosetta Stone to pump hydrogen ions (protons) out the harvest of light energy for growth of biology that allows the information of the cell. This action creates a polar- under conditions where oxygen is in genes to be used as a blueprint for ized cell membrane. A separate pro- scarce. In classic experiments, Stoeck- proteins. The existence of this code is tein complex harnesses the flow of enius and others showed that light one of the strongest lines of evidence protons trying to reenter the cell to could drive the synthesis of adenos- for the unity of all life on our planet. provide energy—similar to the way ine triphosphate or ATP—the cellular

© 2007 Sigma Xi, The Scientific Research Society. Reproduction www.americanscientist.org 2007 May–June 225 with permission only. Contact [email protected]. marine Extreme Genomics cyanobacteria Halobacterium NRC-1 100 Despite its name (more on this later), an organism that was known as “Halobacte- rium species NRC-1” was the first haloarchaeon—and one of the first archaea of any kind—to have its genome studied. W. Ford Doolittle at Dalhousie Univer- sity and my research group (then at the University of Massachusetts Amherst) conducted those early experiments. NRC-1 is in most respects a typical haloarchaeon, widely distributed in hyper- growth rate (percent) saline environments such as the Great saturation seawater Salt Lake. The genome of this species has 0 0 1 2 3 4 5 the unusual property of being spontane- ously unstable, such that entire physio- sodium chloride (moles per liter) logical systems, such as the phototrophic purple membrane and buoyant gas-filled vesicles, are sometimes mutated. This cu- Figure 2. Normal seawater has around 30 grams of sodium chloride (0.5 mole) per liter (left), riosity led us to identify a large number a concentration that supports cyanobacteria, sometimes called blue-green algae. By contrast, of mobile genetic elements—similar to Halobacterium NRC-1 thrives in brine that is roughly eight times saltier. This concentration the “jumping genes” described by pio- borders the saturation point—the point at which solid salt begins falling out of solution. Seen neering geneticist Barbara McClintock in a transmission electron micrograph (right), the gas-filled sacs that buoy NRC-1 cells into the sunlight look like the holes in Swiss cheese. (Micrograph courtesy of the author.) in maize. These elements were the first to be discovered in any archaeon. We also found that NRC-1 carries a pair of smaller DNA molecules alongside its chromosome. The sequence of the NRC-1 genome was completed in the summer of 2000. light It was the first complete genome to be hydrogen ions sequenced with funds from the U.S. National Science Foundation. The genome consists of a large, circular chromosome (2,014 kilobases) and the two smaller DNA hoops, called plasmids or replicons: pNRC100 (191 kilobases) and pNRC200 (365 kilobases). The pNRC replicons contain many of the DNA repeats that enable genomic rear- rangements, including 69 of 91 mobile elements, 33 to 39 kilobases of so-called bacteriorhodopsin ADP inverted repeats, which can flip or in- vert portions of the circles, and 145 ki- P lobases of sequence that are identical in phosphate both plasmids. All this repetition confounded the computer programs tasked with orga- nizing the overlapping fragments of ATP DNA sequence into a seamless genome. ATP synthase The solution to this problem required extensive knowledge of the structure of pNRC100—knowledge that we had Figure 3. Purple-tinted bacteriorhodopsin harnesses light energy to make cellular fuel. Photons painstakingly acquired in the 1980s and of light cause changes in the shape of the bacteriorhodopsin protein that pump hydrogen ions (protons) out of the cell. Another protein, ATP synthase, uses the flood of protons trying to rush ‘90s by cutting the genome in specific back into the cell to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP). places and analyzing the parts. Later in ATP in turn provides energy for other molecular machines to do useful work. 2000, we summoned an international consortium of 12 laboratories to meet currency for energy transactions—in organelles present in eukaryotic cells. in Amherst over the winter holidays reconstituted spheres that contained This work provided irrefutable proof to identify familiar elements in the ge- bacteriorhodopsin and ATP synthase. of chemiosmotic coupling, the mecha- nome. Such an analysis was a Hercu- The latter is a key enzyme found in nism of energy generation used by all lean task in those days. The computa- mitochondria, those “powerhouse” cellular life. tional tools used to analyze genomes

