Sequence analysis of an archaeal virus isolated from a hypersaline lake in Inner Mongolia, China

BioMed Central Page 1 of 13 (page number not for citation purposes) BMC Genomics Open Access Research article Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China Eulyn Pagaling†1, Richard D Haigh†1, William D Grant1, Don A Cowan2, Brian E Jones3, Yanhe Ma4, Antonio Ventosa5 and Shaun Heaphy*1 Address: 1Department of Infection Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 9HN, UK, 2Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa, 3Genencor International B, V., Archimedesweg 30, 2333 CN Leiden, The Netherlands, 4State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China and 5Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, 41012, Spain Email: Eulyn Pagaling - eulyn_pa[email protected]; Richard D Haigh - rx[email protected]; William D Grant - [email protected]; [email protected]; BrianEJones - [email protected]; Yanhe Ma - [email protected]; Antonio Ventosa - ventos[email protected]; Shaun Heaphy* - [email protected] * Corresponding author †Equal contributors Abstract Background: We are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus. Results: Haloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content of ~65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained. Conclusion: The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system. Background The three domain description of cellular life on earth, Eukarya, Bacteria and Archaea is a firmly established biological tenet [1]. Each domain has an associated, probably vastly diverse, virus population [2-6]. Thousands of viruses infecting representatives of the domain Eukarya have been described and many of their DNA/RNA genomic sequences determined [7]. Something like 5– Published: 9 November 2007 BMC Genomics 2007, 8:410 doi:10.1186/1471-2164-8-410 Received: 12 July 2007 Accepted: 9 November 2007 This article is available from: http://www.biomedcentral.com/1471-2164/8/410 © 2007 Pagaling et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 2 of 13 (page number not for citation purposes) 6000 viruses (bacteriophages) infecting representatives of the domain Bacteria have been described, at least morphologically, although rather fewer DNA/RNA genomic sequences have been determined [8]. In contrast we are largely ignorant about viruses infecting representatives of the domain Archaea. Just 40 or so have been described and the genomic sequences of only a few have been determined, sixteen being listed in Genbank. All archaeal viruses so far discovered have dsDNA genomes, both linear and circular [8,9]. Archaeal viruses having an RNA genome have not yet been identified and perhaps do not exist [9]. The domain Archaea is divided into four established kingdoms, the Crenarchaeota, the Euryarchaeota, the uncultivated Korarchaeota and the very recently identified Nanoarchaeota [10,11]. Virus particles associated with the first two phyla have been identified, recently reviewed in [9]. About 24 viruses of crenarchaeotes have been identified, often with unusual shapes, e.g. droplets and bottle shapes never observed elsewhere; these viruses have no obvious relationship to phage infecting members of the domain Bacteria [8,9]. Similarly about 20 viruses infecting members of the Euryarchaeota have been identified of which 15 infect haloarchaea, recently reviewed in [12]. These are mostly head/tail viruses of the order Caudovirales, including myoviruses and siphoviruses that may be distantly related to those infecting the domain Bacteria [8,9]; although other morphotypes have also been observed [12]. Only six viruses of the haloarchaea have been sequenced. All were isolated by the Dyall-Smith laboratory in Melbourne, from hypersaline sources in Australia, except for φ Ch1. φ Ch1, a temperate myovirus with a 58.5 kb linear genome, the host of which is the haloalkaliphile Natrialba magadii [13] was isolated from a laboratory strain and presumably originates, like the host, from