The Family Halobacteriaceae

Abstract

The family Halobacteriaceae, first proposed by Gibbons in 1974, is affiliated with the archaeal phylum Euryarchaeota. Currently (August 2012) it encompasses 40 genera: Halobacterium [type genus], Haladaptatus, Halalkalicoccus, Halarchaeum, Halarchaeobius, Haloarcula, Halobaculum, Halobellus, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halogranum, Halolamina, Halomarina, Halomicrobium, Halonotius, Halopelagius, Halopenitus, Halopiger, Haloplanus, Haloquadratum, Halorhabdus, Halorientalis, Halorubrum, Halosarcina, Halosimplex, Halostagnicola, Haloterrigena, Halovenus, Halovivax, Natrialba, Natrinema, Natronoarchaeum, Natronobacterium, Natronococcus, Natronolimnobius, Natronomonas, Natronorubrum, and Salarchaeum, with a total of 137 species. All members of the family have a high requirement for salt, and most grow optimally at salt concentrations above 150–200 g/l. Most species are pigmented red-pink by carotenoid pigments and have an aerobic chemoheterotrophic metabolism. Some have the ability to grow anaerobically by fermentation, anaerobic respiration, or using bacteriorhodopsin to absorb light as an energy source.

Taxonomy, Historical and Current

Family Halobacteriaceae Gibbons 1974, 269^AL

Ha.lo.bac.te.ri.a.ce’ae. N.L. neut. n. Halobacterium, type genus of the family; -aceae, ending to denote a family; N.L. fem. pl. n. Halobacteriaceae, the Halobacterium family.

Type genus: Halobacterium.

The mol% G + C of the DNA varies between 46.9 and 71.2.

The family Halobacteriaceae (order Halobacteriales; Grant et al. 2001a) was circumscribed on the basis of the high salt requirement of its members, their physiological and chemotaxonomic features, and their phylogenetic affiliation with the Euryarchaeota phylum of the Archaea (Grant et al. 2001b). At the time of writing (August 2012), the family contained 40 genera with a total of 137 species whose names have standing in the nomenclature (Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22): Halobacterium [type genus; three-letter abbreviation Hbt.] (3 species), Haladaptatus (Hap.) (3 species), Halalkalicoccus (Hac.) (2 species), Halarchaeum (Hla.) (1 species), Halarchaeobius (Hab.) (1 species), Haloarcula (Har.) (9 species), Halobaculum (Hbl.) (1 species), Halobellus (Hbs.) (3 species), Halobiforma (Hbf.) (3 species), Halococcus (Hcc.) (7 species), Haloferax (Hfx.) (11 species), Halogeometricum (Hgm.) (2 species), Halogranum (Hgn.) (4 species), Halolamina (Hlm.) (1 species), Halomarina (Hmr.) (1 species), Halomicrobium (Hmc.) (3 species), Halonotius (Hns.) (1 species), Halopelagius (Hpl.) (1 species), Halopenitus (Hpt.) (1 species), Halopiger (Hpg.) (2 species), Haloplanus (Hpn.) (3 species), Haloquadratum (Hqr.) (1 species), Halorhabdus (Hrd.) (2 species), Halorientalis (Hos.) (1 species), Halorubrum (Hrr.) (25 species), Halosarcina (Hsn.) (2 species), Halosimplex (Hsx.) (1 species), Halostagnicola (Hst.) (3 species), Haloterrigena (Htg.) (9 species), Halovenus (Hvn.) (1 species), Halovivax (Hvx.) (2 species), Natrialba (Nab.) (7 species), Natrinema (Nnm.) (7 species), Natronoarchaeum (Nac.) (1 species), Natronobacterium (Nbt.) (1 species), Natronococcus (Ncc.) (3 species), Natronolimnobius (Nln.) (2 species), Natronomonas (Nmn.) (2 species), Natronorubrum (Nrr.) (5 species), and Salarchaeum (Sar.) (1 species). The three-letter abbreviations for the genus names within the family have been endorsed by the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteriaceae (http://www.the-icsp.org/taxa/halobacterlist.htm; accessed October 29, 2012). At the time of writing, descriptions were in press of Hla. salinum sp. nov. (Yamauchi et al. 2012) and Hbl. magnesiiphilum (Shimoshige et al. 2012). The names Hrr. sfaxense (Trigui et al. 2011) and Salinarchaeum laminariae gen. nov., sp. nov. (Cui et al. 2011c) were effectively published, but were not yet validated. These taxa were not included in Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22.

Table 7.1 Comparison of selected characteristics of the members of the genera Halobacterium, Haladaptatus, and Halalkalicoccus

Table 7.2 Comparison of selected characteristics of the members of the genera Halarchaeum, Haloarchaeobius, Halobaculum, and Halobellus

Table 7.3 Comparison of selected characteristics of the members of the genus Haloarcula

Table 7.4 Comparison of selected characteristics of the members of the genera Halobiforma, Halogeometricum, and Halogranum

Table 7.5 Comparison of selected characteristics of the members of the genus Halococcus

Table 7.6 Comparison of selected characteristics of the members of the genus Haloferax (part A)

Table 7.7 Comparison of selected characteristics of the members of the genus Haloferax (part B)

Table 7.8 Comparison of selected characteristics of the members of the genera Halolamina, Halomarina, Halomicrobium, Halonotius, and Halopelagius

Table 7.9 Comparison of selected characteristics of the members of the genera Halopenitus, Halopiger, Haloplanus, and Haloquadratum

Table 7.10 Comparison of selected characteristics of the members of the genera Halorhabdus, Halorientalis, Halosarcina, and Halosimplex

Table 7.11 Comparison of selected characteristics of the members of the genus Halorubrum (part A)

Table 7.12 Comparison of selected characteristics of the members of the genus Halorubrum (part B)

Table 7.13 Comparison of selected characteristics of the members of the genus Halorubrum (part C)

Table 7.14 Comparison of selected characteristics of the members of the genus Haloterrigena

Table 7.15 Comparison of selected characteristics of the members of the genera Halostagnicola, Halovenus, and Halovivax

Table 7.16 Comparison of selected characteristics of the members of the genus Natrialba

Table 7.17 Comparison of selected characteristics of the members of the genus Natrinema

Table 7.18 Comparison of selected characteristics of the members of the genera Natronoarchaeum, Natronobacterium, and Natronococcus

Table 7.19 Comparison of selected characteristics of the members of the genera Natronolimnobius, Natronomonas, and Salarchaeum

Table 7.20 Comparison of selected characteristics of the members of the genus Natronorubrum

Table 7.21 Properties of the sequenced genomes of members of the Halobacteriaceae (as of August 2012)

Table 7.22 List of Haloviruses described as of August (2010)^a

DasSarma and DasSarma (2008) proposed to rename the family as “Haloarchaeaceae.” This name is not validly published, and the proposed change is in violation of the General Considerations, Principles, and Rules of the International Code of Nomenclature of Prokaryotes (Oren 2008).

When in the late 1970s 16S rRNA sequence information was first used to obtain phylogenetic information on the prokaryotes, it was quickly realized that the Halobacteriaceae belong to the newly defined group of the Archaea (Magrum et al. 1978). A short history of the taxonomy of the family, documenting how our concepts on the systematics of the group have changed over the years with the advance of new methods for the characterization of prokaryotes, was given by Oren (2012). Until the late 1970s, the diversity of the group was considered to be low (Colwell et al. 1979), but the use of more varied growth media and culture conditions, together with improved methods for the taxonomic characterization of strains, has led to our current insight that a great physiological and chemotaxonomic diversity exists within the family.

Phylogenetic Structure of the Family and Its Genera

Phylogenetically the family Halobacteriaceae is affiliated with the Euryarchaeota. Figure 7.1a shows a grouped neighbor-joining tree based on 16S rRNA sequence comparisons, showing the genera of the family; an unfolded tree showing the type strains of species of the family is shown in Fig. 7.1b.

Fig. 7.1

Phylogenetic reconstruction of the family Halobacteriaceae based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. (a) presents the genera in a grouped tree, and (b) is an unfolded tree showing the type strains of species of the family. The sequence datasets and alignments were used according to the All-Species Living Tree Project (LTP) database (Yarza et al. 2010; http://www.arb-silva.de/projects/living-tree). The tree topology was stabilized with the use of a representative set of nearly 750 high-quality type strain sequences proportionally distributed among the different bacterial and archaeal phyla. In addition, a 40 % maximum frequency filter was applied in order to remove hypervariable positions and potentially misplaced bases from the alignment. Scale bar indicates estimated sequence divergence

Most genera are well separated within the tree. A major exception is formed by the genera Haloterrigena, Natrialba, Natrinema, and Natronorubrum (see also Tindall 2003 and Wright 2006). The genus Halomicrobium may require a taxonomic reassessment. Alkaliphilic genera are found throughout the tree; there is no single haloalkaliphilic lineage such as was proposed in an early 16S RNA sequencing study (McGenity and Grant 1993).

A major problem encountered when constructing 16S rRNA-based phylogenetic trees of the family Halobacteriaceae is the fact that many species contain multiple 16S rRNA genes, and their sequences can differ by as much as 5 % or more. Mylvaganam and Dennis (1992) first documented the phenomenon for the rrnA and rrnB genes of Har. marismortui, which showed substitutions in 74 positions. Both genes can be expressed in each individual cell (Amann et al. 2000). rrnB appears to be preferentially expressed at higher growth temperatures (López-López et al. 2007). Har. marismortui has three divergent rRNA genes. Other Haloarcula species also show 16S rRNA polymorphism, and so do the members of the genus Halomicrobium (Cui et al. 2009). Halosimplex carlsbadense possesses three different 16S rRNA genes (Vreeland et al. 2002).

Genes encoding 23S rRNA have seldom been used for the reconstruction of the phylogenetic relationships within the Halobacteriaceae. In a study of six species for which both 16S and 23S rRNA gene sequences were known at the time, Briones and Amils (2000) obtained trees with different topologies for the two phylogenetic markers.

Other genetic markers have been proposed in recent years for phylogenetic tree reconstruction for the members of the family by multilocus sequence analysis (Dennis and Shimmin 1997; Enache et al. 2007a; Minegishi et al. 2010a, 2012a; Papke et al. 2007). Use of the markers atpB, EF-2, radA, rpoB′, and secY enabled differentiation of individual strains within species, as well as the delineation of species and genera, including the identification of potential novel species and even family-like relationships (Papke et al. 2011).

Genome Analysis

At the time of writing (August 2012), information was available on the genome sequences of 27 isolates of Halobacteriaceae, 21 of which are type strains of species (Table 7.21). Detailed information can be found in the specialized databases HaloLex (http://www.halolex.mpg.de;) (Pfeiffer et al. 2008b) and HaloWeb (http://halo4.umbi.umd.edu) (DasSarma et al. 2010), as well as in the UCSC Archaeal Genome Browser http://archaea.ucsc.edu (all accessed November 1, 2012). Based on genomic information, comparative studies are possible, e.g., of carbohydrate and amino acid degradation pathways (Anderson et al. 2011).

The chromosomes are between 2.0 and 4.5 Mbp in length and contain between 2,630 and 4,682 protein-coding genes. The number of rRNA operons encoded by these genomes varies between 1 and 3. Many species of Halobacteriaceae contain, in addition to the main chromosome, additional DNA in “minichromosomes,” “megaplasmids,” or plasmids. An extreme case is Har. marismortui with nine circular replicons: two “chromosomes” (3.1 and 0.28 MBp) and 7 plasmids. In many species 25–30 % of the genetic material is found outside the main chromosome. The presence of more than one kind of DNA in representatives of the family was first reported in a CsCl density gradient centrifugation of Hbt. salinarum DNA, which yielded two fractions with 67–68 % and 58–59 % mol% G+C (Joshi et al. 1963). The distinction between minichromosomes, megaplasmids, and plasmids, to be based on copy number, replication control, and evolutionary history, is not always clear (DasSarma et al. 2008; Ng et al. 1998). Out of 65 strains of haloarchaea tested, 75 % had at least one megaplasmid (Gutiérrez et al. 1986).

The paper describing the Hrd. utahensis genome (Bakke et al. 2009) is of special interest as it shows how three different genome annotation services (IMG, Joint Genome Institute Integrated Microbial Genome system; RAST, Rapid Annotation using Subsystems Technology server of the National Microbial Pathogen Data Resource NMPDR; and JCVI, J. Craig Venter Institute Annotation Service) differ considerably in gene calls and different other features. Based on the same raw sequence data, the number of predicted genes ranged from 2,898 to 3,254, and the average gene length ranged between 845 and 942 bp.

At the time of writing, the type strain of the type species of the family (Hbt. salinarum) had not yet been sequenced. For two related strains, the complete genome sequences are available: Halobacterium strain NRC-1, a strain that can be classified within the species (Gruber et al. 2004), and strain R1 (Pfeiffer et al. 2008a). The Halobacterium strain NRC-1 genome was the first complete Halobacteriaceae genome sequenced (Ng et al. 2000), and the data have been extensively used for computational analysis and functional genomics and transcriptomics studies (Kennedy et al. 2001; Soppa et al. 2008). The chromosome of strain R1 is completely colinear and virtually identical to that of NRC-1, but in addition, it possesses not 2 but 4 megaplasmids. A portion of 210 kb of sequence occurs only in strain R1. Pfeiffer et al. (2008a) concluded that the two strains were descendents of one isolate and that the differences observed were the result of rapid evolution in the laboratory. Further information about the origin and the relations between the two strains was provided by Ng et al. (2008) and Pfeiffer et al. (2008b).