© 2007 Sigma Xi, The Scientific Research Society. Reproduction 226 American Scientist, Volume 95 with permission only. Contact [email protected]. were still in their infancy, so we had to write our own computer scripts and manually inspect much of the data. One of the most exciting discoveries gleaned from that sequence was that the 2,630 predicted proteins were, on average, much more acidic than those of other organisms. The average isoelectric l point (a measure of acidity) for predict- a tin ed Halobacterium proteins is only 4.9. By re ll contrast, the values for nearly all non- y h p haloarchaeal species are close to neutral, ro ca lo rotenoids a 7 on this scale. Acidic proteins carry ch strong negative charges, which ought

to repel other negatively charged mole- absorbance cules in the cell, such as DNA and RNA. But even proteins tasked with binding DNA (itself an acid)—including pieces 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 of the complex machine called the tran- wavelength (nanometers) scription apparatus, which creates RNA from DNA—turned out to be acidic. Figure 4. The retinal pigment in bacteriorhodopsin absorbs light of a wavelength that human Our recent experiments have shown beings perceive as green, but reflects light at the red and, to a lesser extent, the violet ends of the that acidic transcription factors bind spectrum, a pattern that yields a purple appearance (purple line). By contrast, photosynthetic chlo- DNA with ease in the hypersaline en- rophyll pigments absorb indigo and red and reflect green (green line). This mirror-image relation suggests that chlorophyll evolved to exploit parts of the spectrum left unused by the purple pig- vironment inside haloarchaeal cells or ment. The carotenoid pigments (orange line) shield haloarchaea from high-energy violet and ul- isolated in test tubes filled with saline traviolet light waves but reflect lower-energy orangish-red colors, giving rise to the scarlet shades solution. This attraction is remarkable seen in many salterns. The absorbance spectra have been scaled for comparison. because these proteins and DNA—both negatively charged—ought to repel insights into the evolutionary history, people who name microbes for a liv- each other. One possible explanation or phylogeny, of haloarchaea. Although ing. They had recently renamed sev- is that the proteins and DNA work the data confirmed NRC-1 as a true ar- eral Halobacterium species based solely together by sandwiching positively chaeon, the gene that most scientists on DNA sequence, a move that wasn’t charged ions between nearby, negative- use as an evolutionary chronometer, strictly possible for NRC-1 given the ly charged side groups. Mutual repul- the so-called 16S ribosomal RNA, had ambiguity. Thus, the name “Halobac- sion between acidic groups helps acidic a unique sequence that prevented easy terium species NRC-1” has become halophilic proteins to remain in solution categorization of the species. This con- controversial, fueling ongoing debate under conditions in which neutral, non- tradiction presented a challenge to the about the meaning and significance of halophilic proteins would precipitate or “salt out.” Thus, haloarchaea require 10 extremely acidic proteins to maintain 9 cellular functions in a supersaturated- 8 salt environment. In recent years, the full sequence of 7 the NRC-1 genome has been followed 6 by the publication of five additional 5 haloarchaeal genomes: Haloarcula maris- 4 mortui, a metabolically versatile species from the Dead Sea; Natronomonas 3 pharaonis, an alkali (high pH)-loving 2

species from the alkaline soda lakes of protein-coding regions (percent) 1 the Sinai; Haloferax volcanii, a moder- 0 ately halophilic species from Dead Sea mud; Haloquadratum walsbyi, a square- 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 shaped organism common in salterns; predicted isoelectric point and Halorubrum lacusprofundi, a cold- Bsu Eco Hal Mja Mth Sce adapted species from an Antarctic lake. These six genomes currently provide us Figure 5. NRC-1 and other haloarchaea contain extremely acidic proteins, as indicated by low val- with an excellent view of haloarchaeal ues for a quantity called the isoelectric point, or pI. Based on sequences predicted from genomic diversity. data, the isoelectric profile of NRC-1 proteins (red line) is much more acidic than those of other single-cell organisms, including Bacillus subtilis (Bsu), Escherichia coli (Eco), Methanococcus jan- Evolutionary Relics naschii (Mja), Methanothermobacter thermautotrophicus (Mth), and Saccharomyces cerevisiae The unveiling of the NRC-1 genome (Sce). The highly acidic side chains on NRC-1 proteins probably enable them to stay in solution spawned questions about as well as even in concentrated brines.

