Aerobic heterotrophic metabolism of haloarchaea
The Halobacteriaceae typically lead an aerobic heterotrophic life style. However, in spite of their common requirement for high salt concentrations for growth, their nutritional demands and metabolic pathways are quite diverse. Some species possess complex dietary needs that can be met in culture by including high concentrations of yeast extract or other rich sources of nutrients to their growth medium (e.g. Halobacterium salinarum). By contrast, some species grow well on single carbon sources while using ammonia as a nitrogen source. Haloferax mediterranei can grow on simple compounds such as acetate, succinate, etc. while supplying its need for nitrogen, sulfur, and other essential elements from inorganic salts. Such simpler growth demands are generally detected in species of the genera Haloferax and Haloarcula (Oren, 2002b). An even more extreme case is Halosimplex carlsbadense, an organism that only grows in defined medium with acetate and glycerol, acetate and pyruvate, or pyruvate alone. Carbohydrates, amino acids, fats, and proteins do not support its growth (Vreeland et al., 2002). Interestingly, pyruvate is also a preferred substrate of the flat square Haloquadratum walsbyi (Burns et al., 2007).
The members of Halobacteriaceae use the tricarboxylic acid cycle (TCA) in the process of aerobic degradation of carbon and, if necessary, a combination of the glyoxylate cycle and the respiratory electron transport. Haloarchaeal genomes encode the complete set of enzymes of the TCA cycle (Falb et al., 2008). Furthermore, activity of all enzymes of the cycle was detected in Hbt. salinarum (Aitken & Brown, 1969). Field studies on a hypersaline cyanobacterial mat have shown metabolic interactions between haloarchaea and the primary producer Coleofasciculus (Microcoleus) chthonoplastes. This cyanobacterium excretes acids of the citrate cycle into the medium, and aerobic halophilic Archaea further utilizes these as the major carbon and energy source (Zvyagintseva et al., 1995). The existence of a functional glyoxylate cycle has been demonstrated in Haloferax volcanii (Serrano et al., 1998) and in Natronococcus occultus (Kevbrina & Plakunov, 1992). Inquiries effectuated on the 13 complete halophilic genomes present in the HaloWeb data base (DasSarma et al., 2010) did not find any simultaneous positive matches for the glyoxylate cycle key enzymes: isocitrate lyase and malate synthase (with the exception of previous mentioned species Hfx. volcanii). A blastp (Altschul et al., 1997) search made on NCBI using the amino acid sequences of the Hfx. volcanii isocitrate lyase and malate synthase showed that these enzymes are present also in Haladaptatus paucihalophilus strain DX253. Recently, a novel pathway for the synthesis of malate from acetyl-CoA was discovered in Hfx. volcanii and in Har. marismortui, in which acetyl-CoA is oxidized to glyoxylate via methylaspartate as key intermediate (Khomyakova et al., 2011).
Although most halophilic Archaea preferentially use amino acids as carbon and energy source, there are carbohydrate-utilizing species such as Haloarcula marismortui, Halococcus saccharolyticus, and Hfx. mediterranei. These species have the capacity to metabolize pentoses (arabinose, xylulose), hexoses (glucose, fructose), sucrose, and lactose (Rawal et al., 1988; Altekar & Rangaswamy, 1992; Johnsen et al., 2001). Comparative analysis of ten haloarchaeal genomes showed that Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each and may break down complex carbohydrates. Hrb. utahensis has specialized in growth on carbohydrates and has few amino acid degradation pathways. It uses the nonoxidative pentose phosphate cycle and a transhydrogenase instead of the oxidative pathway, giving it a great deal of flexibility in the metabolism of pentoses (Anderson et al., 2011). Hrb. utahensis degrades xylan and can grow on xylose (Wainø & Ingvorsen, 2003). Many species of Halobacteriaceae also produce exoenzymes such as proteases, lipases, DNAses, and amylases to degrade organic polymeric substances extracellularly, making small organic molecules available as carbon and energy source.