The genome of Hqr. walsbyi, an unusually shaped square archaeon, which also has the lowest G+C content of all (47.9 mol%), is of special interest because of its low coding density (76 %) as compared to 86–91 % in other haloarchaea, a phenomenon caused by a very large average intergenic spacing (average 289 bp) and a high number (>1,000) intergenic regions. It also encodes the largest protein identified in the group: the 9,159 amino acid long halomucin.

In some cases, phage-like elements were encountered within the sequenced genomes. Thus, the Nab. magadii genome contains a phage-like element—halovirus ФCh1.

Phages

Since the first viruses lysing Halobacterium strains were described (Torsvik and Dundas 1974; Wais et al. 1975), a large number of viruses tageting different members of the Halobacteriaceae have been isolated and characterized in greater or lesser depth (Dyall-Smith et al. 2003; Pina et al. 2011). They have been classified within the Myoviridae, Siphoviridae, Fuselloviridiae, and “Pleolipoviruses.” Table 7.22 summarizes their properties. These viruses show different morphologies including head-tail, lemon-shaped, and pleomorphic types, and they may contain circular or linear double-stranded or single-stranded DNA. Halorubrum sp. virus HRPV-3 has double-stranded DNA with single-stranded interruptions (Senčilo et al. 2012). Nab. magadii halovirus ФCh1 contains both linear double-stranded 55 kbp DNA and several RNA species (80–700 nt) (Witte et al. 1997). The recently discovered pleomorphic “Pleolipoviruses” are of special interest as they contain lipids derived from the host, as well as glycolipid spikes (Bamford et al. 2005; Pietilä et al. 2012a). Restriction and modification, known from bacteriophages of the Bacteria, have been identified for haloviruses as well (Daniels and Wais 1984).

Comparisons of haloviral genomes have shown that HHPV-1, a pleomorphic double-stranded DNA Har. hispanica virus that is released from the host without cell lysis, has remarkable synteny and amino acid sequence similarity to the single-stranded DNA Halorubrum sp. HRPV-1 virus. A provirus identified in the Hfx. volcanii chromosome is also a member of this group (Roine et al. 2010). Analysis of the genomes of haloviruses HF1 and HF2 yielded evidence for a recent and large recombination event. HF1, a virus with a broad host range (Hbt. salinarum, Hfx. volcanii, Hfx. lucentense), is 94.4 % identical to the Hrr. coriense HF2 genome but about 1.8 kb shorter. Except for a single base change, the first 48 kb are identical. Then there is an abrupt change, suggesting a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb (Tang et al. 2004).

The true diversity of haloviruses in hypersaline ecosystem is probably much larger than that suggested in Table 7.22. A transmission electron microscopy study of the hypersaline Lake Retba, Senegal, showed a tremendous variety of virus like particle morphologies including spindle-shaped, spherical, and linear particles, chains of small globules, hook-shaped particles, reed-shaped particles consisting of a cylindrical body and thin tail, “tadpole” shape, and branched filaments. All these are likely archaeal viruses, although association with bacteria or with eukaryotes cannot be excluded. All these novel types are waiting to be isolated and further characterized. Less than 1 % of the viruslike particles observed had a head-and-tail morphology (Sime-Ngando et al. 2010).

Phenotypic Analyses

The Properties of the Genera and Species of Halobacteriaceae

Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22 summarize the properties of the species of Halobacteriaceae whose names have been validly published until August 2012. A number of additional genus and species names have been effectively published but were not yet validated. These include the genus Halorubellus with species Hrb. salinus and Hrb. litoreus (Cui et al. 2012b) and the genus Halorussus with the species Hrs. rarus (Cui et al. 2010e).

The tables provide a general overview only of the properties of the organisms, and do not list all characters that have been documented in the original species descriptions and in later studies. For example, a positive reaction for nitrate reduction as listed in Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22 can signify reduction of nitrate to nitrite only or true denitrification with production of N₂ and/or N₂O coupled to anaerobic growth. The entry “Organic substrates used” may list compounds that stimulate growth or compounds that can be used as sole carbon and energy source. The species descriptions are not always clear in this respect. Some species are indeed able to grow on single carbon sources without amino acid supplement, examples being Har. hispanica, Hfx. volcanii, Hmn. pharaonis, and Hrd. utahensis (Feng et al. 2012). The list of organic compounds given here is limited to sugars, sugar alcohols, and organic acids. Some species descriptions give information on the use of different amino acids as well. Properties such as the Gram stain are not listed in the tables as, with very few exceptions, all members of the Halobacteriaceae stain Gram negative, when using the special modification of the Gram stain protocol developed for halophilic prokaryotes. Exceptions are Hrr. vacuolatum and Htg. daquinensis, reported as Gram variable. Nearly all members of the family were reported to display catalase activity, exceptions being Hla. acidiphilum, Hns. pteroides, Hst. alkaliphila, Hst. kamekurae, and Nac. mannanilyticum. The oxidase reaction is also generally positive, but in some species, no oxidase activity could be detected: Hbt. noricense, Hac. jeotgali, Hla. acidiphilum, Hcc. hamelinensis, Hcc. qingdaonensis, Hfx. mucosum, Hns. pteroides, Hrd. tiamatea (a rare case of a member of the Halobacteriaceae with an anaerobic life style), Hrr. aquaticum, Hrr. cibi, Hst. kamekurae, Nnm. pellirubrum, Nac. mannanilyticum, and Ncc. jeotgali. When evaluating such results, it should be taken into account that different protocols may have been used in different laboratories. A comparative study using comparable methods and culture of similar age was never yet performed.

A critical evaluation of the tables shows some problematic data published in the species descriptions. For example, the growth of Hmr. oriensis, Hrr. aquaticum, Hrr. kocurii, Hrr. orientale, Hrr. tibetense, Htg. salina, and Hvx. ruber was reported to be stimulated by starch, but amylase activity could not be detected in these species. Also some of the published data on sensitivity to antibiotics need a renewed evaluation. For example, the described sensitivity of Hvx. ruber to ampicillin, an antibiotic that inhibits the formation of the bacterial cell wall but not of the wall of the Archaea, needs to be reassessed.

The short descriptions of the different genera given below only list their distinctive phenotypic and chemotaxomic properties. Since in recent years many new genera have been proposed mainly on the basis of 16S rRNA gene sequences, it has become more difficult to find morphological, physiological, or chemotaxonomic traits that can be used to unequivocally assign a strain to one of these genera. An example can be found in the description below of the genus Halomicrobium, a genus established only on the basis of 16S rRNA gene comparisons, which now contains organisms with greatly different G+C contents of their DNA, with some species possessing and some lacking phosphatidylglycerol sulfate in their polar lipids. Full descriptions of the genera can be found in the original descriptions as cited. A number of emended genus descriptions were given by Oren et al. (2009).

Genus Halobacterium Elazari-Volcani 1957, 207^AL; emend. Kamekura and Dyall-Smith 1995, 344; emend. Oren, Arahal, and Ventosa 2009, 638

Ha.lo.bac.te’ri.um. Gr. n. hals, halos, salt; L. neut. n. bacterium, a small rod; N.L. neut. n. Halobacterium, salt (-requiring) bacterium.

Cells are motile rods of varying length and lyse in distilled water. Colonies are red or pink due to the presence of bacterioruberin carotenoids; purple retinal pigments may be present as well. Some strains possess gas vesicles. Magnesium requirement is moderate (5–50 mM). Amino acids are required for growth. Many strains grow anaerobically in the dark by fermentation of arginine. Sugars are poorly used, and no acid is formed in the presence of sugars. The optimum salt concentration for growth is 3.5–4.5 M NaCl. Neutrophilic. Characteristic lipids are PG, PGP-Me, PGS, and sulfated triglycosyl- and tetraglycosyl diethers.

The mol% G+C of the DNA is 54.3–70.9.

The genus Halobacterium currently contains three species: Hbt. salinarum (type species), Hbt. jilantaiense, and Hbt. noricense.

The main features of the members of the genus are summarized in Table 7.1.

Additional comments:

• Hbt. piscisalsi (Yachai et al. 2008) is not included in the table, as it is now considered a later heterotypic synonym of H. salinarum (Minegishi et al. 2012b).

• Hbt. cutirubrum, Hbt. halobium, and Hbt. salinarium were renamed as Hbt. salinarium nom. corrig. (Ventosa and Oren 1996).

• Hbt. denitrificans (Tomlinson et al. 1986) was reclassified as Hfx. denitrificans comb. nov. (Tindall et al. 1989).

• Hbt. distributum (Zvyagintseva et al. 1987) was reclassified as Hrr. distributum comb. nov. (Oren and Ventosa 1996).

• Hbt. lacusprofundi (Franzmann et al. 1988) was reclassified as Hrr. lacusprofundi comb. nov. (McGenity and Grant 1995; Validation List 57, 1996).

• Hbt. mediterranei (Rodriguez-Valera et al. 1983) was reclassified as Hfx. mediterranei (Torreblanca et al. 1986).

• Hbt. pharaonis (Soliman and Trüper 1982) was reclassified first as Nbt. pharaonis comb. nov. (Tindall et al. 1984) and later as Nmn. pharaonis comb. nov. (Kamekura et al. 1997).

• Hbt. saccharovorum (Tomlinson and Hochstein 1976) was reclassified as Hrr. saccharovorum comb. nov. (McGenity and Grant 1995; Validation List 57, 1996).

• Hbt. sodomense (Oren 1983) was reclassified as Hrr. sodomense comb. nov. (McGenity and Grant 1995; Validation List 57, 1996).

• Hbt. trapanicum (Petter 1931) (Elazari-Volcani 1957) was reclassified as Hrr. trapanicum comb. nov. (McGenity and Grant 1995).

• Hbt. vallismortis (Gonzalez et al. 1978) was reclassified as Har. vallismortis comb. nov. (Torreblanca et al. 1986).

Genus Haladaptatus Savage, Krumholz, Oren, and Elshahed 2007, 23^VP; emend. Cui, Sun, Gao, Dong, Xu, Zhou, Liu, Oren, and Zhou 2010a, 1087; emend. Roh, Lee, and Bae 2010, 1189

Hal.a.dap.ta’tus. Gr. n. hals, halos, salt; L. part. adj. adaptatus, adapted to a thing; N.L. masc. n. Haladaptatus, a bacterium adapted to salt.

Cells are cocci or coccobacilli occurring singly or in pairs and do not lyse in distilled water. Some species possess more than one different 16S rRNA gene sequences. Grow on a wide range of substrates, including single and complex carbon sources. Acid is produced from carbohydrates. Grow at a wide range of NaCl concentrations. Cells contain PG and PGP-Me and two or three glycolipids may be present, one of which is chromatographically identical to S-DGD-1. The presence of PGS is variable.

The mol% G+C of the DNA is 54.0–60.5.

The genus Haladaptatus currently contains three species: Hap. paucihalophilus (type species), Hap. cibarius, and Hap. litoreus.

The main features of the members of the genus are summarized in Table 7.1.

Genus Halalkalicoccus Xue, Fan, Ventosa, Grant, Jones, Cowan, and Ma 2005, 2504^VP

Hal.al.ka.li.coc’cus. Gr. n. hals, halos, salt; Arabic n. alkali (al-qaliy), the ashes of saltwort; N.L. masc. coccus (from Gr. masc. n. kokkos, grain, seed), coccus; N.L. masc. n. Halalkalicoccus, coccus existing in salted and alkaline environment.

Cells are cocci occurring singly, in pairs, or irregular clusters. Stain mainly Gram negative with some cells Gram positive in young cultures. Cells do not lyse in distilled water. Alkaliphilic. Possesses C₂₀C₂₀ and C₂₀C₂₅ diethers. No glycolipids or PGS detected. Isoprenoid quinones are MK-8 and MK-8(H₂).

The mol% G+C of the DNA is 61.5–63.2.

The genus Halalkalicoccus currently contains two species: Hac. tibetensis (type species) and Hac. jeotgali.

The main features of the members of the genus are summarized in Table 7.1.

Genus Halarchaeum Minegishi, Echigo, Nagaoka, Kamekura, and Usami 2010, 2515^VP

Hal.ar.chae’um. Gr. n. hals, halos, salt; N.L. neut. n. archaeum (from Gr. adj. archaios, ancient), ancient one, archaeon; N.L. neut. n. Halarchaeum, a saline archaeon.

Cells are nonmotile, pleomorphic, with triangular and disk morphology. Lipids are C₂₀C₂₀ and C₂₀C₂₅ derivatives of PG and PGP-Me, and four unidentified glycolipids. Cells lyse in distilled water. Cells grow on a wide range of substrates, including simple and complex carbon sources.

The mol% G+C of the DNA is 61.4.

Type species and currently only species: Hla. acidiphilum.

The main features of the members of the genus are summarized in Table 7.2.

Genus Haloarchaeobius Makhdoumi-Kakhki, Amoozegar, Bagheri, Ramezani, and Ventosa 2012, 1024^VP

Ha.lo.ar.chae.o’bi.us. Gr. n. hals, halos, salt; N.L. adj. archaeos from Gr. adj. archaios ancient; N.L. masc. n. bius from Gr. masc. n. bios life; N.L. masc. n. Haloarchaeobius, halophilic ancient (archaeal) life.

Cells are motile strictly aerobic rods, pigmented orange-red. Neutrophilic and mesophilic. Magnesium is not required for growth. Polar lipids include PG, PGP-Me, PGS, three unidentified glycolipids, and one minor phospholipid. MK-8(II-H₂) is the only respiratory lipoquinone present.

The mol% G+C of the DNA is 67.7.

Type species and currently only species: Hab. iranensis.

The main features of the members of the genus are summarized in Table 7.2.