© 2007 Sigma Xi, The Scientific Research Society. Reproduction www.americanscientist.org 2007 May–June 227 with permission only. Contact [email protected]. ies concluded that NRC-1 contained nearly as many bacterial genes as archaeal ones, raising the possibility that these unusual microbes began as a kind of prokaryotic jumble. Despite uncertainties about the pre- cise history of prokaryotic evolution, evidence for the lateral transfer of genes in the haloarchaeal lineage appears con- a b vincing. For example, the genes that enable oxygen-based metabolism in Halo- bacterium are arranged the same way as those found in Escherichia coli bacteria. The gene sequences are similar to bacteria too. The combination of ribosomal genes that resemble anaerobic archaea and metabolic genes that resemble aerobic bacteria suggests that haloarchaea may have adapted to an oxygen atmosphere by stealing pieces of the c d respiration machinery through lateral gene transfers from bacteria. Presum- Figure 6. The shape of a DNA-binding protein complex is similar in Halobacterium NRC-1 (a and ably, such events occurred very early b) and Homo sapiens (c and d), even after hundreds of millions of years of divergent evolution. in Earth’s history when photosynthetic However, the surface charges of the proteins are starkly different. In this image, red indicates the cyanobacteria first began to oxygenate negative charges of acidic parts, blue the positive charges of basic parts. In the center of this protein the atmosphere. However, the possibil- complex (made of two proteins, TBP and TFB) is the DNA molecule (here, green portions indicate the active gene, whereas pink and orange denote noncoding areas). Negative charges present a ity of multiple transfers, including more problem for attracting and binding DNA—which, as an acid, also carries a net negative charge. recent ones, cannot be ruled out. The solution is probably a layer of negative ions sandwiched between the otherwise repellant A relatively modern example of later- molecules. The views in a and c depict the protein complex riding the coil of DNA into the page, al gene transfer in Halobacterium NRC-1 whereas b and d show the same complex coming out of the page. (Images courtesy of the author.) comes from the gene that encodes ar- ginyl-tRNA synthetase (ArgS), an en- the term “species” among prokaryotic zyme essential to the manufacture of organisms. Some scientists go so far proteins. The closest cousins to the ArgS as to suggest that the species concept protein from NRC-1 are found among is inappropriate for prokaryotes and ArgS proteins from bacteria, not those should be ditched in favor of “strain” from other archaea or haloarchaea—a or “phylotype.” puzzling situation, given that NRC-1’s Hoping to find an answer to the phy- presumed archaeal ancestors predate logenetic puzzle, my coworkers and I bacteria. The most plausible scenario is compared the entire NRC-1 genome to that following the capture of a bacterial the few other microbial genomes then gene that encoded ArgS, the archaeal available. This preliminary analysis gene was lost in Halobacterium NRC-1. noted interesting similarities between ArgS isn’t the only example of this Halobacterium NRC-1 and two true bac- kind. Based on predictions from the teria: the gram-positive, spore-forming genome sequence, about 40 genes in Bacillus subtilis and the radiation-resis- the two pNRC replicons encode es- tant Deinococcus radiodurans. But a few sential proteins, including those that years later, after the genomes of dozens make DNA and RNA, and those that of additional species had been added to use oxygen for cellular respiration. the databases, the position of Halobacte- Since the cell can’t do without these rium on the tree of life seemed to have genes, the pNRC replicons that contain moved, either to a spot near the base of them may be more accurately labeled the archaeal branch, or, surprisingly, to minichromosomes than plasmids, a branch of the bacteria. which are defined by their dispens- This relocation disagreed with earlier ability. In NRC-1, the plasmids serve Figure 7. The genome of NRC- Halobacterium analyses that had placed Halobacterium at least two important functions: as 1, shown schematically here, contains three circular pieces of DNA: a large chromosome squarely within the archaea. One pos- reservoirs for captured genes and as of roughly two million bases and two small sible explanation for the discrepancy is vehicles for the lateral transfer of those plasmids of 191,000 and 365,000 bases. The that over time, halophilic archaea may genes. And when those genes are es- plasmids may have helped shuttle DNA be- have acquired many genes from unre- sential for survival, their transfer may tween microorganisms during the evolution of lated bacterial species in a process called lead a prokaryotic replicon to evolve haloarchaea. (Image courtesy of the author.) lateral gene transfer. Indeed, some stud- from a plasmid into a chromosome.

