Home Page Image

  Acridine orange stained cells of a new Haloquadratum isolate (Bajool9) from an australian salt lake.



    Haloarchaeal Genomics and Diversity

Many haloarchaea have been isolated from salt lakes or solar saltern crystalliser ponds, including members of the genera Haloferax, Halogeometricum, Halococcus, Haloterrigena, Halorubrum, Haloarcula and Halobacterium (Oren, 2000). Others have been isolated from athalassohaline lakes (Dead Sea, Soda lakes) or salty soils, or even beach sand. Recently low salt haloarchaea have been isolated, extending the range of this archaeal family to salinities approaching that of sea water (Purdy et al. 2004). However, cultivation of the dominant members of a saltern crystalliser was not demonstrated until the studies published from my lab in 2004.

Laboratory culture of representative members of the dominant populations of salt lakes is a critical first step in understanding the ecology of such systems. The most significant haloarchaeal group is easily recognized by light microscopy - it is a flat square, with gas-vacuoles, first described by Walsby in 1980. It commonly represents 40 – 80% of cells in salt lakes and soltern crystallizer ponds. We originally denoted this group with the acronym SHOW (square haloarchaea of Walsby). Despite many attempts over the years (Oren, 2002) it was only cultivated in 2004, and reported by us (Burns et. al, 2004) [more], and independently by Henk Bolhuis and colleagues in Europe. In a joint study, the organism was formally described and named Haloquadratum walsbyi in 2007. The genome sequence of the spanish isolate (HBSQ001) and metagenomic studies in Spain have provided a wealth of information about the metabolism and evolution of this curious extremophile, and recently the genome sequence of the Australian strain, C23T, has been completed and compared to that of strain HBSQ001 (PLos ONE, in press). In addition to Hqr. walsbyi, the genome sequences of the type species of many genera of halobacteria have recently been reported and deposited in Genbank and the UCSC archaeal genome browser, opening up numerous possibilities for future study.


Like marine waters, hypersaline lakes have virus populations that are 10-100 times greater than the cell population (Wommack and Colwell, 2000), making them by far the predominant organism in the environment. In the Dead Sea, virus particles were estimated to number 7 x 107 VLP/ml (Oren et al., 1997). Some resembled head-tail bacteriophages but many were an unusual spindle-shape. Viral lysis of infected cells is believed to be responsible for the majority of nutrient cycling and population turnover. Grazing of bacteria (by protozoa) is absent in ponds above about 20% salinity, while virus levels increase significantly at salinities above 15% (Pedros-Alio et al., 2000). Viral lysis is estimated to affect 7% of the total population per day, a significant figure given the long doubling time of about 2 days for the in-situ prokaryotic population.

Given the enormous concentration of viruses in fresh, saline and hypersaline environments, it is not surprising that these parasites play a key role in the evolution and dynamic population structure of their prokaryotic hosts (eg. Proctor et al., 1993; Wommack and Colwell, 2000).  At a genetic level, examples of host resistance mechanisms (eg. restriction-modification), lysogenic conversion (eg. virulence factors in pathogenic bacteria), and transduction spreading genes throughout a population (eg. antibiotic resistance genes) are well known in bacteria.  Genome sequences of viruses and their hosts show that significant fractions of the total host DNA can be integrated phage genomes (eg. about 10% in B. subtilis) and recombination events between viruses (eg. Desiere et al., 1998), or between virus and host (eg. Nakayama et al., 1999) are common and widespread. The commercial spin-offs of phage research are many (e.g., enzymes, vectors, peptide display, phage therapy, etc.)

Why, after decades of work, have only about 21 haloviruses been reported, a number that is miniscule compared to over 5,000 known bacteriophages ? Most of the 15 halovirus isolates have head-tail morphology, linear dsDNA genomes, and grow on members of one genus, Hbt. salinarum. The true diversity of haloviruses is not known but, in hindsight, the low success rate of halovirus isolation is probably due to the fact that the laboratory strains of haloarchaea used as hosts are now known to make up very minor fractions of natural populations and are unlikely to be infected by the dominant viruses. The best studied halovirus, PhiH, is a temperate, head-tail virus of about 59 kb linear dsDNA (Reiter et al., 1988; Stolt and Zillig, 1994a), that shows similarities to P-type coliphages, such as P1. PhiH has provided very useful insights into the control of lysogeny (Stolt and Zillig, 1994b) and was instrumental in establishing the first transformation system in Archaea (Cline et al., 1989), but both virus and host suffer from extraordinarily high mutation rates (caused by active insertion sequences) and work stopped several years ago (W. Zillig, 1999, personal communication).  A related halovirus, PhiCh1 (Witte et al., 1993; Klein et al., 2002), isolated from a haloalkiliphile (Nab. magadii) continues to be studied (Prof. Witte's laboratory, Austria). The PhiCh1 genome, genome plasticity, and function of several of its genes have been reported. Unfortunately, many of the early haloviruses, including PhiH, may now be lost. See my table of described haloviruses.