Genus Haloarcula Torreblanca, Rodriguez-Valera, Juez, Kamekura, and Kates 1986b, 573^VP (Validation list 22); Effective Publication: Torreblanca, Rodriguez-Valera, Juez, Kamekura, and Kates 1986a, 98; emend. Oren, Arahal, and Ventosa 2009, 638

Ha.lo.ar’cu.la. Gr. n. hals, halos, salt; L. fem. n. arcula small box; N.L. fem. n. Haloarcula, salt (-requiring) small box.

Cells are extremely pleomorphic and lyse in distilled water. Irregular disks, flat triangles, and other irregular shapes are commonly found. Some species are motile. Magnesium requirement is moderate (5–50 mM). Amino acids are not required for growth. The optimum salt concentration for growth is 2–3 M NaCl. Neutrophilic. Characteristic lipids are PG, Me-PGP, and a triglycosyl diether lipid (S-TGD-2).

The mol% G+C of the DNA is 60.1–64.7.

The genus Haloarcula currently contains 9 species: Har. vallismortis (type species), Har. amylolytica, Har. argentinensis, Har. hispanica, Har. japonica, Har. marismortui, Har. quadrata, Har. salaria, and Har. tradensis.

The main features of the members of the genus are summarized in Table 7.3.

Additional comments:

• Har. mukohataei (Ihara et al. 1997) has been transferred to the genus Halomicrobium as Hmc. mukohataei comb. nov. (Oren et al. 2002).

Genus Halobaculum Oren, Gurevich, Gemmell, and Teske 1995, 752^VP

Ha.lo.ba’cu.lum. Gr. n. hals, halos, salt; L. neut. n. baculum stick; N.L. neut. n. Halobaculum, salt stick.

Cells are motile rods of varying length and lyse in distilled water. Magnesium requirement is moderate (5–50 mM). Amino acids are required for growth. The optimum salt concentration for growth is 3.5–4.5 M NaCl. Neutrophilic. Characteristic lipids are PG, Me-PGP, and a sulfated diglycosyl diether. PGS is absent.

The mol% G+C of the DNA is 70.

Type species and currently only species: Hbl. gomorrense.

The main features of the members of the genus are summarized in Table 7.2.

Additional comment:

• The description of Hbl. magnesiiphilum, a species that grows optimally at 5 % NaCl only and can grow at salt concentrations as low as 1 % is currently in press (Shimoshige et al. 2012).

Genus Halobellus Cui, Yang, Gao, and Xu 2011d, 2687^VP

Ha.lo.bel’lus. Gr. n. hals, halos, salt; L. masc. adj. bellus, beautiful; N.L. masc. n. Halobellus, beautiful salt organism.

Cells are rod shaped under optimal growth conditions and lyse in distilled water. Sugars are metabolized, in some cases with formation of acids. The major polar lipids are PG, PGP-Me, PGS, and one major glycolipid chromatographically identical to S-DGD-1.

The mol% G+C of the DNA is 61.5–69.2.

The genus Halobellus currently contains three species: Hbs. clavatus (type species), Hbs. limi, and Hbs. salinus.

The main features of the members of the genus are summarized in Table 7.2.

Genus Halobiforma Hezayen, Tindall, Steinbüchel, and Rehm 2002, 2278^VP; emend. Oren, Arahal, and Ventosa 2009, 640

Ha.lo.bi.for’ma. Gr. n. hals, halos, salt; L. adv. num. bis, twice; L. fem. n. forma, form; N.L. fem. n. Halobiforma, the halophile with two different shapes.

Cells are rod shaped, coccoid or pleomorphic, motile. Cells are red or pink and lyse in distilled water. Neutrophilic or alkaliphilic with growth up to pH 10.5. Grow by aerobic respiration; some species grow also by anaerobic respiration in the presence of nitrate. No growth on single substrates. Some species produce acids from sugars. The major polar lipids are C₂₀C₂₀ and C₂₀C₂₅ glycerol diether derivatives of PG and PGP-Me. Glycolipids may be present in some species; when present, the glycolipids are a triglycosyl diether and its sulfated derivative.

The mol% G+C of the DNA is 63.8–66.9.

The genus Halobiforma currently contains three species: Hbf. haloterrestris (type species), Hbf. lacisalsi, and Hbf. nitratireducens.

The main features of the members of the genus are summarized in Table 7.4.

Genus Halococcus Schoop 1935, 817^AL; emend. Oren, Arahal, and Ventosa 2009, 639

Ha.lo.coc’cus. Gr. n. hals, halos, salt; N.L. masc. n. coccus (from Gr. masc. n. kokkos a berry), coccus; N.L. masc. n. Halococcus, salt (-requiring) coccus.

Cells are coccoid, nonmotile, occurring in pairs, tetrads, or irregular clusters. Most cells stain Gram negative. Do not lyse in distilled water. Oxidase positive or negative. Magnesium requirement is moderate (1–40 mM). The optimum salt concentration for growth is 3.5–4.5 M NaCl. Neutrophilic. Some species require amino acids for growth. Possess both C₂₀C₂₀ and sometimes C₂₀C₂₅ core lipids. Characteristic lipids are PG, Me-PGP, and a sulfated diglycosyl diether.

The mol% G+C of the DNA is 59.5–66.

The genus Halococcus currently contains seven species: Hcc. morrhuae (type species), Hcc. dombrowskii, Hcc. hamelinensis, Hcc. qingdaonensis, Hcc. saccharolyticus, Hcc. salifodinae, and Hcc. thailandensis.

The main features of the members of the genus are summarized in Table 7.5.

Additional comments:

• Hcc. turkmenicus (Zvyagintseva and Tarasov 1987; Validation List 31, 495, 1989) was reclassified as Htg. turkmenica comb. nov. (Ventosa et al. 1999).

Genus Haloferax Torreblanca, Rodriguez-Valera, Juez, Kamekura, and Kates 1986b, 573^VP (Validation list 22); Effective Publication: Torreblanca, Rodriguez-Valera, Juez, Kamekura, and Kates 1986a, 98; emend. Oren, Arahal, and Ventosa 2099, 639

Ha.lo.fe’rax. Gr. n. hals, halos, salt; L. neut. adj. ferax fertile; N.L. neut. n. Haloferax, salt (-requiring) and fertile.

Cells are extremely pleomorphic and lyse in distilled water. Flat disks and pleomorphic rods are commonly found. Colonies have a mucoid appearance. Pigmentation often depends on the salinity of the medium. Some species are motile; some possess gas vesicles. Most species are oxidase positive, but oxidase-negative and oxidase-variable species have been reported. Magnesium requirement is high (20–50 mM). Amino acids are not required for growth. Acids are produced from sugars. The optimum salt concentration for growth is 2–3 M NaCl. Neutrophilic. Characteristic lipids are PG, Me-PGP, and a sulfated diglycosyl diether. PGS is absent.

The mol% G+C of the DNA is 59.5–66.3.

The genus Haloferax currently contains 11 species: Hfx. volcanii (type species), Hfx. alexandrinus, Hfx. denitrificans, Hfx. elongans, Hfx. gibbonsii, Hfx. larsenii, Hfx. lucentense, Hfx. mediterranei, Hfx. mucosum, Hfx. prahovense, and Hfx. sulfurifontis.

The main features of the members of the genus are summarized in Tables 7.6 and 7.7.

Additional comments:

• The name Hfx. alexandrinus is illegitimate because the epithet must be in the neuter gender (alexandrinum).

• The original spelling of the specific epithet lucentensis (Gutierrez et al. 2002) has been corrected to lucentense on validation.

Genus Halogeometricum Montalvo-Rodríguez, Vreeland, Oren, Kessel, Betancourt, and López-Garriga 1998, 1310^VP; emend. Cui, Yang, Gao, Li, Xu, Zhou, Liu, and Zhou 2010f, 2615

Ha.lo.ge.o.me’tri.cum. Gr. n. hals, halos, salt; L. neut. adj. geometricum geometrical; N.L. neut. n. Halogeometricum, salty geometrical shape.

Cells are extremely pleomorphic (short and long rods, squares, triangles, ovals, and irregular cocci) under optimal growth conditions, motile, and lyse in distilled water. Sugars are metabolized, in some cases with formation of acids. Neutrophilic. Cells contain PG and Me-PGP. In some species, a yet unidentified non-sulfate-containing glycolipid and S-DGD-1 may be present as a minor component. In other species, the major glycolipid is chromatographically identical to S-DGD-1, and DGD-1 may be present as a minor component. PGS is absent.

The mol% G + C of the DNA is 59.9–64.9

The genus Halogeometricum currently contains two species: Hgm. borinquense (type species), and Hgm. rufum.

The main features of the members of the genus are summarized in Table 7.4.

Genus Halogranum Cui, Gao, Sun, Dong, Xu, Zhou, Liu, Oren, and Zhou 2010b, 1369^VP, emend. Cui, Yang, Gao, and Xu 2011e, 913

Ha.lo.gra’num. Gr. n. hals, halos, salt; L. neut. n. granum, granule; N.L. neut. n. Halogranum, salty granule shape.

Cells are pleomorphic under optimal growth conditions and lyse in distilled water. Sugars are metabolized with the formation of acids. The polar lipids are PG, PGP-Me, traces of PGS, and one major glycolipid and one minor glycolipid chromatographically identical to S-DGD-1 and DGD-1, respectively. Other minor glycolipids may be present.

The mol% G + C of the DNA is 55.7–64.4.

The genus Halogranum currently contains 4 species: Hgn. rubrum (type species), Hgn. amylolyticum, Hgn. gelatinilyticum, and Hgn. salarium.

The main features of the members of the genus are summarized in Table 7.4.

Genus Halolamina Cui, Gao, Yang, and Xu 2011b, 1619^VP

Ha.lo.la’mi.na. Gr. n. hals, halos, salt; L. fem. n. lamina, a thin slice; N.L. fem. n. Halolamina, thin-slice-shaped salt (organism).

Cells are pleomorphic and thin-slice-shaped. Sugars are metabolized, sometimes with the formation of acids. Polar lipids include PGS and 8 yet uncharacterized glycolipids.

The mol% G + C of the DNA is 64.8.

Type species and currently only species: Hlm. pelagica.

The main features of the members of the genus are summarized in Table 7.7.

Genus Halomarina Inoue, Itoh, Ohkuma, and Kogure 2011, 944^VP

Ha.lo.ma.ri’na. Gr. n. hals, halos, salt; L. adj. marinus, marine; N.L. fem. n. Halomarina, a halophile existing in the marine environment.

Cells are mesophilic and neutrophilic. Lipids are C₂₀C₂₀ and C₂₀C₂₅ diether derivatives of PG, PGP-Me, triglycosyl diether, and at least one unidentified glycolipid. Grows on a wide range of substrates, including single and complex carbon sources. Survives at low salt concentrations and can recover after prolonged exposure to distilled water.

The mol% G + C of the DNA is 67.7.

Type species and currently only species: Hmr. oriensis.

The main features of the members of the genus are summarized in Table 7.8.

Genus Halomicrobium Oren, Elevi, Watanabe, Tamura, Ihara, and Corcelli 2002, 1834^VP

Ha.lo.mi.cro’bi.um. Gr. n. hals, halos, salt; N.L. neut. n. microbium (from Gr. adj. micros, small and Gr. n. bios, life), a microbe; N.L. neut. n. Halomicrobium, small, salt life-form.

Cells are rod shaped or pleomorphic, aerobic, or facultatively anaerobic in the presence of nitrate. Some species are motile.

The mol% G + C of the DNA is 52.4–69.1.

The genus Halomicrobium currently contains three species: Hmc. mukohataei (type species), Hmc. katesii, and Hmc. zhouii.

The main features of the members of the genus are summarized in Table 7.8.

Additional comment:

• Har. mukohataei (Ihara et al. 1997) has been transferred to the genus Halomicrobium as Hmc. mukohataei comb. nov. (Oren et al. 2002).

Genus Halonotius Burns, Janssen, Itoh, Kamekura, Echigo, and Dyall-Smith 2010, 1198^VP

Ha.lo.no’ti.us. Gr. n. hals, halos, salt; L. masc. n. notius, southern; N.L. masc. n. Halonotius, a salty southern one.

Cells are flat rods, often with rounded ends. Oxidase and catalase tests are negative.

The mol% G + C of the DNA is 58.4–58.7.

Type species and currently only species: Hns. pteroides.

The main features of the members of the genus are summarized in Table 7.8.

Genus Halopelagius Cui, Li, Gao, Xu, Zhou, Liu, Oren, and Zhou 2010g, 2092^VP

Ha.lo.pe.la’gi.us. Gr. n. hals, halos, salt; L. masc. adj. pelagius, of or pertaining to the sea; N.L. masc. n. Halopelagius, salt organism from the sea.

Cells are pleomorphic under optimal growth conditions and lyse in distilled water. Sugars are metabolized, in some cases with formation of acid. Lipids are PG, PGP-Me, and two main glycolipids chromatographically identical to S-DGD-1 and DGD-1. PGS is absent.

The mol% G + C of the DNA is 59.9–61.0.

Type species and currently only species: Hpl. inordinatus.

The main features of the members of the genus are summarized in Table 7.8.

Genus Halopenitus Amoozegar, Makhdoumi-Kakhki, Shahzedeh Fazeli, Azarbaijani, and Ventosa 2012, 1935^VP

Ha.lo.pe’ni.tus. Gr. n. hals, halos, salt; L. masc. adj. penitus inner, interior; N.L. masc. n. Halopenitus, intended to mean an archaeon isolated from an inland salt lake.

Cells are pleomorphic rods, triangular, or disk-shaped, nonmotile. Neutrophilic. Polar lipids include PG, PGP-Me, one unidentified glycolipid, and three minor phospholipids.

The mol% G + C of the DNA is 66.0.

Type species and currently only species: Hpt. persicus.