Studies on glucose and fructose degradation pathways in Halococcus saccharolyticus showed that glucose is entirely degraded via an Entner–Doudoroff (ED) type pathway, whereas fructose is almost completely degraded (96%) via an Embden–Meyerhof type pathway and only to a small extent (4%) via an ED pathway (Johnsen et al., 2001). This ED pathway, in which the phosphorylation step is postponed, is also probably used by the other members of the carbohydrate-utilizing group. In this pathway, glucose is oxidized via gluconate to 2-keto-3-deoxygluconate and then phosphorylated to 2-keto-3-deoxy-6-phosphogluconate, which is further split into pyruvate and glyceraldehyde-3-phosphate (Tomlinson et al., 1974). In addition, other steps in common metabolic pathways may have special modifications in the halophilic Archaea, such as the production of acetate by an ADP-forming acetyl-CoA synthetase (Siebers & Schönheit, 2005).
Halobacterium does not grow on sugars, but its growth is stimulated by the addition of carbohydrates to the medium (Oren, 2002b), where glucose can be transformed into gluconate (Sonawat et al., 1990). Oxidation of carbohydrates is often incomplete and is usually associated with the production of acids (Hochstein, 1978).
In the presence of glycerol, some species of the genus Haloferax and Haloarcula produce acetate, pyruvate, and d-lactate (Oren & Gurevich, 1994). Production of d-lactate, acetate, and pyruvate from glycerol by the haloarchaeal communities of the Dead Sea and saltern crystallization ponds has also been observed. In these environments, acetate is poorly utilized (Oren, 1995).
Analysis of the genome of the flat square archaeon Hqr. walsbyi showed a few unique features. One of them is the presence of a gene cluster that allows uptake of phosphonates and subsequent cleavage of the carbon–phosphorus bond by a phosphonate lyase. Another is the possible use of dihydroxyacetone as a carbon and energy source after its uptake via a phosphoenol pyruvate-dependent phosphotransferase system (Bolhuis et al., 2006). Growth studies showed that, indeed, Hqr. walsbyi could metabolize dihydroxyacetone (Elevi Bardavid & Oren, 2008). Based on the analysis of its genome, this species can also grow on pyruvate and glycerol (Bolhuis et al., 2006). Its apparent inability to take up glycerol, as shown in an analysis of the natural community in a saltern crystallizer pond in Mallorca (Rosselló-Mora et al., 2003) remains unexplained.
A food chain is thus possible, in which glycerol produced as an osmotic solute by the alga Dunaliella is converted in part to dihydroxyacetone by extremely halophilic bacteria of the genus Salinibacter (Bacteroidetes). Haloquadratum and other members of the Halobacteriaceae (Elevi Bardavid & Oren, 2008; Elevi Bardavid et al., 2008) can then take up the dihydroxyacetone and the remainder of the glycerol.
Some representatives of the family can metabolize aliphatic and aromatic hydrocarbons and long-chain fatty acids, such as hexadecanoic acid (Bertrand et al., 1990; Oren, 2006; McGenity, 2010a). Thus, a study of the biodegradation of crude oil and pure hydrocarbons by extreme halophilic Archaea from hypersaline coasts of the Arabian Gulf yielded two strains of Haloferax, one of Halobacterium, and one of Halococcus, which can grow on crude oil vapor as sole carbon and energy source (Al-Mailem et al., 2010). Hydrocarbon-degrading extremely halophilic Archaea were also isolated from a saltern crystallizer pond in the south of France (Tapilatu et al., 2010).
Degradation of aromatic compounds by haloarchaea was first documented by Emerson et al. (1994) in Haloferax strain D1227 that grew on benzoate, cinnamate, and phenylpropionate. Aerobic degradation of p-hydroxybenzoic acid by a Haloarcula sp. follows an unusual metabolic pathway (Fairley et al., 2002). More halophilic Archaea growing on benzoic acid, p-hydroxybenzoic acid, salicylic acid, and on a mixture of the polycyclic hydrocarbons naphthalene, anthracene, phenanthrene, pyrene and benzo[a]anthracene, with and without 0.05% yeast extract, were isolated from different geographic locations: salt flats in Bolivia, salterns in Chile and Puerto Rico, a sabkha in Saudi Arabia, and the Dead Sea. Most isolates were affiliated with the genus Haloferax (Cuadros-Orellana et al., 2006; Bonfá et al., 2011).