Africa. Lytic viruses HF1 and the closely related HF2, having linear genomes of 75.9 kb and 77.7 kb, infect the haloarchaea Haloferax lucentense and Halorubrum coriense respectively [14,15]. His1 and the distantly related His2 spindle shaped viruses with linear genomes of 14.5 and 16 kb respectively, both have lytic and carrier status in Haloarcula hispanica [16]. Finally a lytic icosahedral virus SH1, having a linear genome of 31 kb infects Har. hispanica [17,18]. We have been studying both archaeal and bacterial prokaryotic diversity in Chinese salt lakes in Inner Mongolia; as part of this study we looked for virus particles associated with haloarchaea. In this report we describe the complete genomic sequence of a ~43 kb virus BJ1. Results Description of site and lake water parameters Lake Bagaejinnor is a hypersaline lake in Inner Mongolia, China [coordinates N45 08 527 E116 36 167]. The lake was sampled in September 2003. It had substantially evaporated over the summer, exposing expanses of [pink salt – encrusted] mud flats and had been reduced to small pools and lagoons of salt – saturated colourless water, pH 8.5. The pink colouration of the salt crystals indicated the presence of haloarchaea. The chemical composition of lake water was determined using laser inductively coupled plasma optical emission spectrometry by the Department of Geology, University of Leicester. Carbonate/bicarbonate concentrations were determined by titration with H2SO4 using a Digital Titrator Model 16900 according to manufacturer's instructions (Hach Systems for Analysis). Chemical concentrations were Na, 5.32 M; Cl, 4.61 M; S 1.07 M; Mg, 0.35 M; K, 33.25 mM; Br, 8.05 mM; HCO3, 7.4 mM; B, 4.25 mM; CO3, 3.3 mM; Ca, 0.77 mM; Li, 0.33 mM. Obviously this is a seasonal chemical analysis of the lake water, the composition of which continually varies, more dilute in spring following the winter thaw and then gradually becoming concentrated by the hot summer winds. We used trial and error techniques to find an appropriate medium where we could pour both top and bottom agars. Medium composition was influenced by very high salt concentrations interfering with agar solidification and causing "salting out" of some of the components. The eventual salt composition of this medium was identical to that determined for the lake above with the following exceptions; Na was at 2.85 M, Cl was at 2.6 M, S was at 0.642 M, Ca and Li were omitted completely. Identification of a haloarchaeal host Virus BJ1 was isolated from the water column of Lake Bagaejinnor and propagated using strain BJ1 B11. The host was characterised by 16S rRNA gene sequence using both forward and reverse primers, giving 1305 bp of sequence [EMBL: AM412370]. Strain BJ1 B11is most closely related, at 98% identity, to Halorubrum saccharovorum with 1289 identical nucleotides. It is also closely related to Hrr. lacusprofundi (1283 identical nucleotides) and the recently described Hrr. aidingense (1286 identical nucleotides). All three species were originally isolated from hypersaline environments, a salt pan in San Francisco, Deep lake Antarctica and Xin-Jiang in China respectively [19,20]. Fig 1 is a phylogenetic tree showing the relationship of the BJ1 B11 isolate to other closely related sequences present in the BLAST database. Plaque morphology Plaques for BJ1 required one to two weeks to appear on plates because the host is slow growing. Plaque size for BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 3 of 13 (page number not for citation purposes) BJ1 was variable between experiments ranging from 1–5 mm in diameter, probably due to slight changes in growth conditions; they were also irregularly shaped and turbid. No attempt was made to optimise plaque formation by modifying temperature, salt concentrations or host strain. Electron microscopy Virus BJ1 has an icosahedral head, collar and tail, (Fig. 2). The icosahedral head usually has an electron dense shadowing in the centre. The sizes of these features are