The main features of the members of the genus are summarized in Table 7.9.

Genus Halopiger Gutiérrez, Castillo, Kamekura, Xue, Ma, Cowan, Jones, Grant, and Ventosa 2007, 1404^VP

Ha.lo.pi’ger. Gr. n. hals, halos, salt; L. masc. adj. piger, lazy; N.L. masc. n. Halopiger, lazy halophile, referring to the slow growth under laboratory conditions.

Cells are strictly anaerobic pleomorphic rods. Polar lipids include C₂₀C₂₀ and C₂₀C₂₅ glycerol diethers of PG, PGP-Me, and the bis-sulfated glycolipid S₂-DGD-1. PGS is absent.

The mol% G + C of the DNA is 65.2–67.1.

The genus Halopiger currently contains two species: Hpg. xanaduensis (type species) and Hpg. aswanensis.

The main features of the members of the genus are summarized in Table 7.9.

Genus Haloplanus Elevi Bardavid, Mana, and Oren 2007, 782^VP; emend. Cui, Gao, Li, Xu, and Zhou 2010c, 1826

Ha.lo.pla’nus. Gr. n. hals, halos, salt; L. adj. planus, flat; N.L. masc. n. Haloplanus, flat salt-life form.

Cells are pleomorphic and flat and contain gas vesicles. In static liquid culture, cells float to the surface. Strictly aerobic. Cells lyse in distilled water. Cells contain PG, PGP-Me, PGS, and one major glycolipid that is chromatographically identical to S-DGD-1.

The mol% G + C of the DNA is 62.1–66.4.

The genus Haloplanus currently contains three species: Hpn. natans (type species), Hpn. aerogenes, and Hpn. vescus.

The main features of the members of the genus are summarized in Table 7.9.

Genus Haloquadratum Burns, Janssen, Itoh, Kamekura, Li, Jensen, Rodríguez-Valera, Bolhuis, and Dyall-Smith 2007, 391^VP

Ha.lo.qua.dra’tum. Gr. n. hals, halos, salt; L. neut. n. quadratum, square; N.L. neut. n. Haloquadratum, salt square.

Cells are flat and square and usually contain gas vesicles and PHA storage granules. Oxidase and catalase tests are negative.

The mol% G + C of the DNA is 46.9–47.9.

Type species and currently only species: Hqr. walsbyi.

The main features of the members of the genus are summarized in Table 7.9.

Genus Halorhabdus Wainø, Tindall, and Ingvorsen 2000, 188^VP; emend. Antunes, Taborda, Huber, Moissl, Nobre, and Da Costa 2008, 218

Ha.lo.rhab.dus. Gr. n. hals, halos, salt; Gr. fem. n. rhabdos, rod, stick; N.L. fem. n. Halorhabdus, salt (-loving) rod.

Cells are extremely pleomorphic, although most are rod shaped. Pigmented red or unpigmented. Motile by a single flagellum or nonmotile. Cells lyse in distilled water. Ferments glucose. Amino acids are not required for growth; grows under aerobic or anaerobic conditions in defined media; some species prefer anaerobic conditions. PHA is produced. Acid is produced from carbohydrates. A limited number of organic substrates are used for growth. The polar lipids are PG, PGP-Me, TGD, S-TGD, and an unknown component. PGS is absent. MK-8 and MK-8(VIII-H₂) are the respiratory lipoquinones.

The mol% G + C of the DNA is 62.0–64.0.

The genus Halorhabdus currently contains two species: Hrd. utahensis (type species), and Hrd. tiamatea.

The main features of the members of the genus are summarized in Table 7.10.

Genus Halorientalis Cui, Yang, Gao, and Xu 2011d, 2687^VP

Hal.o.ri.en.ta’lis. Gr. n. hals, halos, salt; L. fem. adj. orientalis, of the east; N.L. fem. n. Halorientalis, salt-loving organism from the orient.

Cells are pleomorphic and rod shaped under optimal growth conditions and lyse in distilled water. Sugars are metabolized, in some cases with the formation of acids. The polar lipids are PG, PGP-Me, one major glycolipid, chromatographically identical to S-DGD-1, and 3–4 minor unidentified glycolipids.

The mol% G + C of the DNA is 61.5–61.9.

Type species and currently only species: Hos. regularis.

The main features of the members of the genus are summarized in Table 7.10.

Genus Halorubrum McGenity and Grant 1996, 362^VP; Effective Publication: McGenity and Grant 1995, 241; emend. Oren, Arahal, and Ventosa 2009, 639

Ha.lo.ru’brum. Gr. n. hals, halos, salt; L. neut. adj. rubrum red; N.L. neut. n. Halorubrum, salt (-requiring) and red.

Cells are rod shaped or pleomorphic under optimal growth conditions, motile or nonmotile, and lyse in distilled water. Short and long rods, triangles, squares, and oval cells are found. Some species may be almost colorless. Some species possess gas vesicles. Magnesium requirement is moderate (5–50 mM) or low. Amino acids are not required for growth. Some species grow on single carbon sources. Most species use sugars, some with the production of acids. The optimum salt concentration for growth is 2.5–4.5 M NaCl. Neutrophilic and alkaliphilic species exist. The major polar lipids are C₂₀C₂₀ or C₂₀C₂₀ and C₂₀C₂₅ glycerol diether derivatives of PG, PGP-Me, PGS, and a sulfated diglycosyl diether. Alkaliphilic species lack PGS and glycolipids.

The mol% G + C of the DNA is 60.2–71.2.

The genus Halorubrum currently contains 26 species: Hrr. saccharovorum (type species), Hrr. aidingense, Hrr. alkaliphilum, Hrr. aquaticum, Hrr. arcis, Hrr. californiense, Hrr. chaoviator, Hrr. cibi, Hrr. coriense, Hrr. distributum, Hrr. ejinorense, Hrr. ezzemoulense, Hrr. kocurii, Hrr. lacusprofundi, Hrr. lipolyticum, Hrr. litoreum, Hrr. luteum, Hrr. orientale, Hrr. sodomense, Hrr. tebenquichense, Hrr. terrestre, Hrr. tibetense, Hrr. trapanicum, Hrr. vacuolatum, and Hrr. xinjiangense.

The main features of the members of the genus are summarized in Table 7.10.

Additional comments:

• Halorubrobacterium (Kamekura and Dyall-Smith 1995) (Validation list 57, 1996) is a later synonym of Halorubrum (McGenity and Grant 1995). Halorubrobacterium coriense, Halorubrobacterium distributum, and Halorubrobacterium sodomense were transferred to Halorubrum (Oren and Ventosa 1996).

• Strain VKM B-1733 was originally designated as the type strain of Hrr. distributum (Zvyagintseva and Tarasov 1987). Zvyagintseva et al. (1996) later proposed VKM B-1739 as the new type. However, strain VKM B-1733 and JCM 9100 which was derived from it must remain the type strain of the species (Oren et al. 1997).

• NCIMB 13488 was proposed as a neotype of Hrr. trapanicum (basonym: Hbt. trapanicum) as the original isolate is no longer available (Grant et al. 1998). However, the Judicial Commission of the ICSP ruled that strain NCIMB 13488 is derived from strain NRC 34021, which in turn is derived from Petter’s original isolate (Judicial Commission 2003). Therefore NCIMB 13488 may serve as the type strain of the species.

• The name Hrr. sfaxense (Trigui et al. 2011) has been effectively but not yet validly published. The species was therefore not included in Table 7.11, 7.12, and 7.13.

Genus Halosarcina Savage, Krumholz, Oren, and Elshahed 2008, 859^VP; emend. Cui, Gao, Li, Xu, Zhou, Liu, and Zho 2010d, 2464

Ha.lo.sar.ci’na. Gr. n. hals, halos, salt; L. fem. n. sarcina a package; N.L. fem. n. Halosarcina, a salt (-loving) package.

Cells are cocci (sarcina-like clusters) or pleomorphic (rods and deformed cocci) under optimal growth conditions. Cells are motile or nonmotile and lyse in distilled water. Sugars are metabolized, sometimes with formation of acids. Polar lipids are PG and PGP-Me; PGS is absent. The major glycolipid is chromatographically identical to S-DGD-1. DGD-1 or another glycolipid may be present in some species as a minor component.

The mol% G + C of the DNA is 61.2–65.4.

The genus Halosarcina currently contains two species: Hsn. pallida (type species) and Hsn. limi.

The main features of the members of the genus are summarized in Table 7.10.

Genus Halosimplex Vreeland, Rosenzweig, Straight, Krammes, Dougherty, and Kamekura 2002, 450 (Validation list 92, 2003, 936^VP)

Ha.lo.sim’plex. Gr. n. hals, halos, salt; L. adj. simplex; simple, uncomplicated; N.L. neut. n. Halosimplex, the simple halophile.

Cells are rod shaped or pleomorphic, pink to red. Cannot use nitrate or other alternate electron acceptors. Neutrophilic and mesophilic. Extremely fastidious: grows only on pyruvate, pyruvate plus glycerol, or glycerol plus acetate as carbon sources in defined medium. Unable to grow on any other organic carbon compounds tested. Lipids are PG, PGP-Me, and four sulfated glycolipids, two of which have been identified as TeGD and S₂-DGD.

The mol% G + C of the DNA is 64.4.

Type species and currently only species: Hsx. carlsbadense.

The main features of the members of the genus are summarized in Table 7.10.

Genus Halostagnicola Castillo, Gutiérrez, Kamekura, Xue, Ma, Cowan, Jones, Grant, and Ventosa 2006, 1521^VP

Ha.lo.stag.ni’co.la. Gr. n. hals, halos, salt; L. neut. n. stagnum, a piece of standing water, pond, lake; L. suff. –cola (from L. n. incola), inhabitant, dweller; N.L. fem. n. Halostagnicola, a dweller of a saline lake.

Cells are pleomorphic, although most are rod shaped. Strictly aerobic. Polar lipids include C₂₀C₂₀ and C₂₀C₂₅ diethers of PG, PGP-Me, and two unidentified glycolipids.

The mol% G + C of the DNA is 59.8–61.0.

The genus Halostagnicola currently contains three species: Hst. larsenii (type species), Hst. alkaliphila, and Hst. kamekurae.

The main features of the members of the genus are summarized in Table 7.15.

Genus Haloterrigena Ventosa, Gutiérrez, Kamekura, and Dyall-Smith 1999b, 135^VP; emend. Oren, Arahal, and Ventosa 2009, 640

Ha.lo.ter.ri’ge.na. Gr. n. hals, halos, salt; L. fem. adj. terrigena born from the earth; N.L. fem. n. Haloterrigena, salt (-requiring) and born from the earth.

Cells are coccoid or oval or rod shaped under optimal growth conditions. Some species become coccoid in stationary cultures. Cells lyse in distilled water. The optimum salt concentration for growth is 3.5–4.5 M NaCl. Magnesium requirement is moderate (5–50 mM) or low. Amino acids are required for growth. Some species grow on single carbon sources. Most species use sugars, some with the production of acids. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. Characteristic lipids are PG, Me-PGP, and a glycolipid (S₂-DGD in most species or S-DGD). PGS is absent.

The mol% G + C of the DNA is 59.3–67.0.

Type species: Haloterrigena turkmenica.

The genus Haloterrigena currently contains nine species: Htg. turkmenica (type species), Htg. daqingensis, Htg. hispanica, Htg. jeotgali, Htg. limicola, Htg. longa, Htg. saccharevitans, and Htg. salina.

The main features of the members of the genus are summarized in Table 7.14.

Additional comment:

• Phylogenetically the genera Haloterrigena and Natrinema are not well separated (Tindall 2003; see also Fig. 7.1). Based on other phylogenetic markers such as the RNA polymerase subunit B′ (rpoB′) gene, the genera Natrinema (McGenity et al. 1998) and Haloterrigena (Ventosa et al. 1999) might constitute a single genus (Minegishi et al. 2010a).

Genus Halovenus Makhdoumi-Kakhki, Amoozegar, and Ventosa 2012a, 1334^VP

Ha.lo.ve’nus. Gr. n. hals, halos, salt; L. fem. n. venus beauty, grace, elegance; N.L. fem. n. Halovenus, a salt-loving beauty, reflecting the attractive appearance of colonies.

Cells are nonmotile and pleomorphic (rods to triangles, squares, or disk shaped) and lyse in distilled water. Strictly aerobic, growing on a wide range of substrates, including single and complex carbon sources. Polar lipids are PG, PGP-Me, and two minor phospholipids. MK-8(II-H₂) is the only lipoquinone present.

The mol% G + C of the DNA is 61.0.

Type species and currently only species: Hvn. aranensis.

The main features of the members of the genus are summarized in Table 7.15.

Genus Halovivax Castillo, Gutiérrez, Kamekura, Ma, Cowan, Jones, Grant, and Ventosa 2006, 767^VP

Ha.lo.vi’vax. Gr. n. hals, halos, salt; L. adj. vivax, long-lived, tenacious of life; N.L. masc. n. Halovivax, long-living halophile.

Cells are extremely pleomorphic, although most are rod shaped. Colonies are pale pink pigmented. Strictly aerobic. Polar lipids include PG, PGP-Me, two major and one minor glycolipids similar to those of Nnm. pellirubrum, and an unidentified glycolipid.

The mol% G + C of the DNA is 60.3–65.0.

The genus Halovivax currently contains two species: Hvx. asiaticus (type species) and Hvx. ruber.

The main features of the members of the genus are summarized in Table 7.15.

Genus Natrialba Kamekura and Dyall-Smith 1996, 625^VP; Effective Publication: Kamekura and Dyall-Smith 1995, 347 (Validation list 57); emend. Oren, Arahal, and Ventosa 2009, 640

Na.tri.al’ba. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; L. fem. adj. alba, white; N.L. fem. n. Natrialba, sodium white, referring to the high sodium ion requirement and the pigmentless colonies of the type species.