Genomic information revealed that the recently discovered nanohaloarchaeal organisms lead an aerobic heterotrophic life style. The presence of lactate dehydrogenase may point to a potential for fermentative metabolism. The genes encoding the enzymes of the Embden–Meyerhof glycolytic pathway were identified, and both the oxidative (based on glucose-6-phosphate dehydrogenase as the key enzyme) and the nonoxidative branches of the pentose phosphate pathway were present. This is the first case in which the complete pentose phosphate pathway was demonstrated in a member of the Archaea (Narasingarao et al., 2012).
Anaerobic heterotrophic metabolism of the Halobacteria
Oxygen has a low solubility in salt-saturated brines, and therefore, it may easily become a limiting factor for the development of halophilic Archaea. Some produce gas vesicles or posses aerotaxis sensors (e.g. HemAT in Halobacterium) (Hou et al., 2000) that enable them to reach the water–air interface, while others have the capacity to grow anaerobically. Variants of anaerobic growth documented within the Halobacteriaceae include the use of alternative electron acceptors such as nitrate, dimethylsulfoxide, trimethylamine N-oxide or fumarate, fermentation of arginine, and possibly other types of fermentation as well (Oren, 2006).
Considering the low concentrations of nitrate generally encountered in hypersaline brines and the apparent lack of regeneration of nitrate by nitrification at high salt concentrations, the process can be expected to occur only to a limited extent in nature (Oren, 1994). Some halophilic Archaea (e.g. Har. marismortui, Har. vallismortis, Hfx. mediterranei) can grow anaerobically when nitrate is present as the electron acceptor, forming gaseous nitrogen and/or nitrous oxide (Mancinelli & Hochstein, 1986).
The ability to use dimethylsulfoxide and trimethylamine N-oxide, or fumarate as electron acceptors for anaerobic growth, is quite widespread among the halophilic Archaea (Oren & Trüper, 1990; Oren, 1991). Additionally, thiosulfate and elemental sulfur have been suggested to act as potential electron acceptors (Tindall & Trüper, 1986; Elshahed et al., 2004). Nonetheless, information on the nature of these processes is scarce (Oren, 2006).
Fermentation of l-arginine to citrulline can drive anaerobic growth in Hbt. salinarum (Hartmann et al., 1980), but this metabolic pathway does not seem to be widespread among haloarchaea. Thus far, it has only been detected in the genus Halobacterium (Oren & Litchfield, 1999; Oren, 2006). When grown anaerobically, species of the mentioned genus are able to ferment arginine via the arginine deiminase pathway (Ruepp & Soppa, 1996). Throughout this pathway, arginine is converted to ornithine and carbamoylphosphate, which is further split into carbon dioxide and ammonia with concomitant ATP production.
Fermentation is probably the preferred mode of life of Halorhabdus tiamatea, a nonpigmented, extremely halophilic archaeon isolated from the brine–sediment interface of the Shaban Deep, a hypersaline anoxic basin in the northern Red Sea. This species uses yeast extract and starch as carbon and energy sources and grows anaerobically and under microaerophilic conditions, but aerobic incubation was shown to support only a very poor growth (Antunes et al., 2008). A gene encoding lactate dehydrogenase was found in the Hrb. tiamatea genome, and this enzyme might participate in the fermentation pathway (Antunes et al., 2011).