© 2007 Sigma Xi, The Scientific Research Society. Reproduction 228 American Scientist, Volume 95 with permission only. Contact [email protected]. One of the most interesting and still unresolved evolutionary questions about halophiles centers on the M. thermoautotrophicum origin of bacteriorhodopsin, the protein M. thermoautotrophicum component of the purple membrane. D. radiodurans

D. radiodurans Bacteriorhodopsin is related to mam- Synechocystis malian rhodopsins and like them con- Synechocystis tains retinal, a substance that is, as the M. jannaschii A. aeolicus name suggests, important in enabling M. jannaschii A. aeolicus S. pombe the eye’s retina to perceive light. Halo- S. pombeC. albicans bacterium NRC-1 and some other halo- A. fulgidus C. albicans archaea have other retinal-containing S. cerevisiae A. fulgidus S. cerevisiae proteins too, including halorhodopsin, which pumps chloride ions across the D. melanogaster membrane, and photosensory rhodop- P. furiosus D. melanogaster H. sapiens sin, which can discriminate between P. furiosus C. elegansH. sapiens useful and harmful sources of light and P. horikoshii Halobacterium A. thaliana C. elegans control the cell’s swimming behavior. P. horikoshiiP. abyssi Halobacterium A. thaliana P. abyssi S. solfataricus

Scientists still debate the evolution- S. solfataricus ary history of these ancient retinal- A. pernix containing proteins. Although their A. pernix initial discovery in haloarchaea sug- T. acidophilum

gested for a time that such proteins T. acidophilum were unique to this group, recent sur- a veys have found similar proteins in a many other bacteria and in eukaryotes. Therefore, these proteins may have arisen before archaea, bacteria and eukaryotes first diverged hundreds of millions—perhaps more than B. subtilis K12

a billion—years ago. Alternatively, aeruginosa P. B. subtilis K12 the genes encoding retinal-containing aeruginosa P. E. coli proteins may have passed by way of E. coli lateral gene transfers into planktonic bacteria, some fungi and haloarchaea. T. acidophilum Although the phylogeny of retinal- T. acidophilum based pigments remains inconclusive, Halobacterium it’s intriguing to think that they co- M. thermoautotrophicum Halobacterium evolved with chlorophyll-based pig- M. thermoautotrophicum S. pombe ments. Comparing the visible-light ab- S. pombeC. albicans sorption patterns of the pigments, it’s S.C. cerevisiae albicans clear that retinal and chlorophyll have M. jannaschii M. jannaschii S. cerevisiae nonoverlapping, complimentary spec- A. fulgidus D. melanogaster tra. The co-evolution hypothesis ar- A. fulgidus S. solfataricus D. melanogaster gues that simpler, retinal-based use of H. sapiens P. furiosus S. solfataricus sunlight as an energy source evolved P. furiosus A. thaliana C. elegansH. sapiens A. thaliana in microorganisms that dominated P. horikoshii P. abyssi C. elegans

A. pernix during the anaerobic, “purple phase” P. horikoshii P. abyssi A. pernix of the planet’s existence. Later, more complex pigments based on chlorophyll could have evolved to harvest light from regions of the solar spec- b trum not absorbed by the preexisting b species. The success of this strategy led Figure 8. Geneticists who study the evolutionary history of life have a difficult time classifying to the “green phase” of Earth’s evo- Halobacterium NRC-1. Depending on what parts of the genome they study, NRC-1 may appear to be more closely related to archaea (red), as shown in a, or to bacteria (black), as seen in b. Oddly lution and the attendant oxidation of enough, some regions of the NRC-1 genome look most similar to the genes of eukaryotes such as its atmosphere through photosynthe- plants, animals and yeasts (blue). These similarities suggest that NRC-1 incorporated DNA from sis—changes that displaced most reti- other organisms at multiple points during its evolution, a process called lateral gene transfer. nal-based microorganisms. Although speculative, such a scenario suggests Means of Survival can withstand intense solar and ion- that retinal-based nourishment from Haloarchaea endure many stressful izing radiation that in most organisms sunlight is one of the oldest metabolic conditions that kill other microbes. causes widespread damage to DNA. systems on Earth. For instance, Halobacterium NRC-1 This feat is possible because the cells