Cells are rods, cocci, or coccobacilli, sometimes occurring in tetrads. Some species lack pigmentation, while others are pigmented red by bacterioruberin carotenoids. Cells lyse in distilled water. Salt concentration for growth is 1.6–5.3 M NaCl. Neutrophilic or alkaliphilic with growth up to pH 10.5–11. Magnesium requirement is moderate (5–50 mM) or low. No growth on single substrates. Neutrophilic species produce acids from sugars. Amino acids are required for growth. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. Polar lipids are PG and PGP-Me. Neutrophilic species contain S₂-DGD in addition. Unidentified phospholipids found in alkaliphilic species. Glycolipids are absent in alkaliphilic species.

The mol% G + C of the DNA is 61.5–64.3 CHECK.

The genus Natrialba currently contains six species: Nab. asiatica (type species), Nab. aegyptiaca, Nab. chagannaoensis, Nab. hulunbeirensis, Nab. magadii, and Nab. taiwanensis.

The main features of the members of the genus are summarized in Table 7.16.

Additional comments:

• Nab. taiwanensis was originally described as a strain of Nab. asiatica (Hezayen et al. 2001).

• The name Nab. aegyptiaca was changed to Nab. aegyptia by the List Editor of Int. J. Syst. Evol. Microbiol. (Notification List, Int. J. Syst. Evol. Microbiol. 51, 1233, 2001), but Nab. aegyptiaca is correct as well. The International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteriaceae recommends use of Nab. aegyptiaca.

Genus Natrinema McGenity, Gemmell, and Grant 1998, 1194^VP

Na.tri.ne’ma. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; Gr. neut. n. nema, a thread; N.L. neut. n. Natrinema, sodium (-requiring) thread.

Cells are rods of varying length that lyse in distilled water. The optimum salt concentration for growth is 3.4–4.3 M NaCl. Magnesium requirement is moderate (5–50 mM) or low. Amino acids are required for growth. Neutrophilic and slightly alkaliphilic species exist. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids, as well as several unidentified glycolipids.

The mol% G + C of the DNA is 64.2–69.9.

The genus Natrinema currently contains 6 species: Nnm. pellirubrum (type species), Nnm. altunense, Nnm. ejinorense, Nnm. gari, Nnm. pallidum, Nnm. pellirubrum, and Nnm. versiforme.

The main features of the members of the genus are summarized in Table 7.17.

Additional comments:

• Phylogenetically the genera Haloterrigena and Natrinema are not well separated (Tindall 2003; see also Fig. 7.1). Based on other phylogenetic markers such as the RNA polymerase subunit B′ (rpoB′) gene, the genera Natrinema (McGenity et al. 1998) and Haloterrigena (Ventosa et al. 1999) might constitute a single genus (Minegishi et al. 2010a).

Genus Natronoarchaeum Shimane, Hatada, Minegishi, Mizuki, Echigo, Miyazaki, Ohta, Usami, Grant, and Horikoshi 2010, 2532^VP

Na.tro.no.ar.chae’um. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. neut. n. archaeum (from Gr. adj. archaios, ancient), ancient one, archaeon; N.L. neut. n. Natronoarchaeum, the soda archaeon.

Cells are nonmotile and extremely pleomorphic. Aerobic and slightly alkaliphilic. The major polar lipids are PG, PGP-Me, and a disulfated diglycosyl diether (S₂-DGD).

The mol% G + C of the DNA is 63.

Type species and currently only species: Nac. mannanilyticum.

The main features of the members of the genus are summarized in Table 7.18.

Genus Natronobacterium Tindall, Ross, and Grant 1984b, 355^VP; Effective Publication: Tindall, Ross, and Grant 1984a, 41 (Validation list 15)

Na.tro.no.bac.te’ri.um. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. pref. natrono-, pertaining to soda; L. neut. n. bacterium, a small rod; N.L. neut. n. Natronobacterium, soda rod.

Cells are rods of varying length that lyse in distilled water. Motile or nonmotile. The optimum salt concentration for growth is 3.5–4.5 M NaCl. Alkaliphilic, with a very low magnesium requirement. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. Unidentified phospholipids are present; glycolipids and PGS are absent.

The mol% G + C of the DNA is 65.

Type species and currently only species: Nbt. gregoryi.

The main features of the members of the genus are summarized in Table 7.18.

Additional comments:

• Nbt. magadii (Tindall et al. 1984) was transferred to the genus Natrialba as Nab. magadii comb. nov. (Kamekura et al. 1997).

• Nbt. nitratireducens (Xin et al. 2001) was transferred to the genus Halobiforma as Hbf. nitratireducens comb. nov. (Hezayen et al. 2002).

• Nbt. pharaonis (Soliman and Trüper 1982) was transferred to the genus Natronomonas as Nmn. pharaonis comb. nov. (Kamekura et al. 1997).

• Nbt. vacuolatum (Mwatha and Grant 1993) was transferred to the genus Halorubrum as Hrr. vacuolatum comb. nov. (Kamekura et al. 1997).

Genus Natronococcus Tindall, Ross, and Grant 1984b, 355^VP; Effective Publication: Tindall, Ross, and Grant 1984a, 41

Na.tro.no.coc’cus. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. pref. natrono-, pertaining to soda; N.L. masc. n. coccus (from Gr. n. kokkos, grain, seed), coccus; N.L. masc. n. Natronococcus, soda berry.

Cells are coccoid, nonmotile, occurring in pairs, tetrads, or irregular clusters, and do not lyse in distilled water. Alkaliphilic, with a very low magnesium requirement. The optimum salt concentration for growth is 3.0–4.0 M NaCl. Neutrophilic. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. Unidentified phospholipids are present; glycolipids and PGS are absent.

The mol% G + C of the DNA is 63.5–64.0

Type species: Natronococcus occultus.

The genus Natronococcus currently contains three species: Ncc. occultus (type species), Ncc. amylolyticus, and Ncc. jeotgali.

The main features of the members of the genus are summarized in Table 7.18.

Genus Natronolimnobius Itoh, Yamaguchi, Zhou, and Takashina 2005, 1744^VP (Validation list no. 105); Effective Publication: Itoh, Yamaguchi, Zhou, and Takashina 2005, 114

Na.tro.no.lim.no’bi.us. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. pref. natrono-, pertaining to soda; Gr. n. limnos, a pool of standing water, lake; Gr. masc. n. bios, life; N.L. masc. n. Natronolimnobius, organism living in a soda lake.

Cells are rod shaped or pleomorphic flat shaped and strictly aerobic. Cells lyse in distilled water. Mesophilic or thermotolerant. C₂₀C₂₀ and C₂₀C₂₅ core lipids are present; glycolipids are not detected.

The mol% G + C of the DNA is 59–63.

The genus Natronolimnobius currently contains two species: Nln. baerhuensis (type species) and Nln. innermongolicus.

The main features of the members of the genus are summarized in Table 7.19.

Genus Natronomonas Kamekura, Dyall-Smith, Upasani, Ventosa, and Kates 1997, 856^VP, emend. Burns, Janssen, Itoh, Minegishi, Usami, Kamekura, and Dyall-Smith 2010, 1175

Na.tro.no.mo’nas. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. pref. natrono-, pertaining to soda; L. fem. n. monas, monad, unit; N.L. fem. n. Natronomonas, the soda unit.

Cells are rods or pleomorphic shapes of varying length, motile, and lyse in distilled water. Amino acids are required for growth. Alkaliphilic or non-alkaliphilic. Alkaliphilic strains grow at pH 7–10, while non-alkaliphilic strains grow at pH 5.5–8.5. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. PG, PGP-Me, and phosphatidic acid. Unidentified phospholipids or glycolipids are present; PGS are absent, and alkaliphilic strains lack glycolipids.

The mol% G + C of the DNA is 61.2–64.3.

The genus Natronomonas currently contains two species: Nmn. pharaonis (type species) and Nmn. moolapensis.

The main features of the members of the genus are summarized in Table 7.19.

Additional comment:

• Nbt. pharaonis (Soliman and Trüper 1982) was transferred to the genus Natronomonas as Nmn. pharaonis comb. nov. (Kamekura et al. 1997).

Genus Natronorubrum Xu, Zhou, and Tian 1999, 265^VP; emend. Cui, Tothy, Feng, Zhou, and Liu 2006, 1517; emend. Oren, Arahal, and Ventosa 2009, 641

Na.tro.no.ru’brum. N.L. neut. n. natron (arbitrarily derived from the Arabic n. natrun or natron) soda, sodium carbonate; N.L. pref. natrono-, pertaining to soda; L. neut. adj. rubrum, red; N.L. neut. n. Natronorubrum, the red of soda.

Cells are pleomorphic nonmotile rods or pleomorphic flat shaped, which lyse in distilled water. Cells are nonmotile or motile. Amino acids are required for growth. The optimum salt concentration for growth is 3.4–3.8 M NaCl. Alkaliphilic, with a very low magnesium requirement, or neutrophilic. Many sugars are utilized, sometimes with acid production. Possess both C₂₀C₂₀ and C₂₀C₂₅ core lipids. Unidentified phospholipids are present; glycolipids absent in some species; others may contain TGD-1 and additional unidentified glycolipids. PGS is absent.

The mol% G + C of the DNA is 59.9–62.5.

The genus Natronorubrum currently contains 5 species: Nrr. bangense (type species), Nrr. aibiense, Nrr. sediminis, Nrr. sulfidifaciens, and Nrr. tibetense.

The main features of the members of the genus are summarized in Table 7.20.

Genus Salarchaeum Shimane, Hatada, Minegishi, Echigo, Nagaoka, Miyazaki, Ohta, Maruyama, Usami, Grant, and Horikoshi 2011, 2269^VP

Sal.ar.chae’um. L. n. sal, salt; N.L. neut. n. archaeum (from Gr. adj. archaios, ancient), ancient one, archaeon; N.L. neut. n. Salarchaeum, salt-requiring archaeon.

Cells are motile short rods. Does not use sugars as a single carbon source. Slightly acidophilic. The major polar lipids are PG, PGP-Me, S-DGD-1, and five unidentified glycolipids.

The mol% G + C of the DNA is 64.

Type species and currently only species: Sar. japonicum.

The main features of the members of the genus are summarized in Table 7.19.

Isolation, Enrichment, and Maintenance Procedures

A variety of media have been recommended for the growth of different members of the Halobacteriaceae. Useful information can be found in the original papers with the species descriptions and in earlier reviews (Tindall 1991; Oren 2006). The web site of the Deutsche Sammlung von Mikroorganismen und Zellkulturen mbH (DSMZ, http://www.dmsz.de) lists many protocols for the preparation of media. Another useful online resource providing descriptions of growth media and laboratory procedures for use with the halophilic Archaea is the “Halohandbook” prepared by Dyall-Smith (2008) http://www.haloarchaea.com/resources/halohandbook/halohandbook_2008_v7.pdf). For solid media, higher than usual agar concentrations should be used as the high salt concentration of the medium interferes with the solidification of the agar. A concentration of 20 g agar/l generally gives satisfactory results. For the preparation of agar media for the haloalkaliphiles, the agar should be sterilized separately from the sodium carbonate and the other alkaline components of the media.

Media used differ greatly in total salt concentration, ionic composition (e.g., high magnesium concentrations of up to 0.8 M for species isolated from the Dead Sea), and pH (9.5 and higher for the alkaliphilic species, using media very low in divalent cation concentrations). Members of the Halobacteriaceae are generally grown in complex media containing high concentrations of yeast extract, casamino acids, and similar rich sources of nutrients. The use of media with high concentrations of peptides and amino acids reflects environments such as salted fish and hides from which many isolates were obtained. Not all members of the family prefer such rich media; the use of low-nutrient media and a restricted range of organic compounds has in the past decade led to the isolation of a number of interesting species such as Hqr. walsbyi and Hsx. carlsbadense. Notably the use of pyruvate, whether or not combined with the use of agarose instead of agar, has enabled the isolation of the elusive square flat Haloquadratum (Bolhuis et al. 2004; Burns et al. 2004a; Walsby 1980, 2005) and is one of the few substrates that enable growth of Hsx. carlsbadense (Vreeland et al. 2002).

Several brands of peptone, notably Bacto peptone (Difco), are unsuitable for the cultivation of members of the Halobacteriaceae as they cause lysis of the cells. The toxic factor present in Bacto peptone was identified as bile acids (Kamekura et al. 1988), known since 1956 to cause lysis of halophilic Archaea when present at very low concentrations (Dussault 1956a, b). Sugars may stimulate growth of many species. When adding sugars, proper buffering may be required to avoid acidification of the medium to values inhibitory for growth. Though light can be used as an energy source in species containing bacteriorhodopsin, no absolute requirement for light has been demonstrated for any strain, and all known members of the Halobacteriaceae grow well in the dark.

One of the key factors important when trying to recover as many species of Halobacteriaceae as possible as colonies on agar plates is the incubation time. Combined use of cultivation-dependent and cultivation-independent methods showed that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable. Out of the 1.2 × 10⁷ cells/ml detected by microscopy, up to 1.9 × 10⁶ were recovered as colonies on plates containing 0.01 % nutrient broth and salts after 8 weeks of incubation (Burns et al. 2004b). Drying out of agar plates with the formation of salt crystals on the surface of the agar may present a serious problem in view of the often long incubation times required for colonies to appear. Incubation and storage of petri dishes in plastic bags is then recommended.