I and my students have been studying the molecular biology of representatives of the 3 major morphotypes (head-tail, spindle, round). For example, the transcription and gene regulation in HF2, His2 and SH1 are currently being studied. In addition, the structural proteins are beginning to be identified.

The head-tail halovirus HF2

hf2We have isolated a number of Australian haloviruses, and the genome sequences of five have so far been reported (HF1, HF2, His1, His2, SH1 ). We were lucky to isolate pairs of related haloviruses i.e., HF1 / HF2, and His1 / His2. In 2002, we were the first to report a full halovirus genome sequence (HF2; Tang et al., 2002; picture above left), and two years later 2004, we were the first to compare the (complete) genome sequences of two related haloviruses, HF1 and HF2. Interestingly, they share 94% sequence similarity but the differences were all clustered in the late gene region (structural, assembly  and packaging genes).  The first 48kb of their 77kb genomes were identical (bar 1 nt !). We believe this unusual pattern indicates a recent recombination event, suggesting that high rates of recombination occur naturally in hypersaline waters.

The spindle-shaped viruses His1 and His2

Halovirus His1We have isolated the only of examples of spindle-shaped haloviruses, His1 (picture at left) and His2 [see diagram of virus particle]. This is a dominant morphotype in hypersaline lakes, and it was important to ascertain if they were similar to spindle-shaped viruses of thermophilic archaea, such as the Sulfolobus virus, SSV1. As we have discovered, while they look similar, their properties are very different from members of the Fuselloviridae (SSV1 and relatives). They are lytic, and have dsDNA genomes of 15-16 kb, dsDNA, with terminal proteins attached to the 5' ends. His1 and His2 both grow on Haloarcula hispanica, a strain that can be transformed easily with commonly available shuttle plasmids. They have been classified into a new virus group, the Salterprovirus group (Bath et al., 2006; wikipedia)

The round haloviruses SH1 and PH1

The isolation and properties of the first round halovirus, SH1 was reported in 2005. It was isolated from a salt lake on Rottnest Island, a small island just off the coast of Western Australia. Round virus-like particles are also a dominant morphotype in hypersaline lakes, so having a laboratory isolate for study is a significant step forward. It also proved that the round particles seen by EM were not simply dislodged heads from head-tail particles.

SH1 is unusual in having an internal lipid membrane layer, and for having terminal proteins on its linear dsDNA genome (Bamford et al., 2005; Porter et al., 2005). The genome is about 30 kb dsDNA and the sequence showed very few matches to proteins or ORFs in the Genbank database. This low level is quite typical of archaeal viruses, and illustrates just how little we know of the 'sequence space' in natural environments. A related halovirus, PH1, was also isolated and is yet to be formally described. The efforts of the Venter Institute (e.g. Sorecerer II expedition) should greatly assist in filling in some of these gaps in our knowledge.


Anton, J., Llobet-Brossa, E., Rodriguez-Valera, F., and Amann, R. (1999)Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environmental Microbiology 1: 517-523.

Bamford, D.H., Ravantti, J.J., Rönnholm, G., Laurinavi?ius, S., Kukkaro, P., Dyall-Smith, M., Somerharju, P., Kalkkinen, N., and Bamford, J.K. (2005) Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica. J Virol 79: 9097-9107.

Bath, C., and Dyall-Smith, M. L. (1998): His1, an Archaeal Virus Of the Fuselloviridae Family That Infects Haloarcula hispanica. Journal of Virology 72: 9392-9395.

Benlloch, S., Lopez-Lopez, A., Casamayor, E.O., et al. (2002) Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern. Environ Microbiol4: 349-360.

Bowman, JP, McCammon, SA, Rea, SM et al. 2000. The microbial composition of three limnologically disparate hypersaline Antarctic lakes FEMS Microbiology Letters Vol. 183:81-88.

Cline, S. W., Lam, W. L., Charlebois, R. L., Schalkwyk, L. C., and Doolittle, W. F. (1989): Transformation methods for halophilic archaebacteria. Can J Microbiol35, 148-52.

Desiere, F. et al. (1998) Evolution of Streptococcus thermophilus bacteriophage genomes by modular exchanges followed by point mutations and small deletions and insertions. Virology 241:345-356.

Dyall-Smith, ML, Tang, S-L & Bath, C. Haloarchaeal viruses: how diverse are they? Res. Microbiol. (in press, 2003).

Geoscience Australia, http://www.auslig.gov.au/facts/landforms/larglake.htm#saltlake (2002).