Photoheterotrophic metabolism of the Halobacteria
An entirely different mode of anaerobic growth displayed by some halophilic Archaea is photoheterotrophy, which consists in the use of light energy absorbed by retinal-based pigments. The light-driven proton pump bacteriorhodopsin can drive anaerobic growth of Hbt. salinarum (Hartmann et al., 1980; Oesterhelt, 1982). Many members of the Halobacteriaceae and, possibly, the newly described group of Nanohaloarchaea (Ghai et al., 2011) possess the necessary genes for the biosynthesis of the bacteriorhodopsin protein and the retinal prosthetic group, but little is known about the relative importance of light as an energy source to drive growth of the halophilic Archaea in their natural environment. Organic substrates serve as carbon sources, still photoautotrophy has not been demonstrated in the archaeal domain.
Archaeal methanogenesis in hypersaline systems
Methanogenic Archaea acquires the necessary energy for growth and survival by the stoichiometric conversion of a limited number of substrates to methane gas. The major substrates are H2 + CO2, formate (group 1), acetate (group 3) and, in a lesser extent, compounds such as methanol, trimethylamine, dimethylsulfide (group 2), and some alcohols such as isopropanol.
Methane is a major end product of anaerobic degradation of the biomass only in anoxic environments where the concentration of products such as sulfate, nitrate, Mn(IV), or Fe(III) is low. The presence of these substances in the medium allows other organisms to outcompete methanogens in the competition for electron acceptors, mainly because of thermodynamic reasons. For instance, sulfate-reducing bacteria have the ability to utilize H2 at lower concentrations than minimum required by methanogens, in the presence of sulfate. Consequently, sulfidogenesis generally prevails in estuarine, marine, and hypersaline sediments where sulfate diffuses from overlying water (McGenity, 2010b). However, increased salinity in many cases supplies higher concentrations of noncompetitive substrates, which derive from compatible solutes synthesized by the environmental microbiota. Such high-salinity-associated solutes include methylated amines and dimethylsulfide.
At high salt concentration, neither the reduction of carbon dioxide by hydrogen nor the aceticlastic reaction was shown to occur. Acetate splitting methanogens appear to be very little salt tolerant. The upper salt concentration for growth of cultures of methanogenic Archaea greatly depends on the substrate used: 270 g L−1 for group 2 methanogens, 120 g L−1 for group 1 methanogens, and 40 g L−1 for group 3 methanogens (Oren, 1999). These salinities should not be considered as the upper limit of activity in situ, but to be indicative of the relative importance of these substrates at different salinities (McGenity, 2010b). The absence of group 1 and group 3 methanogens at high salinity may be governed by the relative energy gain from different methanogenic reactions per mole of substrate (methylotrophic ≫ hydrogenotrophic ≥ aceticlastic), especially because halophiles must expend a lot of energy to maintain an osmotically balanced and functional cytoplasm via the biosynthesis and/or uptake of organic compatible solutes, and/or uptake of potassium ions (Oren, 1999). This may further explain the predominance of methylotrophic methanogens like Methanohalophilus spp. in hypersaline environments. On the other hand, we must approach this interpretation with caution, because standard Gibbs free energy yields are only one determinant of the total metabolic energy yield, and we must take into consideration the rate of substrate flux/consumption.
Trimethylamine is often found in saline systems, where it is formed from glycine betaine or other osmoprotectants used by the resident organisms to equilibrate the cytoplasmic osmolarity to that of the water. This substance is rapidly transformed by methanogens to methane, CO2, and ammonia, but it is not easily utilized by sulfidogenic bacteria. Trimethylamine-degrading methanogens from saline environments belong to the family Methanosarcinaceae, and all methanogens that have been isolated to date from high-salinity ecosystems use trimethylamine as catabolic substrate (with the exception of M. halotolerans, which uses H2 + CO2 or formate and has a relatively restricted salt tolerance, and does not grow above 120 g L−1 salt).
On the origin of prokaryotic "species": the taxonomy of halophilic Archaea
1University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 East Pratt Street, Baltimore, MD 21202, USA
Priya DasSarma: ude.dmu.ibmu@pmrassad; Shiladitya DasSarma: ude.dmu.ibmu@amrassad
Author information ►Article notes ►Copyright and License information ►
Received 2008 Apr 30; Accepted 2008 May 16.