Recovery of colonies of halophilic Archaea from samples collected from nature may sometimes be enhanced by the addition of natural brine from the sampling site and a whole cell extract of Halobacterium salinarum as a source of stimulatory growth factors (Wais 1988). For the selective isolation of Archaea from natural sources, inclusion of antibiotics such as penicillin or ampicillin has been recommended.

Few procedures have been described for the selective isolation of specific genera and species belonging to the family Halobacteriaceae. Halobacterium can be selectively enriched under anaerobic conditions in medium containing l-arginine (Oren and Litchfield 1999). Halococcus species may possibly be selectively isolated by suspension of the sample in medium with a salt concentration sufficiently low to kill other neutrophilic halophilic Archaea, followed by cultivation in a suitable high-salinity medium. Viable Halococcus cells could even be recovered from seawater: 2–35 Halococcus colonies were obtained from 5 l portions of Mediterranean seawater sampled 5 km off the coast of Spain (Rodriguez-Valera et al. 1979). Members of the genera Haloferax and Haloarcula grow on inorganic media amended with a suitable single carbon and energy source (Rodriguez-Valera et al. 1980).

Maintenance

Cold maintenance at −20 C to −70 C in 10–15 % glycerol + salts in stab cultures kept under liquid paraffin at 4–8 C are sometimes used for preservation of cultures of Halobacteriaceae. For long-term preservation, suspensions can be frozen in liquid nitrogen in salt solutions + 5 % DMSO or by lyophilization with the pre-dried milk method (Tindall 1991).

Chemotaxonomic Properties

The Halobacteriaceae show many interesting chemotaxonomic traits, part of which are connected to their phylogenetic affiliation with the archaeal domain.

Surface Layers

The non-coccoid representatives of the Halobacteriaceae possess an S-layer cell wall, whose main constituent is a high-molecular-weight glycoprotein. This glycoprotein cell wall is responsible for maintaining the native cell shape. The S-layer glycoprotein of Hbt. salinarum (molecular mass ∼120 kDa) consists of a 87 kDa core protein rich in acidic amino acids, containing attached acidic and neutral saccharide chains. The primary structure of the protein backbone and the mode of glycosylation vary among the species. The glycoprotein cell wall requires high NaCl concentrations for stability. Similar to most other proteins of halophilic Archaea (see below), the wall protein denatures when suspended in distilled water, and as a result, the cells of most species lyse in the absence of salt due to the denaturation and dissolution of the cell wall. In some species, relatively high concentrations of magnesium or other divalent cations are required in addition to high NaCl concentrations to maintain the structural stability of the glycoprotein cell wall.

Halococcus species possess a thick sulfated heteropolysaccharide cell wall that does not require high salt concentrations to maintain its rigidity. The polysaccharide wall of Hcc. morrhuae contains glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and different uronic acids; part of the sugar residues are sulfated (Schleifer et al. 1982). The coccoid Ncc. occultus also has a thick cell wall that retains its shape in the absence of salt. Its structure is unlike that of the cell wall polymer of Halococcus, and it consists of repeating units of a poly(l-glutamine) glycoconjugate (Niemetz et al. 1997).

Some species excrete exopolysaccharides that form a slime layers around the cells. This feature is especially prominent in some Haloferax species. The Hfx. mediterranei exopolysaccharide is built of glucose, mannose, and sulfated glucose units; the Hfx. gibbonsii polymer is composed of mannose, galactose, glucose, and rhamnose. Hfx. denitrificans has an exopolysaccharide composed of 2,3-diacetamido-2,3-dideoxy-d-glucopyranosiduronic acid and galactose (Parolis et al. 1999). Further information about the structure of these polysaccharides was reviewed by Oren (2006). Other types of extracellular polymers may occur as well; an interesting example is the poly-(γ-glutamate) layer found outside the cell wall of Nab. aegyptiaca (Hezayen et al. 2001).

Polar Lipids, Neutral Lipids, and Pigments

The Halobacteriaceae possess lipids based on branched 20-carbon (phytanyl) and in some genera also 25-carbon (sesterterpanyl) chains, bound to glycerol by ether bonds. This unusual lipid structure was elucidated long before the Archaea were recognized as the third domain of life (Sehgal et al. 1962; Kates et al. 1966). A variety of polar lipids, including phospholipids, sulfolipids, and glycolipids, can be encountered in the different representatives of the group. The types of polar lipids present are an important characteristic in the taxonomic classification of genera and species (see Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22). The diether core lipid that forms the basis for most of the polar lipid structures is 2,3-di-O-phytanyl-sn-glycerol (C₂₀,C₂₀). Many genera (Halalkalicoccus, Halarchaeum, Halobiforma, Halococcus, Halomarina, Haloterrigena, Natrialba, Natrinema, and others) contain in addition 2-O-sesterterpanyl-3-O-phytanyl-sn-glycerol (C₂₅,C₂₀). Sometimes 2,3-di-O-sesterterpanyl-sn-glycerol (C₂₅,C₂₅) is encountered as a minor component as well (De Rosa et al. 1982, 1983). The ratio between C₂₀,C ₂₀ and C₂₅,C₂₀ lipids may depend on growth conditions: increasing medium salinity leads to an increased proportion of C₂₅,C₂₀ in the alkaliphiles Nbt. gregoryi and Nab. magadii (Morth and Tindall 1985). In most cases, the hydrophobic chains are fully saturated, but the occurrence of unsaturated phytanyl (“phytenyl”) side chains was documented in Hrr. lacusprofundi isolated from Deep Lake, Antarctica, and able to grow at temperatures down to 4 C (Franzmann et al. 1988). Introduction of double bonds in the carbon chains is important in the regulation of membrane fluidity in this cold-adapted species. When grown at 25 C, the polar lipids (PG, PGP-Me, PGS, sulfated and non-sulfated glycolipids; see below) have fully saturated hydrophobic chains; cells grown at 12 C have unsaturated analogues with up to six double bonds (Gibson et al. 2005).

All Halobacteriaceae contain diether derivatives of phosphatidyl glycerol (PG) and the methyl ester of phosphatidyl glycerophosphate (PGP-Me) (Kates et al. 1993). The diether derivatives of phosphatidyl glycerosulfate (PGS) is present in many species. Its presence or absence is an important chemotaxonomic property that can be used to discriminate between different genera and species. Cardiolipins (bis-phosphatidyl glycerol) with different combinations of C₂₀ and C₂₅ isopranoid chains were found in the membranes of the haloalkaliphiles Ncc. occultus and Ncc. amylolyticus (Angelini et al. 2012). Other yet unidentified phospholipids have been detected in the genus Natrinema (McGenity et al. 1998). In Ncc. occultus, a phospholipid with a cyclic phosphate group has been identified: 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3′-sn glycerol-1,2-cyclic phosphate (Lanzotti et al. 1989).

Glycolipids are present in most neutrophilic species, but are generally absent in the alkaliphilic members of the family. Di-, tri-, and tetraglycosyl diether lipids occur, part of them carrying one or more sulfate groups bound to the sugar moieties. Not all have yet been fully characterized. Some of the better known and widespread glycolipids are:

• S-DGD-1 (1-O-[α-d-mannose-(6′-SO₃H)-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), the major glycolipid in the genus Haloferax. Chromatographically identical sulfated diglycosyl diether lipids have been identified in many other genera.

• DGD-1 (1-O-[α-d-mannose-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), found in minor amounts in Haloferax species.

• DGD-2, a minor diglyceride lipid of unknown structure, containing mannose and glucose, found as a minor component in Haloarcula species.

• S-DGD-3 (1-O-[α-d-mannose-(2′-SO₃H)-α-d-(1 → 4)-glucose]-2,3-di-O-phytanyl-sn-glycerol), the glycolipid of some Halorubrum species.

• S₂-DGD-1, a bis-sulfated glycolipid (1-O-[α-d-mannose-(2′,6′-SO₃H)-α-d-(1′ → 2′)-glucose]-2,3-di-O-phytanyl- or phytanyl sesterterpenyl-sn-glycerol), first characterized from Natrialba asiatica.

• TGD-1 (1-O-[β-d-galactose-(1′ → 6′)-α-d-mannose-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), a minor glycolipid of Hbt. salinarum.

• TGD-2 (1-O-[β-d-glucose-(1′ → 6′)-α-d-mannose-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), the sole or major glycolipid of most Haloarcula species.

• S-TGD-1 (1-O-[β-d-galactose-(3′-SO₃H)-(1′ → 6′)-α-d-mannose-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), found in the genus Halobacterium.

• TeGD (1-O-[β-d-galactose-(1′ → 6′)-α-d-mannose-(3′ ← 1′)-α-d-galactofuranose-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), a minor glycolipid of Hbt. salinarum.

• S-TeGD (1-O-[β-d-galactose-(3′-SO₃H)-(1′ → 6′)-α-d-mannose-(3′ ← 1′)-α-d-galactofuranose)-(1′ → 2′)-α-d-glucose]-2,3-di-O-phytanyl-sn-glycerol), found in Halobacterium.

The chemical structures of many of the above-listed lipids were given by Kamekura and Kates (1999) and Oren (2006).

Neutral lipids may represent about 10 % of the total lipid content of the Halobacteriaceae (Kamekura and Kates 1988; Kushwaha and Kates 1979). They include:

• C₂₀ isoprenoid lipids: geranylgeraniol.

• Neutral phytanyl ethers of glycerol: dl-O-phytanyl-sn-glycerol and 2,3-di-O-phytanyl-sn-glycerol.

• C₃₀-isoprenoid compounds: squalene, dihydrosqualene, tetrahydrosqualene, and dehydrosqualene.

• Carotenoids. Most species are pigmented red-orange due to a high content of carotenoid pigments in their cell membrane. Rare exceptions are Nab. asiatica and Hrb. tiamatea, which lack substantial amounts of carotenoids. The pigment content of the cells may depend on their nutritional status (Gochnauer et al. 1972; Kushwaha and Kates 1979) and on the salinity of the growth medium: certain Haloferax species are pigmented when grown at low salinity (e.g., 15 %), while at higher salt concentrations (e.g., 25 %), they may be almost colorless (Kushwaha et al. 1982; Rodriguez-Valera et al. 1980). The most abundant carotenoids of the Halobacteriaceae are the 50-carbon compounds α-bacterioruberin and its derivatives monoanhydrobacterioruberin and bis-anhydrobacterioruberin (Kelly et al. 1970; Kushwaha et al. 1974, 1975). C₄₀ carotenoids may be present in small amounts, lycopene and β-carotene being the most abundant (Kushwaha et al. 1982; Tindall 1992). Accumulation of canthaxanthin, in addition to bacterioruberin carotenoids, was reported in Hfx. alexandrinus (Asker and Ohta 2002b). Carotenoid pigments protect the cells against photodamage as shown in competition experiments in which wild-type cells of Hbt. salinarum and a carotenoid-less mutant were incubated at high light intensities (Dundas and Larsen 1962).

Respiratory Quinones and Polyamines

The major respiratory quinones in the Halobacteriaceae are MK-8 and MK-8(VIII-H₂) (Collins et al. 1981; Tindall and Collins 1986). Quinones may amount to about 9 % of the total neutral lipid content of the cells (Kamekura and Kates 1988). The relative abundance of MK-8 and MK-8(VIII-H₂) depend on the growth conditions (Tindall 1992; Tindall et al. 1991). Nbt. gregoryi also contains monomethylated and dimethylated menaquinones (Collins and Tindall 1987).

Polyamines are found in the Halobacteriaceae in very small amounts, if at all. If present, agmatine appears to be the most common polyamine (Hamana et al. 1995; Kamekura et al. 1987).

Physiological Properties

To provide osmotic balance of the cytoplasm with the extremely high salt concentrations in their environment, the Halobacteriaceae accumulate molar concentrations of KCl. Organic osmotic solutes are generally not used. An exception is the accumulation of 2-sulfotrehalose in Ncc. occultus (Martin et al. 1999). Most proteins require high salt for structural stability and activity and are characterized by an exceptionally high content of the acidic amino acids glutamate and aspartate. Accordingly they have a large net negative charge at the physiological pH (Mevarech et al. 2000; Lanyi 1974; Reistad 1970). In addition to the high salinity required by all species, many are obligate alkaliphiles. Some have temperature optima above 50 °C (Bowers and Wiegel 2011; Robinson et al. 2005).

Most species lead an aerobic heterotrophic life style (Tindall and Trüper 1986). They generally prefer complex media with amino acids as main organic nutrient. Hbt. salinarum, the type species of the type genus of the family, is unable to grow on sugars, but carbohydrates can be used by a variety of other species. Carbohydrate utilization was first demonstrated in Hrr. saccharovorum (Tomlinson and Hochstein 1972a, b; Tomlinson and Hochstein 1976). Breakdown of glucose follows a modified Entner-Doudoroff pathway in which the phosphorylation step is postponed: glucose is oxidized via gluconate to 2-keto-3-deoxygluconate, followed by phosphorylation to 2-keto-3-deoxy-6-phosphogluconate, which is then split into pyruvate and glyceraldehyde-3-phosphate (Tomlinson et al. 1974). Use of carbohydrates is often associated with the production of acids, as oxidation of such substrates is incomplete. When grown on glucose, Hrr. saccharovorum excretes acetic acid and pyruvic acid; galactose, lactose, and other sugars are converted to the corresponding aldonic acids (Tomlinson and Hochstein 1972b; Tomlinson et al. 1978). Acetate, pyruvate, and d-lactate were identified in cultures of several Haloferax and Haloarcula species grown in the presence of glycerol (Oren and Gurevich 1994).

While the species of Halobacterium and many other genera require complex media for growth, other members of the family can grow on defined media with single organic compounds (simple sugars, organic acids, amino acids) as carbon and energy source (Rodriguez-Valera et al. 1980). Many species produce exoenzymes (proteases, amylases, lipases, nucleases) enabling them to use proteins, starch, lipids, and nucleic acids as sources of nutrients.