shown in the schematic diagram Fig 2. The length of a single vertex is 28 nm. The average length of an entire virus particle is about 127 nm. The virus appears to be non-contractile and can be tentatively assigned to the Siphoviridae family, (see the Discussion). Characterisation of virus genome The genomic nucleic acid was tested for susceptibility to various nucleases (Fig 3) Control experiments showed that no virus – associated nucleases were responsible for the degradation observed in these experiments. Fig. 3, lanes 1 and 4 show undigested genome controls, lane 2 shows that the genome was sensitive to DNase I digestion and lane 3 shows that the genome was insensitive to RNAse A. Susceptibility to a wide range of double strand – specific endonucleases i.e BamHI, HaeIII, SstI and XhoI, confirmed that the DNA was double stranded e.g. (Fig 3, panel c). Exonuclease III, specific for linear or nicked circular dsDNA, failed to cut circular double stranded DNA plasmid DNA controls (not shown) but substantially degraded virus genomic DNA (Fig 3 panel a, lane 5). Thus BJ1 probably has a linear dsDNA genome, although the possibility that it is a nicked circular genome cannot be completely ruled out. Genomic nucleic acid ran on 1.2% TAE agarose gels as a discrete single band larger than a 23 kb DNA marker band. (data not shown). PFGE also suggested a genomic size greater than 23 kb but less than 48 kb (Fig 3, panel b). BamH1 digestion of the genomic DNA gave 21 discrete bands ranging in size from 6.5 kb to ~500 bp (Fig 3, panel c). From the size of these fragments we estimated a genome size of 42.7 kb, remarkably close to the size eventually determined by sequencing (42.271 kb, see below). In silico digestion of the determined sequence with BamHI showed that it would generate 20 different fragments i.e. 4949, 4661, 3762, 3235, 3185, 2952, 2434, 2406, 2004, 1949, 1679, 1617, 1505, 1314, 1275, 1094, 816, 781, Unrooted phylogenetic tree showing the relationship of the environmental archaeal strain host BJ1B11 for the virus BJ1, to other closely related environmental strains isolated by us and Halorubrum speciesFigure 1 Unrooted phylogenetic tree showing the relationship of the environmental archaeal strain host BJ1B11 for the virus BJ1, to other closely related environmental strains isolated by us and Halorubrum species. The scale bar represents the number of inferred nucleotide substitutions per site. Values at nodes indicate >50% percentage occurrence in 500 bootstrapped trees. BJ1A4 BJ1B11 BJ1A12B BJ1C11 SH1D13 Halorubrum sp. 31-hong (DQ355813) Halorubrum saccharovorum (U17364) Halorubrum lacusprofundi (X82170) EN1C10A Halorubrum sp. F100 (DQ309090) EJ3B3B Halophilic archaeon NaxosII (AJ400624) EN1D10 Haloarcula sp. AJ4 (AY208973) 100 100 91 100 100 91 60 67 99 67 0.02 0.02 substitutions/site Halorubrum aidingense strain 31-hong (DQ355813) Halorubrum saccharovorum (U17364) Halorubrum lacusprofundi (X82170) Halorubrum sp. F100 (DQ309090) Haloarcula sp. AJ4 (AY208973) BJ1A4 BJ1B11 BJ1A12B BJ1C11 SH1D13 Halorubrum sp. 31-hong (DQ355813) Halorubrum saccharovorum (U17364) Halorubrum lacusprofundi (X82170) EN1C10A Halorubrum sp. F100 (DQ309090) EJ3B3B Halophilic archaeon NaxosII (AJ400624) EN1D10 Haloarcula sp. AJ4 (AY208973) 100 100 91 100 100 91 60 67 99 67 0.02 0.02 substitutions/site Halorubrum aidingense strain 31-hong (DQ355813) Halorubrum saccharovorum (U17364) Halorubrum lacusprofundi (X82170) Halorubrum sp. F100 (DQ309090) Haloarcula sp. AJ4 (AY208973) BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 4 of 13 (page number not for citation purposes) 563, and 90 bps, with sizes in close agreement to those we observed. Thus the genomic DNA is not subject to methylation at BamHI sites. Genome sequence of BJ1 See Figure 4 and Table 1. The double stranded genomic DNA isolated from virus particles is shown as a circular sequence 42, 271 bp long with a G+C content of 64.8 mol% [EMBL: AM419438]. Exonuclease III susceptibility showed that the DNA is