© 2007 Sigma Xi, The Scientific Research Society. Reproduction www.americanscientist.org 2007 May–June 229 with permission only. Contact [email protected]. produce additional pigments, such as lyase reverses the damage caused by homologous recombination (using a the red-orange carotenoids, to help ultraviolet radiation during the day, good copy of the gene to fix the dam- shield the genome. NRC-1 also con- a process known as “light repair.” Ex- aged copy). A third enzyme, exinucle- tains a double dose—both the bacte- posure to UV light also triggers the ase, snips out bits of damaged DNA rial and eukaryotic versions—of the production of a recombinase enzyme altogether. Recombinase and exinu- enzymes used for DNA repair. Photo- that aids DNA repair by promoting clease are critical for “dark repair,” which takes place at night when cells can spend their energy fixing sun- damaged genomes. High-energy ra- Haloarchaea in the Classroom diation and desiccation produce a different type of genetic damage in which both strands of the DNA back- he model halophile Halobacte- high schools. In addition to helping bone are broken. Such irradiated cells Trium species NRC-1 is an ideal keep their results clean, the hyper- also make copies of the so-called rep- microorganism for teaching basic saline conditions prevent the growth lication protein A, which binds to ex- microbiology to high school and col- of potentially pathogenic strains of posed, single-stranded DNA, as well lege students. The primary reason microorganisms. as to recombinase, and helps repair is that the high-salt medium neces- When grown in the lab, liquid cul- genetic damage. sary for growing NRC-1 prevents tures of NRC-1 have a vibrant pink Toxic metals and fluctuating ion color that gets students’ attention concentrations also elicit changes in (bottom right), and the presence of gene expression that promote survival pink, red-orange, or even sectored in Halobacterium NRC-1. For example, colonies (bottom left) intrigues them. cells resist the toxic effects of arsenic Students can see and explore the link by turning on a cluster of genes en- between genotype and phenotype coding enzymes that alter the metal’s through the presence or absence of oxidation state and prepare it for active organelles that enable the cells to transport out of the cell. The identity float when suspended in liquid cul- of the pump itself remains a mystery, tures. These gas-filled organelles are which suggests the existence of a novel easy to isolate even in a rudimentary mechanism in archaea. Another gene laboratory, and students can test their near the arsenic cluster seems to be functional properties with a series of part of a second detoxification system, quick experiments. which transforms arsenite to a volatile Among its other user-friendly form, trimethylarsine. properties, NRC-1 grows in culture NRC-1 responds to low oxygen lev- media that are inexpensive to make els in several ways. One is to induce and safe to handle, and its moder- the production of gas-filled vesicles ate growth rate (a three- to seven-day that enable the cells to float to aerobic culture period) can be adapted to a zones in the water column. But un- weekly or semiweekly lab schedule. der strictly anaerobic conditions, cells contamination from other types of More advanced students can use shift to an alternate means of energy bacteria or fungi. (The same princi- NRC-1 to learn basic techniques of conversion that uses dimethyl sulfox- ple enables salt to act as a food pre- molecular biology and genetic manip- ide, produced by other microbes, and servative by preventing the growth ulation. And when the experiments trimethylamine N-oxide, produced by of microbes that cause spoilage.) As are over, NRC-1 can be stored in salt fish, to carry out anaerobic respiration. a result, students can conduct their crystals (above left) at room temper- NRC-1 also responds to oxygen star- experiments without needing strict ature for extended periods of time. vation by increasing the production adherence to sterile protocols—a dif- Instructors won’t need to keep their of bacterio-opsin, the protein compo- ficult enough challenge in a profes- haloarchaea cultures for thousands of nent of the bacteriorhodopsin used for sional research laboratory and one years, but it’s interesting to know that phototrophic growth. The synthesis frequently beyond the reach of most they could.— Priya DasSarma of bacterio-opsin is linked to levels of the retinal pigment, and the two molecules react to form a single complex. Indeed, the genes that encode the first and last steps of retinal synthesis and the nearby sensor-activator gene are all coordinately induced under low- oxygen conditions. Scientists don’t completely understand how NRC-1 regulates different suites of genes in response to environmental stresses, but my coworkers and I recently proposed a mechanism to ex-