Recent advances in genomic and systems biology have enabled an in-depth reconstruction of the metabolism of model organisms, notably strains of Hbt. salinarum, Har. marismortui, Hqr. walsbyi, and Nmn. pharaonis (Falb et al. 2008; Gonzalez et al. 2008). Recently a novel anaplerotic pathway for the incorporation of acetate into cellular carbon was identified in Har. marismortui and Hfx. volcanii: rather than the classic glyoxylate cycle, a methylaspartate cycle is operative (Khomyakova et al. 2011).

Some isolates can degrade aliphatic hydrocarbons (tetradecane, hexadecane, heptadecane, eicosane, heneicosane), aromatic hydrocarbons (acenaphthene, phenanthrene, anthracene, 9-methylanthracene), and other aromatic compounds (benzoate, cinnamate, 3-phenylpropionate, p-hydroxybenzoate). Most hydrocarbon and aromatic compounds degrading isolates belong to the genera Haloferax and Haloarcula (Bertrand et al. 1990; Cuadros-Orellana et al. 2006.; Emerson et al. 1994; Fu and Oriel 1999; Kulichevskaya et al. 1991; Tapilatu et al. 2010).

Due to the low solubility of oxygen in salt-saturated brines, oxygen may easily become a limiting factor for development of halophilic Archaea. One possible strategy to avoid oxygen limitation is the use of gas vesicles to buoy the cells toward the surface of the brine. Hbt. salinarum, Hfx. mediterranei, Hgm. borinquense, Hpn. aerogenes, Hpn. natans, Hpn. vescus, Hqr. walsbyi, and Hrr. vacuolatum can form gas vesicles. How effective gas vacuoles are to enable the cells to reach layer richer in oxygen is uncertain; for a critical discussion of this topic, see Oren et al. (2006).

One member of the family preferentially leads an anaerobic life style: Hrd. tiamatea, found in deep brines near the bottom of the Red Sea. It probably obtains its energy by fermentation (Antunes et al. 2008). Many other species have limited possibilities to survive and even grow in the absence of molecular oxygen, strategies based on anaerobic respiration with different electron acceptors, fermentation, or the use of light as an energy source when respiration cannot supply sufficient energy.

The ability to reduce nitrate is widespread among the members of the Halobacteriaceae. A few species (e.g., Hfx. denitrificans, Har. marismortui, Har. vallismortis, Hgm. borinquense) can grow anaerobically using nitrate as electron acceptor. Nitrate is generally reduced to N₂, but N₂O formation has also been observed in several cases (Hochstein and Tomlinson 1985; Mancinelli and Hochstein 1986; Tomlinson et al. 1986). The ecological relevance of anaerobic growth on nitrate has never yet been ascertained.

Other alternative electron acceptors for respiration are dimethylsulfoxide (DMSO), trimethylamine N-oxide (TMAO), and fumarate. Reduction of DMSO and TMAO is coupled with growth in several species. Hbt. salinarum, Hfx. mediterranei, Har. marismortui, and Har. vallismortis grew anaerobically in the presence of DMSO or TMAO; in Hfx. volcanii, DMSO supported anaerobic growth, while TMAO did not (Oren and Trüper 1990). The genes enabling Halobacterium strain NRC-1 to grow on DMSO and TMAO have been characterized (Müller and DasSarma 2005). Fumarate-driven anaerobic growth was reported in some Hbt. salinarum strains, in Hfx. denitrificans, and in Hfx. volcanii (Oren 1991). The ecological relevance of anaerobic growth of members of the Halobacteriaceae on DMSO, TMAO, or fumarate is unknown. However, TMAO may be available as an electron acceptor in salted fish, a well-known habitat for species of Halobacterium and Halococcus. TMAO can be present in high concentrations within fish tissues as an osmotic solute. A recent addition to the list of electron acceptors that can drive anaerobic respiration in members of the Halobacteriaceae is thiosulfate. It is used by the pleomorphic rod-shaped strain HG, isolated from hypersaline lakes in the Kulunda steppe, Altai, Russia, phylogenetically affiliated with the genus Natronorubrum. It grows on acetate with reduction of thiosulfate to tetrathionate (Sorokin et al. 2005).

An entirely different strategy for anaerobic energy generation is fermentation of l-arginine to citrulline, ammonia, and CO₂. This process can drive anaerobic growth in Hbt. salinarum (Hartmann et al. 1980; Ruepp and Soppa. 1996). Anaerobic growth on arginine is not widespread among the haloarchaea; a wide variety of neutrophilic strains tested belonging to genera other than Halobacterium gave negative results (Oren 1994; Oren and Litchfield 1999). A specific enrichment procedure for members of the genus Halobacterium could thus be developed, based on their ability to grow anaerobically in the presence of l-arginine (Oren and Litchfield 1999).

Light can be used as an energy source to drive anaerobic growth in Hbt. salinarum, provided the cells contain the light-driven proton pump bacteriorhodopsin, a membrane-bound 27 kDa protein carrying retinal as a prosthetic group (Hartmann et al. 1980; Oesterhelt 1982; Oesterhelt and Krippahl 1983). As the biosynthesis of retinal from β-carotene is oxygen-dependent, either trace concentrations of oxygen must still be present, or retinal or retinaloxime should be supplied to the medium to enable sustained light-driven anaerobic growth (Oesterhelt and Krippahl 1983). Four retinal-containing proteins have been identified in members of the family: bacteriorhodopsin, an outward light-driven proton pump; halorhodopsin, an inward light-driven chloride pump; and two sensory rhodopsins, involved in light sensing for phototaxis. Bacteriorhodopsin was first identified in Hbt. salinarum, where it is localized in specialized patches of the cell membrane (“purple membrane”) (Oesterhelt and Stoeckenius 1971; for a historic overview, see also Grote and O’Malley 2011). Upon excitation by light (absorption maximum 570 nm), protons are extruded from the cytoplasm to the outside of the cell (for a review, see Lanyi 2004). The proton gradient thus formed is used to drive energy-requiring processes in the cell, including the phosphorylation of ADP to ATP (Danon and Stoeckenius 1974). Not all members of the family produce bacteriorhodopsin; among those that do are Hrr. sodomense and Hqr. walsbyi. Studies of the bacteriorhodopsin gene cluster of Hbt. salinarum have shown that expression of the protein is induced by low oxygen tension and by light (Betlach et al. 1986; Shand and Betlach 1991). A second retinal protein, also first discovered in Hbt. salinarum, is halorhodopsin. The structure of this protein is similar to that of bacteriorhodopsin, but it acts as a chloride pump: excitation by light (absorption maximum 580 nm) causes the inward transport of chloride ions (Schobert and Lanyi 1982). Chloride transport is important to maintain the proper ionic balance and is essential for cell growth. Halorhodopsin was also found in several haloalkaliphilic Archaea, and the halorhodopsin of Nmn. pharaonis has been studied in detail (Lanyi et al. 1990). Hbt. salinarum contains two sensory rhodopsins, involved in light sensing for phototaxis. Sensory rhodopsin I is a green light receptor (light to which the cells are attracted), and sensory rhodopsin II, also termed phoborhodopsin, is a blue light receptor (light that acts as a repellent).

Sensitivity to Antibiotics

Members of the Halobacteriaceae typically are resistant to such Bacteria-specific antibiotics as penicillin, ampicillin, cycloserine, kanamycin, neomycin, polymyxin, and streptomycin (Bonelo et al. 1984; Hilpert et al. 1981; Pecher and Böck 1981). Sensitivity to chloramphenicol is variable (see also Tables 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, and 7.22). Most species are sensitive to novobiocin and bacitracin. Novobiocin is a DNA gyrase inhibitor (Gadelle and Forterre 1994, Sioud et al. 1988) and acts on the same target in the Archaea as in sensitive Bacteria. Bacitracin inhibits incorporation of the high-molecular-weight saccharide into the cell wall glycoprotein of non-coccoid halophilic Archaea and may also inhibit lipid biosynthesis in these organisms.

Members of the Halobacteriaceae are sensitive to a number of additional antibiotics and other antibacterial compounds:

• Anisomycin, a protein synthesis inhibitor of ribosomes of eukaryotes, also inhibits protein synthesis of nearly all members of the Halobacteriaceae tested, both in vivo and in vitro (Pecher and Böck 1981). Four species were reported as resistant: Hrr. chaoviator, Hsx. carlsbadense, Nmm. moolapensis, and Sar. japonicum.

• Cerulenin, an inhibitor of the synthesis of the straight-chain fatty acids by the fatty acid synthetase complex, inhibits Hbt. salinarum (and possibly other members of the order as well) (Dees and Oliver 1977). This finding suggests that the production of straight-chain fatty acids is essential even in an organism possessing archaeal type lipids. It is now known that fatty acids are used to acylate certain membrane proteins (Pugh and Kates 1994).

• Rifampicin inhibits many members of the Halobacteriaceae (Bonelo et al. 1984; Hilpert et al. 1981; Pecher and Böck 1981). The target of rifampicin action in the halophilic Archaea is probably not the DNA-dependent RNA polymerase, but the inhibition may be due to its detergent effect on the cell membrane, causing cell lysis.

• The DNA polymerase inhibitor aphidicolin prevents cell division and often causes the formation of elongated cells (Forterre et al. 1984, 1986; Schinzel and Burger 1984).

• Inhibition of halophilic Archaea by coumarin and quinolone antibacterial compounds presented evidence for the presence of DNA gyrase-like enzymes (Sioud et al. 1988). The quinolone compound ciprofloxacin inhibited most Haloferax and Haloarcula species at concentrations between 25 and 60 μg/ml. Halobacterium cells proved less sensitive. Sensitivity to ciprofloxacin and other quinolone derivatives (norfloxacin, perfloxacin) was decreased at increased magnesium concentrations (Oren 1996; Sioud et al. 1988). Hfx. volcanii became more resistant when the magnesium concentration in the growth medium was increased. Alkaliphiles such as Nmn. pharaonis are very sensitive (Oren 1996).

• Bile acids at low concentrations cause lysis of the cell envelopes of non-coccoid halophilic Archaea and are potent growth inhibitors (Dussault 1956a, b; Kamekura et al. 1988).

Halocins

Many species of Halobacteriaceae excrete “halocins,” protein antibiotics that inhibit the growth of other related species (Meseguer and Rodriguez-Valera 1986; Meseguer et al. 1986; Rodriguez-Valera et al. 1982; Torreblanca et al. 1989, 1990, 1994). Different modes of action have been suggested for different halocins (Shand et al. 1999). Halocin H4, excreted by the type strain of Hfx. mediterranei and halocin H1 of Hfx. mediterranei strain Xai3 cause membrane permeability changes and ionic imbalance in Hbt. salinarum (Meseguer and Rodriguez-Valera 1986; Meseguer et al. 1991; O’Connor and Shand 2002). Halocin H7, excreted by Hfx. gibbonsii, targets the Na⁺/H⁺ antiporter activity of sensitive strains (Alberola et al. 1998; Meseguer et al. 1995). More than 10 halocins have been wholly or partially characterized (Li et al. 2003; O’Connor and Shand 2002). The size of the mature proteins (after cleavage of signal sequence of the pre-protein) varies between 3.6 kDa, the 36-amino acid long “microhalocin” S8 excreted by a further uncharacterized strain isolated from Great Salt Lake, Utah (Price and Shand 2000), and 34.9 kDa—halocin H4 of Hfx. mediterranei (Cheung et al. 1997). Although the ability to excrete halocins may be expected to be of considerable ecological advantage, no data are available as yet that prove that halocins are excreted by natural communities of halophilic Archaea in concentrations sufficient to inhibit the development of competitor strains, thus substantiating their ecological role (see Kis-Papo and Oren 2000).

Ecology

Members of the Halobacteriaceae can be found worldwide in hypersaline environments with salt concentrations above 10–15 % (Norton 1992; Oren 1994, 2011). As most species lyse when exposed to lower salinities even for short times, a stable high-salt environment is a prerequisite for their development. We find selected species at high pH in soda lakes, and an acidophilic species (Hla. acidiphilum) that grows optimally at pH 4.4 was recently characterized (Minegishi et al. 2008). Curiously, it was isolated from a sample of Chinese salt imported to Japan, that in saturated solution yielded an alkaline pH. Some can grow at low temperatures (Hrr. lacusprofundi from Deep Lake, Antarctica) (Franzmann et al. 1988), and temperatures up to 55 °C are tolerated by many species. Thus, as long as the salinity is suitably high, members of the Halobacteriaceae can be expected to grow.

Lakes with salt concentrations approaching saturation may show red hues, such as documented for the north arm of the Great Salt Lake, the Dead Sea, and hypersaline alkaline lakes such as Lake Magadi, Kenya. Red-colored brines are also typically present during the final stages of the evaporation of seawater in solar saltern ponds (Oren 1994). Archaea are generally responsible for most of the color of the saltern brines. Numbers of 10⁷ to 10⁸ cells and higher per ml of brine are not unusual. Members of the Halobacteriaceae have also been recovered from saline and hypersaline soils (Zvyagintseva and Tarasov 1987).

The concentration of divalent cations, especially magnesium and calcium, in the environment is of considerable ecological importance. Thus, the Dead Sea is dominated by divalent cations (presently around 2 M Mg²⁺ and 0.5 M Ca²⁺, in addition to about 1.5 M Na⁺ and 0.2 M K⁺). Halophilic Archaea isolated from the Dead Sea are characterized by a relatively low requirement for Na⁺ and an extraordinarily high tolerance toward and often also a requirement for high Mg²⁺ concentrations, such as was shown for Hfx. volcanii (Mullakhanbhai and Larsen 1975), Hrr. sodomense (Oren 1983), and Hbc. gomorrense (Oren et al. 1995).

