Kate Porter

Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds

Haloquadratum walsbyi is frequently a dominant member of the microbial communities in hypersaline waters. 16S rRNA gene sequences indicate that divergence within this species is very low but relatively few sites have been examined, particularly in the southern hemisphere. The diversity of Haloquadratum was examined in three coastal, but geographically distant saltern crystallizer ponds in Australia, using both culture-independent and culture-dependent methods. Two 97%-OTU, comprising Haloquadratum- and Halorubrum-related sequences, were shared by all three sites, with the former OTU representing about 40% of the sequences recovered at each site. Sequences 99.5% identical to that of Hqr. walsbyi C23T were present at all three sites and, overall, 98% of the Haloquadratum-related sequences displayed ≤2% divergence from that of the type strain. While haloarchaeal diversity at each site was relatively low (9–16 OTUs), seven phylogroups (clones and/or isolates) and 4 different clones showed ≤90% sequence identity to classified taxa, and appear to represent novel genera. Six of these branched together in phylogenetic tree reconstructions, forming a clade (MSP8-clade) whose members were only distantly related to classified taxa. Such sequences have only rarely been previously detected but were found at all three Australian crystallizers.

PH1: An Archaeovirus of Haloarcula hispanica Related to SH1 and HHIV-2

Halovirus PH1 infects Haloarcula hispanica and was isolated from an Australian salt lake. The burst size in single-step growth conditions was 50–100 PFU/cell, but cell density did not decrease until well after the rise (4–6 hr p.i.), indicating that the virus could exit without cell lysis. Virions were round, 51 nm in diameter, displayed a layered capsid structure, and were sensitive to chloroform and lowered salt concentration. The genome is linear dsDNA, 28,064 bp in length, with 337 bp terminal repeats and terminal proteins, and could transfect haloarchaeal species belonging to five different genera. The genome is predicted to carry 49 ORFs, including those for structural proteins, several of which were identified by mass spectroscopy. The close similarity of PH1 to SH1 (74% nucleotide identity) allowed a detailed description and analysis of the differences (divergent regions) between the two genomes, including the detection of repeat-mediated deletions. The relationship of SH1-like and pleolipoviruses to previously described genomic loci of virus and plasmid-related elements (ViPREs) of haloarchaea revealed an extensive level of recombination between the known haloviruses. PH1 is a member of the same virus group as SH1 and HHIV-2, and we propose the name halosphaerovirus to accommodate these viruses.

Transfection of haloarchaea by the DNAs of spindle and round haloviruses and the use of transposon mutagenesis to identify non-essential regions

Spindle‐shaped halovirus His2 and spherical halovirus SH1 represent ecologically dominant virus morphotypes in high‐salt environments. Both have linear dsDNA genomes with inverted terminal repeat sequences and terminal proteins, and probably replicate using protein priming. As a first step towards conventional genetic analyses on these viruses, we show that purified viral DNAs can transfect host cells. Intact terminal proteins were essential for this process. Despite the narrow host ranges of these viruses, at least under laboratory conditions, their DNAs were able to transfect a wide range of haloarchaeal species, demonstrating that the cytoplasms of diverse haloarchaea possess all the factors necessary for viral DNA synthesis and virion assembly. Transposon mutagenesis of viral DNAs was then used in conjunction with transfection to produce recombinant viruses, and to then map the insertion sites to identify non‐essential genes. The inserts in 34 His2 mutants were mapped precisely, and most clustered in a few, specific regions, particularly in the inverted terminal repeats and near the ends of ORFs. The results are consistent with the small genome size and densely packed, often overlapping ORFs that are transcribed as long operons. This study is the first demonstration of transfection and transposon mutagenesis in protein‐primed archaeal viruses.

28 The Isolation and Study of Viruses of Halophilic Microorganisms

Publisher Summary This chapter discusses viruses of the extremely halophilic Archaea (family halobacteriaceae), since members of the latter group make up the great majority of microbes living in the salt-saturated waters of salt lakes and saltern crystallizer ponds. Haloarchaeal viruses (haloviruses) were studied for some decades, but much of the early work was severely hampered, as in other areas of environmental microbiology, by the inability to culture the dominant haloarchaea present in salt lakes. The ecological importance of haloviruses is demonstrated by direct electron-microscopy of salt lake waters, where high levels of virus-like particles are observed, many with unusual morphologies (lemon-shaped or round particles). Many of the haloviruses that are studied intensively at the molecular level are temperate and are isolated from lysogenic laboratory strains of haloarchaea. Methods for some of the more basic, virological parameters, particularly those that are more specific for haloviruses are described. One of the most important tests is to determine whether isolates can infect one (monovalent) or more than a single species of host (divalent, polyvalent). Examination of halovirus preparations by negative-stain transmission electron microscopy (TEM) is widely used to determine particle morphology, purity, and quality.

Viruses Infecting Euryarchaea

The viruses and virus-like particles of the methanogens, extreme halophiles, and hyperthermophiles of the archaeal kingdom Euryarchaeota are represented by relatively few isolates, but show a remarkable degree of diversity, with three broad morphological categories. Head-and-tail viruses with similar genome structures to head-and-tail bacteriophages infect both extreme halophiles and methanogens. Of these viruses, the methanophage ψM1 and relatives show some sequence similarity to Bacteria and bacteriophages, while the genomes of haloviruses HF1 and HF2 show little similarity to known sequences but are closely related to one another and provide evidence of high recombination rates. Haloviruses ΦCh1 and ΦH form another related group, even though they originate from different environments and infect different hosts, suggesting that similar viruses are widespread. The spindle-shaped haloviruses such as His1 and His2, and the hyperthermophilic VLP PAV1, morphologically resemble archaeal fuselloviruses such as SSV1, but are genetically distinct and form novel virus groups. Finally, spherical viruses such as halovirus SH1 contain an internal lipid layer, similar to tectiviruses (including bacteriophage PRD1) and the crenarchaeal virus STIV, but share no sequence similarity with these viruses and little similarity with other known sequences. Ongoing studies of these unusual viruses continue to reveal unexpected and interesting characteristics.

A strange family, or how a new pleolipovirus reveals its friends and relatives

A new virus of halophilic Archaea is reported by Liu et al., and is remarkable in many ways. SNJ2 is the first temperate, pleomorphic virus (pleolipovirus) that integrates into the genome of its host. Analyses of the virus structure and its genome have provided an unexpected puzzle while at the same time solving another. On the one hand, the study shows a curious relationship exists between SNJ2 and an unrelated provirus (SNJ1) found as a plasmid in the same cell. The presence of SNJ1 appears to allow much higher levels of SNJ2 virus to be produced, although the mechanism involved remains unclear. On the other hand, the curious occurrence of a conserved cluster of pleolipovirus‐related genes found widely distributed among haloarchaeal genomes and known for almost 10 years, now appears to correspond to SNJ2‐related proviruses.