Grant, S. et al. (1999) Novel archaeal phylotypes from an East African alkaline saltern. Extremophiles.3, 139-145.

Grant, W et al. (2001) Class III. Halobacteria class nov. In Bergey’s Manual of Systematic Bacteriology. Vol. 1. Boone, D., Castenholz, R. and Garrity, G. (eds). New York: Springer-Verlag, pp. 294-334.

Gregor, D., and Pfeifer, F. (2001) Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea. Microbiology147: 1745-1754.

Gutierrez, M.C., Kamekura, M., Holmes, M.L., Dyall-Smith, M.L., and Ventosa, A. (2002) Taxonomic characterization of Haloferax sp. (" H. alicantei") strain Aa 2.2: description of Haloferax lucentensis sp. nov. Extremophiles6: 479-483.

Hengstmann U et al. (1999) Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil. Appl Env Microbiol 65: 5050-8

Holmes, M and Dyall-Smith, M (2000) Sequence and expression of a halobacterial beta-galactosidase gene. Molec. Microbiol. 36: 114-122.

Hugenholtz, P., Goebel, B.M., and Pace, N.R. (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180: 4765-4774.

Janssen, P et al. (2002) Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol 68: 2391-2396.

Klein, R et al. (2002) Natrialba magadii virus phiCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon. Mol Microbiol45: 851-863.

Lee, S.J. et al. (2003) TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis. J Biol Chem278: 983-990..

Meile, L., P. Abendschein, and T. Leisinger. 1990. Transduction in the archaebacterium Methanobacterium thermoautotrophicum Marburg. J. Bacteriol. 172:3507-8.

Nakayama, K et al. (1999) The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of P. aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages. Molec Microbiol 31: 399

Noble, R.T., and J.A. Fuhrman. 1998. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14: 113-118

Nuttall, S & Dyall-Smith, M (1993) HF1 and HF2:Novel bacteriophages of halophilic archaea. Virology197: 678-84

Nuttall, S & Dyall-Smith, M (1995) Halophage HF2:genome organization and replication strategy. J Virol 69: 2322-7

Ochsenreiter, T., Pfeifer, F., and Schleper, C. (2002) Diversity of Archaea in hypersaline environments characterized by molecular-phylogenetic and cultivation studies. Extremophiles: 267-274.

Oren, A.  et al. 1997. Occurrence of virus-like particles in the Dead Sea. Extremophiles. 1:143-149.

Oren, A., Ventosa, A., and Grant, W.D. Proposal of minimal standards for the description of new taxa in the order Halobacteriales.  Int. J. Syst. Bacteriol. 47: 233-238, 1997.

Oren, A. (2002) Molecular ecology of extremely halophilic Archaea and Bacteria. FEMS Microbiol Ecol 39: 1-7.

Patenge, N., Haase, A., Bolhuis, H., and Oesterhelt, D. (2000) The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Hbt. salinarum. Molec Microbiol36: 105-113.

Pedros-Alio, C., et al. (2000) The microbial food web along salinity gradients. FEMS Micro Ecol32: 143-155.

Porter, K., Kukkaro, P., Bamford, J.K., Bath, C., Kivelä, H.M., Dyall-Smith, M.L., and Bamford, D.H. (2005) SH1: A novel, spherical halovirus isolated from an Australian hypersaline lake. Virology 335: 22-33.

Proctor, L et al. (1993) Calibrating estimates of phage-induced mortality in marine bacteria. Micro. Ecol. 25:161-182.

Reiter, W., Zillig, W., and Palm, P. (1988) Archaebacterial Viruses. Adv. Virus Res.34: 143-188.

Stolt, P. & Zillig, W. (1994a). Gene Regulation In Halophage Phi-H - More Than Promoters. System. & Appl. Microbiol. 16: 591-596.

Stolt, P., and Zillig, W. (1994b): Transcription of the halophage PhiH repressor gene is abolished by transcription from an inversely oriented lytic promoter. FEBS Letters344, 125-128.

Tang, S.L., Nuttall, S., Ngui, K., Fisher, C., Lopez, P., and Dyall-Smith, M. (2002) HF2: a double-stranded DNA tailed haloarchaeal virus with a mosaic genome. Mol Microbiol44: 283-296.

Walsby, A.E. (1980) A square bacterium. Nature (London)283: 69.

Witte, A. et al. (1997). Characterization of Natronobacterium magadii phage Phi-Ch1, a Unique Archaeal Phage Containing DNA and RNA. Molec. Micro. 23: 603-616.

Woese, C et al. (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eukarya. PNAS (USA) 87: 4576-4579.

Wommack, K & Colwell, R (2000) Virioplankton: viruses in aquatic ecosystems. Microbiol Mol Biol Rev64:69-114.