linear but sequence assembly indicated it to be circular. This indicates that the genome is terminally redundant (and may be circularly permuted). It is unclear if the BJ1 genome ever forms a circular molecule but if it does then cos sites are unlikely to be involved as digests with three infrequent cutting restriction enzymes (HindIII, EcoRV and EcoRI) followed by melting at 80°C failed to show any change in the number of bands compared to un-melted digests (data not shown). In the absence of an obvious end for the genome from our sequencing experiments we analysed the cumulative GC skew of the sequence (Fig. 4). Skew minima and maxima often represent initiation and termination points of DNA replication in prokaryotes and viruses with a cumulative increase in skew related to the direction of replication and transcription [21]. A clear maximum was observed at about 43000 followed by a sharp change with the minima from 1–8000. This in conjunction with the ORF map and pattern of operons was used to designate a +1 start of the genome (Fig. 4). The cumulative GC skew is consistent with the reading direction of most ORFs and a rolling circle pattern of DNA replication. A single tRNA for phenylalanine (GAA anticodon recognising a UUC codon) was identified using the tRNAscan-SE program. Potential ORFs were assigned using the programs FGENESB and GeneMark.hmm v2.5a (set for prokaryotes); these analyses predicted 63 and 66 ORFs, respectively, encoding polypeptides larger than 30 amino acids. We further analysed the regions upstream and downstream of these predicted ORFs for putative ribosome binding sites and overlapping start and stop codons, and found several additional ORFs. BLAST searches using the amino acid sequences of all predicted ORFs were used to differentiate between possible genes e.g. ORFs 5 and 6 have matches (see below), so putative ORFs in the opposite strand with no BLAST matches have been discounted. By combining all of the data we conclude that BJ1 probably contains 70 ORFs (Fig 4 and Table 1). [If we only count ORFs greater than 60 aa in size then the number of ORFs drops to 55]. Taking the upper estimate of 70 gives an ORF density of 1.65/kb. This is fairly close to the figure of 1.7 ORFs/kb observed for other archaeal virus genomes (17). The majority of the ORFs have initiation codons of ATG (62) and the rest are GTG (8). The Shine/Dalgarno sequence from Halobacterium (Halorubrum) saccharovorum 16S rRNA gene sequence (Accession HSU17364), which is the closest phylogenetic match to the phage host was complemented (AGGAGGUGA) and used to search 5–15 bp upstream of each putative start site for the presence of putative ribosome binding sites (RBS). 51 of the 70 ORFs had sequences suggestive of a RBS, (Table 1). One particular stretch of 6 predicted ORFs (ORF43-ORF48) showed no obvious RBSs at all. A lack of a RBS for some genes is not surprising as archaeal transcription/translation is a mosaic of prokaryotic and eukaryotic mechanisms and the first gene of an operon, or a singly transcribed gene often lacks a RBS [22-24]. The majority of the ORFs (59/70) had a low calculated isoelectric point (pI < 5), which is similar to the acidic proteins of halophilic organisms [15,25]. Just three small ORFs (less than 74 aa) were predicted to be extremely basic (pI > 10). No ORF larger than 100 aa had a pI above 6.3. 63 ORFs and the tRNA are coded on one strand (designated forward) and 7 are on the reverse strand. One ORF, 30 (13255–14700 bp) overlaps entirely with Electron micrograph images of BJ1; the scale bar is 500 nm, top panel and 200 nm bottom panelFigure 2 Electron micrograph images of BJ1; the scale bar is 500 nm, top panel and 200 nm bottom panel. A schematic diagram of BJ1 annotated with discernible features and the size of these features is also shown. The standard deviation (SD) of measurements from twenty six different particles was determined. BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 5 of 13 (page number not for citation