Copyright © 2008 DasSarma and DasSarma; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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The consistent use of the taxonomic system of binomial nomenclature (genus and species) was first popularized by Linnaeus nearly three-hundred years ago to classify mainly plants and animals. His main goal was to give labels that would ensure that biologists could agree on which organism was under investigation. One-hundred fifty years later, Darwin considered the term species as one of convenience and not essentially different from variety. In the modern era, exploration of the world's niches together with advances in genomics have expanded the number of named species to over 1.8 million, including many microorganisms. However, even this large number excludes over 90% of microorganisms that have yet to be cultured or classified. In naming new isolates in the microbial world, the challenge remains the lack of a universally held and evenly applied standard for a species. The definition of species based on the capacity to form fertile offspring is not applicable to microorganisms and 70% DNA-DNA hybridization appears rather crude in light of the many completed genome sequences. The popular phylogenetic marker, 16S rRNA, is tricky for classification since it does not provide multiple characteristics or phenotypes used classically for this purpose. Using most criteria, agreement may usually be found at the genus level, but species level distinctions are problematic. These observations lend credence to the proposal that the species concept is flawed when applied to prokaryotes. In order to address this topic, we have examined the taxonomy of extremely halophilic Archaea, where the order, family, and even a genus designation have become obsolete, and the naming and renaming of certain species has led to much confusion in the scientific community.
An important challenge in the classification of microorganisms is ensuring that scientists can follow the pedigree of isolates in the literature. This laudable goal is however especially difficult to achieve for microbes that have a long history and where variants have been isolated and re-isolated from an abundance of niches in the laboratories of many different investigators. For example, the group of extremely salt-loving halophilic microbes which produce red, pink, and purple hues in hypersaline ponds used to make salt from the sea, were among the earliest microorganisms to be recognized and described. They were, not surprisingly, originally identified as agents of food spoilage, before the advent of refrigeration when salting was widely used for preserving fish and meats . Some early isolates from dried and salted codfish (Klippfisch) were documented in a 1919 review on causative agents of fish reddening by the German botanist, Klebahn . Notably, he isolated and named "Bacillus halobius ruber", aware that these bright vermillion halophilic microbes were not spore formers, but his isolate was subsequently lost. A dozen years later, halophilic isolates thought to be similar to Klebahn's, were named "Bacterium halobium" by Petter in the Kluyver laboratory in Delft, Holland . In the 1940s–1960s, additional halophilic microorganisms were isolated from different countries and reported in the scientific literature with names such as Halobacterium halobium, H. salinarium, and H. cutirubrum . Many of these isolates were deposited in US, Canadian, and European culture collections, but a substantial number of them were subsequently lost or renamed. Revision of the taxonomy of these extremely halophilic genera from Bacillus to Bacterium to Halobacterium reflected the increasing sophistication of our knowledge of the microbial world during this period, and these changes were generally accepted by the scientific community. In the modern era, there have been many more proposals for taxonomic revisions among these halophiles, some of which have been readily acceptable, and others that have since been challenged or refuted [5,6].
A proposal to modernize haloarchaeal taxonomy and terminology
Among extremely halophilic microorganisms, the distinction of halophilic Archaea from halophilic Bacteria became apparent in the 1970's through the molecular phylogenetic work of Woese, who proposed the three-domain view of life. While halophilic microorganisms represented many different taxonomic groups in the bacterial domain, those in the archaeal domain fell into a single order (Halobacteriales) and family (Halobacteriacae) . Our understanding of the existence of the three domains has created ambiguity in the terminology used, since 'halobacteria' traditionally referred to all extremely halophilic microorganisms, including both halophilic Bacteria and halophilic Archaea. In order to clarify the definitions, we propose that the term halobacteria be reserved only for halophiles that are members of the bacterial domain, while haloarchaea be used only for halophiles that are members of the archaeal domain. In addition, on a taxonomic level, the order Halobacteriales should be designated as Haloarchaeales and the family Halobacteriaceae should be as Haloarchaeaceae. Finally, the Halobacterium genus would be better named Haloarchaeum to reflect its membership in the archaeal rather than the bacterial domain. These revisions would help update the taxonomy and terminology of halophilic microorganisms to be consistent with our current understanding of the microbial world.