Alkaliphilic members of the family such as Hrr. vacuolatum (Mwatha and Grant 1993), Nmn. pharaonis (Soliman and Trüper 1982), Nab. magadii, Nbt. gregoryi, Ncc. occultus (Tindall et al. 1984), and Ncc. amylolyticus (Kanai et al. 1995) are characteristic inhabitants of hypersaline soda lakes in which the pH reaches 9–10 and higher. In accordance with the low solubility of the divalent cations Mg²⁺ and Ca²⁺ at high pH, the alkaliphilic halophiles do not require significant concentrations of magnesium for growth (Tindall et al. 1980).

Halobacteriaceae were also recovered from rock salt deposited millions of years ago. Such isolates include the type strains of Hsx. carlsbadense, Hcc. dombrowskii, Hcc. salifodinae, and Nbt. noricense. Much has been speculated about the longevity of such organisms within salt crystals. Sequences of haloarchaeal 16S rRNA fragments have been retrieved from salt samples as ancient as the Permian or the Triassic era (Fish et al. 2002; McGenity et al. 2000; Radax et al. 2001).

The first isolates of Halobacteriaceae described—strains of Hbt. salinarum and Hcc. morrhuae—were obtained from red growth on salted food products (fish, meat) and salted hides, nutrient-rich environments where colonization by haloarchaea may cause economic damage.

In view of the extremely high salt requirement of most species of Halobacteriaceae, it is somewhat surprising to find reports of their isolation (and/or the recovery of their 16S rRNA genes in culture-independent studies) from low-salt environments. The isolation of Halococcus sp. from seawater (Rodriguez-Valera et al. 1979) was possible due to the fact that in contrast to most other genera that lyse at low salt concentrations, Halococcus has a rigid cell wall. Halococcus cells may be dispersed by birds: strains of Hcc. morrhuae and Hcc. dombrowskii could be cultivated from the salt-excreting glands in the nostrils of the seabird Calonectris diomedea (Brito-Echeverría et al. 2007). A strain related to Haladaptatus, isolated from a traditional Japanese-style salt field, showed a high survival rate after 9 days incubation in 0.5 % seawater salts (Fukushima et al. 2007). In recent years, several reports were published on the existence of strains of Halobacteriaceae with a relatively low salt requirement for growth. Isolates of haloarchaea were obtained from a salt marsh in Essex, UK, an environment containing ∼2.5 % salt only. One isolate designated strain HA Gp1 (unfortunately not characterized further) was reported to grow optimally at 10 % salt with growth being even possible at 2.5–3 % salt (Purdy et al. 2004). Hap. paucihalophilus was isolated from a low-salt sulfur spring in Oklahoma, and it grows at salt concentrations as low as 4.7 % NaCl with an optimum at 15–18 % (Savage et al. 2007). The recovery of Haloarcula sp. 16S rRNA gene sequences from a deep-sea black smoker chimney at a hydrothermal vent area near Papua New Guinea (Takai et al. 2001) is interesting as well. Whether hypersaline areas may be present within such chimney structures remains to be ascertained. An intriguing environment from which Haloarcula-related strains were recovered is the geothermal steam vent aerosols emerging from fumaroles in Kamchatka, Hawaii, New Mexico, California, and Wyoming. A single fumarole was estimated to emit >10⁹ cells of haloarchaea per year (Ellis et al. 2008). The finding of 16S rRNA gene sequences of Halobacteriaceae within the intestine of humans is discussed in the next section.

Pathogenicity: Clinical Relevance

As the Halobacteriaceae require high salt concentrations for growth, much higher than the physiological salt concentration of ∼9 g/l of the body fluids, members of the family cannot be expected to be associated with the human body. Still there are two interesting observations showing that some species may indeed colonize humans. A culture-independent study of the intestinal microflora of Korean people yielded 16S rRNA gene sequences related to Halorubrum and Halococcus (Nam et al. 2008). The finding may be related to the local diet: species such as Hrr. alimentarium and Hrr. koreense were isolated from salt-fermented seafood based on shrimps. Haloarchaeal 16S rRNA gene sequences were also recovered from the intestinal mucosa of patients with inflammatory bowel disease in the UK. Eight out of the 39 biopsy samples gave positive PCR results. Some phylotypes were affiliated to the genera Halobacterium and Halorubrum; others belonged to a new lineage not yet represented by cultures. Thus, members of the Halobacteriaceae may under certain conditions be present in the mucosal microbiota (Oxley et al. 2010).

Application

Members of the Halobacteriaceae have a number of useful applications, and potential new applications in biotechnological processes are being investigated (Galinski and Tindall 1992; Litchfield 2011; Margesin and Schinner 2001; Oren 2010; Rodriguez-Valera 1992; Ventosa and Nieto 1995). The following list presents some of the current and potential future applications of the group:

• The positive effect of the presence of dense communities of red halophilic Archaea in saltern crystallizer ponds has been recognized for a long time. By trapping solar radiation, they raise the temperature of the brine and the rate of evaporation, thereby increasing salt production. To improve salt production in salterns that do not develop a sufficiently dense archaeal community, fertilization with nutrients may increase the red color of the ponds (Jones et al. 1981).

• Production of fermented fish sauce in the Far East (e.g., nam pla in Thailand) involves participation of halophilic Archaea. The product is traditionally made by adding two parts of fish and one part of marine salts. The mixture is covered with concentrated brine (4.4–5.1 M NaCl) and left to ferment for about a year. Red halophilic Archaea (identified as Halobacterium and Halococcus) reach their maximum density in the liquor after 3 weeks and persist throughout the fermentation period. The halobacterial proteases probably take part in the fermentation process (Thongthai and Siriwongpairat 1990; Thongthai and Suntinanalert 1991; Thongthai et al. 1992). Addition of starter cultures of Halobacterium sp. SP1(1) may accelerate fish sauce fermentation (Akolkar et al. 2010). Use of different strains of halophilic Archaea could improve the safety and quality of salted anchovies by reducing the content of histamine (Aponte et al. 2010). Immobilized cells of Natrinema gari strain BCC 24369 may be highly effective for the removal of histamine from salted food products (Tapingkae et al. 2010a, b).

• Different species of halophilic Archaea synthesize potentially useful products such as bacteriorhodopsin, exopolysaccharides, and poly-β-hydroxyalkanoate (PHA). Halophilic Archaea have distinct advantages in biotechnological processes as they are relatively easy to grow, danger of contamination is minimal, and culture size can be upscaled to the use of large fermenters (Kushner 1966). However, because of the low solubility of gases in concentrated brines, the supply of sufficient amounts of oxygen may cause problems. In addition, the aggressive nature of the salts should be taken into account when planning the construction of large fermenters with metal parts exposed to the medium. A corrosion-resistant bioreactor made of polyetheretherketone was developed for the production of PHA and poly(γ-glutamic acid) by Natrialba sp. (Hezayen et al. 2000).

• Bacteriorhodopsin, the light-driven proton pump of Hbt. salinarum, has considerable biotechnological potential (Oesterhelt et al. 1991). It may be used as a biological material for information processing. Other suggested uses include conversion of sunlight to electricity, ATP generation, desalination of seawater, use in chemo- and biosensors, and ultrafast light detection. The photochromic effect—shifts between the purple ground state of the molecule (the “B state”) and the yellow “M state”—may be used for information storage, including holographic storage, and may enable development of powerful computer memories and processors (Birge et al. 1999). Bacteriorhodopsin offers many advantages since it is a very stable molecule, functioning well between 0 °C and 45 °C and pH 1–11 (Chen and Birge 1993), is easy to immobilize on solid substrates, and produces very reproducible photoelectric signals. Holographic bacteriorhodopsin films are suitable for the construction of computer memories enabling parallel processing, and the developing technology may lead to a new generation of computers (Hong 1986). To turn bacteriorhodopsin into a light sensor, it is spread in a thin film sandwiched between an electrode and an electrically conductive gel. Changes in the shape of the molecule create a displacement of charge, generating an electrical signal. The suitability of bacteriorhodopsin for all these applications can be greatly enhanced by mutation; using genetically engineered bacteriorhodopsin, up to 700-fold improvement has been realized in volumetric data storage (Hampp 2002a, b; Wise et al. 2002). Bacteriorhodopsin-based photo-electrochemical cells can be constructed (Chu et al. 2010). Use of Hbt. salinarum for light-driven hydrogen production was also explored (Lata et al. 2007; Zabut et al. 2006).

• A microbial fuel cell operating at high ionic strength conditions was designed, using Nab. magadii or Hfx. volcanii as biocatalyst at the fuel cell anode. Such a system may be favorable as high-salt media have a high electrical conductivity so that the internal resistance is diminished (Abrevaya et al. 2011).

• The extracellular polysaccharide produced by Haloferax has considerable biotechnological potential (Antón et al. 1988; Rodriguez-Valera et al. 1991). The bacterium produces up to 3–8 g/l of an acidic heteropolysaccharide with a high apparent viscosity at relatively low concentrations and resistance to extremes of salts, temperature, and pH. The structure of this polymer was elucidated (Parolis et al. 1996). Hfx. volcanii and Hfx. gibbonsii also produce exopolysaccharides. Such polymers may be used to modify rheological properties of aqueous systems, for viscosity stabilization as thickening agents, gelling agents, and emulsifiers, and may find applications in microbially enhanced oil recovery (Ventosa and Nieto 1995). Here, a salt-resistant surfactant is advantageous as high-salinity brines are often encountered associated with oil deposits. Whole cell preparations may be used, as the lipids liberated upon lysis of halophilic Archaea may also act as surfactants to improve the oil-carrying properties (Post and Al-Harjan 1988).

• Use of Haloferax species (Hfx. mediterranei, Hfx. denitrificans) was proposed for the bioremediation of saline and hypersaline waters containing nitrate (Cyplik et al. 2007, 2010) or chlorate and perchlorate (Martinez-Espinosa and Bonete 2007). Hbt. salinarum ATCC 43214 (misnamed as Halobacter (sic) halobium) was used to supplement activated sludge in a rotating biological disk system for COD removal from saline wastewater (Dinçer and Kargi 2001; Kargi 2002; Kargi and Dinçer 1996). However, the salinity of the wastewater treated (up to 5 % or 10 % salt) is too low for growth or even for survival of the halophilic Archaea, and therefore, the mechanism of the reported improvement in performance of the system is not clear.

• Hfx. mediterranei cells may contain considerable amounts of poly-β-hydroxyalkanoate (PHA) (Fernandez-Castillo et al. 1986; Rodriguez-Valera et al. 1991). PHA is used for the production of biodegradable plastics. Though halophilic Archaea are not yet being used commercially for PHA production, they have certain obvious advantages over an organism such as Alcaligenes eutrophus, which is already being exploited for the purpose. Hfx. mediterranei can be grown on a cheap substrate such as starch. Moreover, downstream processing and purification of the product should be relatively simple as the cells are easily lysed in water (Ventosa and Nieto 1995). Also the high genomic stability of the organism and the reduced danger of contamination are clear assets. PHA production is maximal when grown on sugars (glucose or starch) and in the presence of low phosphate concentrations (Lillo and Rodriguez-Valera 1990; Rodriquez-Valera and Lillo 1992). PHA production was demonstrated in certain other halophilic Archaea as well, such as Hfx. volcanii and Har. marismortui. It has been argued that Hfx. mediterranei is economically better than several non-halophilic producers for the production of PHA from whey: the strain is robust and stable, and danger of contamination is minimal (Koller et al. 2007). Processes for the use of crude oil, petrochemical wastewater, or glycerol for the production of PHA by Haloarcula sp. IRU1 were recently described (Taran 2011a, b, c). However, the proposed addition of 0.8 % tryptone or 0.4 or 0.8 % yeast extract as source of nitrogen and other nutrients will make such a process economically unfeasible.

• Exoenzymes such as amylases, amyloglucosidases, proteases, and lipases that function at high salinity may be useful in biotechnological processes requiring degradation of macromolecules in the presence of high salt concentrations (Chaga et al. 1993; Eichler 2001; Ventosa and Nieto 1995).

• Canthaxanthin can be produced using Hfx. alexandrinus (Asker and Ohta 1999, 2002b).

• Halophilic Archaea degrading aliphatic and aromatic hydrocarbons may prove useful for the reduction of the chemical oxygen demand of hypersaline petroleum-produced water and the bioremediation of oil spills (Bertrand et al. 1990; Bonfá et al. 2011; Kulichevskaya et al. 1991).

• Members of the Halobacteriaceae growing on brine-cured hides (a phenomenon known as “red heat”) negatively affect the quality of the product (Bailey and Birbir 1996). To prevent damage of brine-cured hides by growth of proteolytic halophilic Archaea, gelatinase-negative halocin producers could be applied against damaging proteolytic strains (Birbir et al. 2004).

• A 84 kDa protein from Hbt. salinarum has been used as an antigen to detect antibodies against the human c-myc oncogene product in the sera of cancer patients, and therefore, the protein may be useful for the detection of certain forms of cancer (Ben-Mahrez et al. 1988, 1991).

• Gas vesicles of Halobacterium can be used as a Chlamydia vaccine display and delivery system. Haptenes incorporated in recombinant GvpC protein showed strong antibody response in mice in the absence of further adjuvants (Childs and Webley 2012; Stuart et al. 2001). Lipid vesicles (“archaeosomes”) made of Hrr. tebenquichense total polar lipids can be developed into vaccine delivery vehicles (Gonzalez et al. 2009).

• Site-specific endonucleases of halophilic Archaea (Schinzel and Burger 1986) may find uses in molecular biological research.