purposes) another, ORF31 (13270–14487 bp), running in the opposite direction. It seems probable that both ORFs are coding, ORF30 because it overlaps with the start and stop codons of the ORFs before and after it i.e. 29 and 32, with a good consensus RBS; ORF31 because it shows significant homology to integrases, (see below). BJ1 ORF analysis BlastN analysis of the whole virus genome showed significant matches (E 10-9 to 10-4) to small segments of several haloarchaeal sequences i.e. Natronomonas pharaonis, Halobacterium sp. NRC-1 and Har. marismortui. BlastX analysis identified four regions of the genome having significant matches to data-base proteins either from haloviruses or haloarchaea, discussed below. The putative ORFs were individually analysed using BlastX and BlastP. InterPro was also used to search for functional domains. Using these approaches we were unable to ascribe any match or function to 50 of the 70 ORFs i.e. E values were greater than 0.05. Of the 20 we could match i.e. E value less than 0.05, most were to haloarchaeal virus entries or to haloarchaea. These results are summarised in Table 2. Of these 20, 4 were matches to data-base entries with no identifiable function, i.e.: ORF9, ORF10, ORF17, ORF55 and ORF 24. The remaining 15 ORFs could have functions tentatively ascribed to them on the basis of amino acid similarity, (Table 2). We place them into three groups. (i) Those probably concerned with DNA replication, gene expression and possibly integration, i.e. ORFs 5, 6, 16, 20, 21, 31, 35, 39 and 43. (ii) Those proteins likely to be involved in virus assembly, i.e. ORFs 48, 49, 50, 52 and 53. (iii) Those proteins with other identifiable functions, i.e. ORF1. Nucleotide features Nine direct repeats were observed greater than 13 nucleotides; the largest was 17 nucleotides, i.e. GGCGGCATCCAACTCGG repeated at positions 34076 and 34120. All of the repeats were located in putative ORFs and we can infer nothing of significance for them. A number of per- Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleasesFigure 3 Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleases. Lanes 1 and 4 , undigested controls; Lane 2, DNAse treated; Lane 3 RNase treated; Lane 5, exonuclease III treated. Panel b, 1% agarose 0.5× TBE pulse field gel; lanes 1 and 4 size markers (kbps), lanes 2 and 3 BJ1 virus genome. Panel c, BamH1 enzyme digest of virus BJ1 genomic DNA, DNA size markers are shown on the left (kbps). The image has been overexposed to show the smaller bands. 1 2 3 4 51 2 3 4 5 a bc BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 6 of 13 (page number not for citation purposes) Table 1: Predicted ORFs in virus BJ1 ORF Start Stop aa Mr pI RBS/distance 1130 990 286 33 4.6 - 21146 1805 219 25 4.9 - 3- 1980 2093 37 3.7 8.5 GGAGGTG-5 42207 2425 72 8.1 7.0 - 5- 2541 3191 216 24 4.7 GAGG-10 6- 3178 3393 71 8.2 4.3 - 7 V 3547 3993 148 16 4.1 GAG-6 83993 4463 156 18 4.3 AGGAGGTGA-8 94456 4851 131 15 4.2 AGGAGGTGA-7 10 4844 5218 124 14 4.7 GGAGGT-6 11 5208 5357 49 5.2 3.8 GAGGTG-8 12 5350 5574 74 8.3 4.6 AGGAGGT-6 13 5571 5744 57 6.1 10.4 GGAGG-5 14 5741 5986 81 8.9 5.8 GGAGG-8 15- 5998 6417 139 15 4.3 GAGG-7 16 6637 7713 358 40 5.0 AGGTG-9 17- 7919 8560 213 24 4.3 AGGA-8 18 8689 8949 86 9.3 4.9 - 19 8950 9153 67 7.9 5.2 GGTG-10 20- 9159944695114.6GGAG-4 21 9660 10022 120 14 4.6 GGA-7 22 10022 10153 43 4.6 4.0 GGTG-8 23 10153 10890 245 28 3.9 GGAGG-8 24 10880 11806 308 34 4.3 GGAGG-9 25 V 11803 11946 47 5.2 4.1 GGTGA-7 26 11946 12671 241 27 4.7 GGTGA-7 27 V 12668 12760 30 3.3 4.5 GGAGGTG-6 28 12757 13092 111 12.2 5.8 GAGGTGA-5 29 13092 13262 56 6.2 3.8 GGAGG-8 30 13255 14700 481 52 6.2 AGGAGG-6 31- 13270 14487 405 46 5.0 - 32 14701 14826 41 4.3 4.0 GGAGGTGA-9 33 14819 15307 162 18 4.6 GAGGTGA-7 34 15310 15531 73 83 11.6 AGGAGGTG-9 35 15489 17603 704 78 4.7 (GAAAA) 36 17606 18058 150 17 4.4 GGAGG-9 ORF Start Stop aa Mr pI RBS/distance 37 18055 18519 154 18 4.3 (GGGGG) 38 V 18512 18817 101 11 5.0 GAGGTG-8 39 V 18814 19074 86 9.9 6.1 GAGGTG-9 40 V 19071 19241 56 5.9 10.3 GGAGG-8 41 V 19129 19806 225 26 6.3 - 42 19803 19982 59 6.4 4.0 GAGGTG-6 tRNA 19973 20046 - - - - 43 20365 21843 492 55 4.9 - 44 21840 21998 52 5.9 4.3 - 45 22001 22111 36 3.9 4.8 - 46 22108 22416 102 11 4.3 - 47 22416 22577 53 6.1 4.2 - 48 22574 23083 169 19 4.3 - 49 23080 24423 447 50 4.9 GAGG-8 50 24427 26382 651 73 4.5 - 51 26461 26586 41 4.4 4.4 GAG-9 52 26590 27933 447 47. 