Taxonomic ambiguity among species
While our proposed revision of haloarchaeal taxonomy is relatively simple, disentangling the taxonomy and pedigree within the original genus, Halobacterium (Haloarchaeum), is considerably more complex. Over the past twenty-five to fifty years, this genus witnessed a contraction in the number of recognized species from over a dozen to just a single species. While some acquired new genus designations (e.g. Halobacterium volcanii changed to Haloferax volcanii and Halobacterium marismortui to Haloarcula marismortui), in 1990, Grant and Larsen proposed combining three common species, Halobacterium halobium, H. salinarium, and H. cutirubrum, into a single one, H. salinarium . However, this proposal was not fully accepted by the community since the changes were not fully in accordance with the rules of the Bacteriological Code . In particular, halobium predated salinarium in the literature and, by convention, the former name should have taken precedence over the latter. To complicate matters further, in 1996, Ventosa and Oren proposed renaming of H. salinarium to H. salinarum , removing an "i", in their opinion, for linguistic reasons. However, many investigators dissented and continued to use the original species designations. In Euzéby's List of Prokaryotic Names with Standing in Nomenclature , he reported that salinarium, is derived from the Latin adjective salinarius a um, meaning "of salt works", while salinarum is derived from salinae arum, meaning "salt works", and concluded that salinarium was indeed correct. There is no doubt that naming and renaming of these species has left the taxonomy of Halobacterium (Haloarchaeum) species in disarray in the literature and in the haloarchaeal community.
Perhaps the worst case of taxonomic ambiguity is for the first Halobacterium (Haloarchaeum) isolate sequenced and also the most widely used haloarchaeal strain, which was published under the name H. halobium strain NRC-1 . The origin of strain NRC-1 is uncertain, though it likely appeared from the 1960's collection of W. Stoeckenius and was disseminated via W.F. Doolittle (personal communications) to S. DasSarma in the 1980's. In 2000, the NRC-1 strain was deposited by the DasSarma laboratory in the American Type Culture Collection (ATCC no. 700922) for standardization and distribution in the research community and has since been used by Carolina Biological Supply Company in the educational sphere (Carolina no. 154777) . Stocks of the original culture used for sequencing are also maintained in the DasSarma laboratory. As a result of uncertainties regarding the origin of this strain, the authors of the complete genome sequencing paper  dropped the species designation, reverting to "Halobacterium sp. strain NRC-1", while its pedigree was being rigorously established and the relationships within this group of organisms fully clarified. However, despite the lack of appearance of definitive information on the identity of NRC-1, Gruber et al.  published a paper in 2004 reclassifying the wild-type isolate as a strain of the 'H. salinarum' species. In so doing, these authors ignored a variety of differences between strains, including their own pulsed-field gel patterns, as well as variations in restriction maps of the unstable resident megaplasmids. Overreliance on phylogenetic trees based on 16S rRNA sequences, some of which are already known to be divergent even within single haloarchaeal species , was a serious shortcoming of this study.
Reexamination of the available data on Halobacterium (Haloarchaeum) isolates at the phylogenetic and taxonomic levels confirms the existence of serious complications. The 16S rRNA sequences vary in nearly all of the originally distinct species, with that of NRC-1 and H. salinarium differing in several positions. Differences also exist between the NRC-1 and H. halobium 23S rRNA sequences, as well as between NRC-1 and H. cutirubrum 5S rRNA. In fact, a recent publication even reported a major deletion in the 16S rRNA promoter region of 'H. salinarum' (originally H. halobium) strain R-1 in comparison to strain NRC-1 and other similar strains . The most compelling case for the existence of substantial taxonomic diversity among Halobacterium (Haloarchaeum) isolates is from the recent genotyping analysis of Cleland et al. using the DiversiLab repPCR system . In this study, some of the seven Halobacterium (Haloarchaeum) strains in the ATCC collection show differences quantitatively similar to haloarchaea that are classified as different genera of the Halobacteriaceae (Haloarchaeaceae) family. Their examination of NRC-1 by this method (Figure 1) showed that this strain fell below 70% similarity compared to other 'H. salinarium' strains in ATCC, suggesting that NRC-1 should be given an entirely new species designation using that criterion. This analysis supports our viewpoint that it is premature to reclassify all of these Halobacterium (Haloarchaeum) isolates as a single species, especially without an existing consensus in the community on the definition of what constitutes a "species" among these organisms.