© 2007 Sigma Xi, The Scientific Research Society. Reproduction 230 American Scientist, Volume 95 with permission only. Contact [email protected]. plain some of that complexity. Our hy- Agency found that a strain of Halo- Bibliography pothesis is that two general transcrip- arcula survived for several weeks in Bayley, S. T., and R. A. Morton. 1978. Recent tion factors, TBP and TFB—which are deep space—longer than any other development in the molecular biology of sometimes overlooked as rather dull, organism that was still capable of di- extremely halophilic bacteria. CRC Critical Reviews in Microbiology 6:151–205. invariant backdrops to more dynamic viding. This finding is consistent with DasSarma, S., F. T. Robb, A. R. Place, K. R. Sow- processes—are themselves acting as the observed capacity of Halobacterium ers, H. J. Schreier and E. M. Fleischmann. regulators. Although the TBP and TFB NRC-1 to withstand desiccation and 1995. Archaea: A Laboratory Manual–Halo- proteins are usually encoded by single radiation in laboratory studies. It also philes. Cold Spring Harbor, New York: Cold genes in archaea and eukaryotes, Halo- supports the findings of geologists Spring Harbor Laboratory Press. bacterium NRC-1 carries six genes for who have isolated haloarchaeal DNA DasSarma, S., and P. DasSarma. 2006. Halo- philes. In Encyclopedia of Life Sciences. Chich- TBP and seven for TFB, suggesting the similar to that of NRC-1 from halite ester: John Wiley & Sons, Ltd. possibility of many different combi- (salt) deposits more than 10 million DasSarma, S. 2004. Genome sequence of an nations of TBP and TFB during tran- years old. extremely halophilic archaeon. In Microbial scription. Specific pairs may activate Some of the chunks of Martian rock Genomes, ed. C. M. Fraser, T. Read and K. E. sets of genes that are intended to work that have fallen to Earth as meteorites, Nelson. Totowa, New Jersey: Humana Press. in concert, by way of regulatory se- including the one that fell on Shergotty, DasSarma, S. 2006. Extreme halophiles are mod- quences found near the genes in ques- India, and the one that fell on Nakhla, els for astrobiology. Microbe 1:120–127. tion. Other scientists have proposed a Egypt, have contained halite salt crys- DasSarma, S., B. R. Berquist, J. A. Coker, P. Das- Sarma and J. A. Müller. 2006. Post-genomics similar mechanism to explain the cho- tals. Thus, it’s conceivable that meteor- of the model haloarchaeon Halobacterium sp. reography of organ development in ites could act as vehicles for the inter- NRC-1. Saline Systems 2:3. some higher organisms. We recently planetary transport of haloarchaea. In Stoeckenius, W., and R. A. Bogomolni. 1982. saw evidence of just such a novel reg- this context, it’s understandable that Bacteriorhodopsin and related pigments of ulatory mechanism for haloarchaea, the public has been keenly interested in halobacteria. Annual Review of Biochemistry which likely evolved to deal with the recent reports that halophilic microbes 51:587–616. The HaloEd Project. http://halo.umbi.umd. stresses and dynamics encountered in were still alive after being trapped in edu/~haloed their hypersaline environment. tiny pockets of brine for hundreds of Woese, C. R., O. Kandler and M. L. Wheelis. millions of years—reports that are cur- 1990. Towards a natural system of organisms: Out of This World rently beyond the reach of rigorous Proposal for the domains Archaea, Bacteria, Because haloarchaea tolerate so many scientific validation. Yet the range of and Eucarya. Proceedings of the National Acad- forms of environmental stress, I have haloarchaeal adaptations to daunting emy of Sciences of the U.S.A. 87:4576–4579. proposed, along with other scientists, conditions suggests that it is still pre- that they are candidate “exophiles”— mature to dismiss the idea of Martian organisms that might survive on Mars life out of hand. There is little data that or other planets. The same durabil- either support or refute the hypothesis For relevant Web links, consult this issue ity could enable haloarchaea to sur- that Earth has exchanged forms of mi- of American Scientist Online: vive the period of deep-space travel crobial life with other celestial bodies http://www.americanscientist.org/ between planets, perhaps encased at some point in its history. For now, IssueTOC/issue/961 in salt crystals, which would shield the fact that Earthly forms of life could them from some of the damaging ra- exist on other planets is remarkable diation. Recently, the European Space enough all by itself.