3.9 AGGAGG-9 53 27949 29031 360 40 4.2 GTGA-8 54 29040 29219 59 6.4 3.8 GAGGTGA-4 55 29222 29572 116 12 3.9 - 56 29576 30451 291 33 4.6 GGAGG-9 57 30444 30761 105 11 4.1 - BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 7 of 13 (page number not for citation purposes) fect and imperfect inverted repeat/stemloop structures were identified, often having loops 100 s–1000 s of nucleotides in size. One perfect palindrome is located at nucleotides 14226GTCCGCTGGA/TCCAGCGGAC14247 in ORF31, the putative integrase gene. Another palindrome separated by 3 nucleotides (lower case) is 42048ACTATCCGACtggGTCGGATAGT42070; again both are present in putative ORFs and their significance is unclear although the last palindrome is located 209 nucleotides from the 3' end of the genome. The BJ1 genome has a low incidence of CTAG and GATC sequences, just three of each of these palindromes being present. This incidence is low, both compared to the statistically expected incidence, (every 256 base pairs) and compared to the related tetramers CGAG and GCTC which were both found 36 times. CTAG and GATC sequences appear to be selected against by many haloviruses e.g. these palindromes are absent from the genomes of HF1, HF2, His2 and SH1 [6]. This selection pressure is thought to be due to the avoidance of restriction-modification systems in the host cells [26], and there is evidence that CTAG and GATC palindromes are used by haloarchaeal systems [27,28]. Sequence heterogeneity BamHI digests of virion DNA gave rise to a fragment of about 3.5 kb, as judged by agarose gel electrophoresis, present in sub-stoichiometric amounts relative to the other bands, indicated by the black arrow in Fig 3c. This was fully sequenced and found not to fit into our genome assembly. Primers derived from this sequence were used with virus sequence primers and virus genomic DNA as a template. Products were observed with primers derived from the 3' end of ORF 32, suggesting that a minor subfraction of virion DNA did contain this BamHI fragment. Sequencing showed that the site of insertion was at nucleotide 14790 in ORF 32 and showed that this part of ORF 32 was rich in CGX repeats, (Table 3). We have not yet been unable to derive PCR products defining either the location or 5' end of the insertion/substitution. Instead we have primer walked out from the defined 3' end of the insertion. ~8.7 kb of sequence has been determined [EMBL: AM491333] having a G+C content of 72.6 mol%, notably higher than the 64 mol% determined for the rest of the virus genome and close to that reported for Hrr. saccharovorum (71 mol%). Predicted ORFs have much higher homologies to known haloarchaeal proteins than the other viral ORFs, (Table 3). We think it most likely this sequence is derived from the host genomic DNA due to an integration/excision event. Discussion Morphological criteria used for virus classification is outlined by the International Committee for Taxonomy of Viruses [7]. Virus BJ1 is an icosahedral head/tailed virus and as such is assigned to the order Caudovirales with examples infecting members of both the domains Bacteria and Archaea. BJ1 can also be assigned to the Bradley classification group B and might tentatively be assigned to the family Siphoviridae due to the apparent absence of a contractile tail, base plate and tail fibres and the presence of striations in the tail fibre. If we assume that this classification is phylogenetically justified then it could indicate that the Caudovirales originated before the divergence of the Bacteria and Archaea [29]. An alternative explanation is