Genotyping of Halobacterium (Haloarchaeum) isolates using the DiversiLab repPCR system by Cleland et al. at the American Type Culture Collection . The two sequenced Halobacterium species are included, the model strain NRC-1 (ATCC 700922) and strain...
Phenotypes and pedigree
At the phenotypic level, Halobacterium (Haloarchaeum) strains have some clear-cut differences. An especially striking difference is the absence, in 'H. salinarum' (e.g. strain R-1), of gas vesicles, which are characteristic of Halobacterium sp. NRC-1. Gas vesicles permit NRC-1 to move vertically in the water column in response to oxygen, light, and temperature, and the corresponding expression of gas vesicle protein genes can be clearly seen in DNA microarray experiments [17,18]. The lack of gas vesicles in 'H. salinarum' indicates that these organisms exist in significantly different environments from NRC-1, with the latter inhabiting dynamic ones, and the former in more constant environments. This notable phenotypic difference is easily visible even to the naked eye. Some other Halobacterium (Haloarchaeum) species contain two different morphological types of gas vesicles in the same cell, those which are narrower and diagnostic of species inhabiting deep habitats, as well as those which are wider and found in shallow brines (like NRC-1), suggesting the widespread environmental distribution of these species. Examined more broadly, isolate-specific differences have been shown to exist at the level of antibiotic-resistance markers, measured cations in cells, and protein, lipid, and sugar content in the cell envelopes .
The pedigree of the various strains of Halobacterium (Haloarchaeum) being studied in laboratories worldwide is also confounding. One example is the unclear relationship between NRC-1 and R-1, and another similar strain S-9, a purple membrane overproducer. While some investigators reported that NRC-1, R-1, and S-9, were very close relatives , others indicated otherwise [20,21]. The stable gas vesicle-deficient strain, R-1, may be the parent of S-9, a strain which was isolated after extensive chemical mutagenesis, but R-1 is probably not a descendent of NRC-1. Not surprisingly, the genome sequencing results showed at least 200 kb of additional DNA in R-1 compared to NRC-1 and very little similarity in their resident megaplasmids . Many additional examples of incongruent taxonomy and pedigree among Halobacterium (Haloarchaeum) species are reviewed by Grant and Larsen  and Tindall . Clearly, the frequent and questionable revisions in the taxonomy of these interesting microbes, and at times, the lack of careful maintenance and documentation of their pedigree, are a serious impediment to advancing the field.
The incredible precision of the genomic era has empowered microbiologists with the genetic blueprints of more than a thousand microorganisms and allowed for the development of many new approaches for the interrogation of their biology. Unfortunately, studies of certain microbes, such as the haloarchaea, have been made exceedingly difficult by the arbitrary and unnecessary renaming of strains, poor record keeping of pedigree, and the lack of a universal definition of species. All of these shortcomings make it likely that future generations will not be able to fully interpret and utilize the current literature, ultimately diminishing the contributions of both past and present generations. While rigorous genetic studies and complete genome sequences are destined to make a permanent contribution to the field, taxonomic rearrangements based on inadequate experimentation and flawed logic hold it back. Although these points resonate especially true among the haloarchaea, we suspect that similar taxonomic issues and challenges are quite widespread among other prokaryotes as well and are deserving of further scrutiny.
The authors declare that they have no competing interests.
Both authors contributed to the development of the concepts and writing of the manuscript.
We thank Dr. David Cleland for Figure 1 and helpful discussions and Professor Martin Bloomer for his expertise with Greek and Latin language issues.
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