that the Caudovirales originally infected members of the domain Bacteria but that horizontal gene exchange from mesophilic Bacteria to the Archaea and the subsequent stabilisation of these genes in the Archaea allowed the Caudovirales to spread into the domain Archaea [Certainly we have detected diverse bacterial populations in the water of Lake Bagaejinnor, SH unpublished] [9]. As described in the Introduction, very few viruses infecting the domain Archaea have been described and as yet we have little idea as to the extent of virus diversity in this domain. The virus we describe here may not be a common or dominant member of the virus community infecting haloarchaea in saline waters. We screened for lytic virus 58 30758 31210 150 17 4.8 AGG-10 59 31207 31734 175 20 4.5 GGAGGT-5 60 V 31766 32680 304 32 3.8 GAGGTGA-7 61 32680 33177 165 18 4.0 AGGAGGTGA-8 62 33281 34408 375 38 4.1 - 63 34444 34731 95 10 4.8 - 64 34771 35439 222 24 4.0 - 65 35446 36633 395 42 4.1 TGA-7 66 36634 38226 530 52 3.7 AGGAGGTG-10 67 38229 40979 916 100 4.0 GGAGGTG-15 68 41059 41400 113 12 3.8 GGAG-6 69 41403 41843 146 16 4.6 AGGTG-9 70 41840 42151 103 11 3.9 GGTGA-4 Orfs are in the forward direction unless indicated by a -ve sign. v indicates a valine start. aa indicates the number of amino acids. Mr is the molecular mass × 10-3, rounded to the nearest 100. pI is the isoelectric point rounded to one decimal place. rbs/distance is the ribosome binding site sequence and its distance from the start codon. Table 1: Predicted ORFs in virus BJ1 (Continued) BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 8 of 13 (page number not for citation purposes) particles forming plaques on archaeal lawns. These requirements for host culturability, good lawn formation and plaque formation are probably extremely restrictive. As pointed out by others, there is a genuine need to develop other isolation and culture techniques to study both the dominant virus populations and the true extent Top panelFigure 4 Top panel. Diagram of the BJ1 genome drawn in a circular form. The major features are shown including the predicted ORFs, blue arrows in the forward direction, green arrows in the reverse. The tRNA gene is in red. ORFs mentioned in the text are numbered. The outer scale bar is in base pairs. The inner curved arrows indicate entirely hypothetical operons. The bottom panel shows the cumulative GC skew. BMC Genomics 2007, 8:410 http://www.biomedcentral.com/1471-2164/8/410 Page 9 of 13 (page number not for citation purposes) of archaeal virus variation in samples such as these – perhaps using a combination of electron microscopy and metagenomic sequence studies. The GC content of BJ1 at 65 mol% is quite close to that reported for Hrr. spp aidingense, lacusprofundi and saccharovorum, varying from about 63–71 mol% [19,20]. The host strain for BJ1 clearly belongs to the genus Halorubrum having 98% 16SrRNA gene sequence identity to these Halorubrum species. Its precise taxonomic relationship to these species, in particular if it belongs to a new Halorubrum species is the subject of current studies. Of the ORFs identified in BJ1 described in the results, all of the statistically significant matches are recorded, (Table 2). Six of the ORFs (9, 20, 50, 52, 53, 55) are most closely related to the haloarchaeal temperate, isometric head/ contractile tail viruses φ Ch1 [13] and the intensively studied, φ H [30]. These two viruses are closely related to each other, the completed genome of φ Ch1 shows 97% homology to the genome of φ H, which is about 60% complete. ORF 43 is most closely related to a gene from the haloarchaeal isometric head/contractile tail virus HF1. There are no similarities with the ORFs from either the spindle (His1, His2) or icosahedral (SH1) shaped haloarchaeal viruses described in the Introduction. The most significant matches were ORFs 16, 31, 35, which are almost certainly the origin of replication complex, integrase and helicase functions respectively of the virus, having highly significant matches to full length proteins in Har. marismortui. ORF50 was also closely related to the putative portal protein (NP_665924) of Nab. magadii virus φ Ch1. Speculatively, almost all ORFs are in the forward strand in the same direction consistent with a rolling circle mechanism of DNA replication. The 7 ORFs on the reverse strand including the integrase may be poorly expressed. A few ORFs had GTG starts (but with good RBS sequences) and the other ORFs lacked RBS sequences altogether, presumably both coding features control/reduce expression levels. The fact that putative Int gene is coded for on the minor strand with no RBS and that it overlaps with ORF 30 on the major strand may indicate that its expression is tightly controlled; perhaps most infections are lytic with a small proportion of lysogenic events. The suggestion of operons indicated in Fig 4 is also entirely speculative and based on the presence of overlapping stop and start signals, one run of ORFs from 43–48 has no RBS at all. Proteins with putative functions involved in DNA replication and transcription are found in ORFs 1–43, putative structural proteins are found after ORF48 consistent with early and late expression of operons. Although BJ1 stocks are clonal in origin, the genomic DNA preparation is obviously and necessarily derived from a virus pool. Genome sequence projects often therefore give rise to heterogeneous sequences. We found one substantial region of heterogeneity in ORF 32 at nucleotide 14790 involving either a large insertion or more probably a substitution event (since terminally redundant virus genomes usually package genomes in a 'head full' mechanism). To distinguish between these possibilities Table 2: BJ1 ORFs with identifiable BlastX matches to data base entries. ORF Homologs (% Identity) 9 59% similarity (E 10-8) to ORF58 halovirus φ Ch1 (AAM88732) 10 54% similarity (E10-5) to protein Haloquadratum walsbyi (CAJ52235) 17 similarity (E 10-13) to protein from Natronomonas pharaonis (CR936257.1) 55 similarity (E 10-7) to a protein of φ Ch1 (NP 665930.1) 24 No significant match to any described protein. InterPro suggests DNA binding protein 5 similarity (E 10-3) to bacterial proteins with DnaJ domain; role in DNA replication? 6 65% similarity (E 10-6) to protein (AAG20925) of Halobacterium sp. NRC-1. A metal regulated homodimeric repressor with a 'winged helix' DNA binding domain 16 60% similarity (E 10-67) to a Har. marismortui protein YP_136906; member of the ORC1/CDC-6 superfamily of NTPases involved in DNA replication 20 54% similarity (E 10-13) to halovirus φ H1 repressor protein(AAV47198.1) with a winged helix DNA binding domain 21 66% similarity (E 10-17) to the Hqr. walsbyi PadR transcriptional regulator (CAJ51359.1) 31 similarity is to a Har. marismortui phage integrase (E 10-66) 45% ID (AAV47153 the λ bacteriophage recombinase family, pfam00589 35 DNA helicase? 62% similarity (E 10-128) to Har. marismortui protein (AAV47142) of the Cdc-46/Mcm family of DNA dependent ATPases. 39 68% similarity (E 0.05) to ArsR-like transcriptional regulator (CAJ51299) from Hqr. Walsbyi (92 amino acids long); the similarity being from amino acids 15–68 in ORF39 with 20–72 in CAJ51299 43 56% similarity (E 10-37), to halovirus HF1 protein (AAO61337.1) which may be a YonJ like, small subunit of the DNA polymerase, (COG1311) 48 54% similarity to Listonella pelagia phage phiHSIC small terminase subunit (YP_224235.1 49 43% similarity (E 0.01) to Streptococcus pneumoniae bacteriophage EJ-1 large terminase (CAE82121) 50 54% similarity (E 10-77) to the putative portal protein (NP_665924) of Nab. magadii virus φ Ch1. 52 49% similarity (E 10-13) to the capsid protein gpD (AAM88683) of halovirus φ Ch1 53 48% similarity (E 10-29) to hp32 (CAA56442) of Hbt. salinarum virus φ H and 47% similarity (E 10-24) to the capsid protein gpE (AAG32163) of halovirus φ Ch1 51 51% similarity (E 10-15) to Enterococcus faecium glycosyl transferase (EAN10921). LPS biosynthesis protein.