Global Distribution and Diversity of Haloarchaeal pL6-Family Plasmids.

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts.

The Novel Halovirus Hardycor1, and the Presence of Active (Induced) Proviruses in Four Haloarchaea.

The virus Hardycor1 was isolated in 1998 and infects the haloarchaeon Halorubrum coriense. DNA from a frozen stock (HC1) was sequenced and the viral genome found to be 45,142 bp of dsDNA, probably having redundant, circularly permuted termini. The genome showed little similarity (BLASTn) to known viruses. Only twenty-two of the 53 (41%) predicted proteins were significantly similar to sequences in the NCBI nr protein database (E-value ≤ 10-15). Six caudovirus-like proteins were encoded, including large subunit terminase (TerL), major capsid protein (Mcp) and tape measure protein (Tmp). Hardycor1 was predicted to be a siphovirus (VIRFAM). No close relationship to other viruses was found using phylogenetic tree reconstructions based on TerL and Mcp. Unexpectedly, the sequenced virus stock HC1 also revealed two induced proviruses of the host: a siphovirus (Humcor1) and a pleolipovirus (Humcor2). A re-examination of other similarly sequenced, archival virus stocks revealed induced proviruses of Haloferax volcanii, Haloferax gibbonsii and Haloarcula hispanica, three of which were pleolipoviruses. One provirus (Halfvol2) of Hfx. volcanii showed little similarity (BLASTn) to known viruses and probably represents a novel virus group. The attP sequences of many pleolipoproviruses were found to be embedded in a newly detected coding sequence, split in the provirus state, that spans between genes for integrase and a downstream CxxC-motif protein. This gene might play an important role in regulation of the temperate state.

Insights into gene regulation of the halovirus His2 infecting Haloarcula hispanica.

Gene expression in Haloarcula hispanica cells infected with the gammapleolipovirus His2 was studied using a custom DNA microarray. Total RNA from cells sampled at 0, 1, 2, 3, and 4.5 hr postinfection was reverse-transcribed into labeled cDNA and hybridized to microarrays, revealing temporal and differential expression in both host and viral genes. His2 gene expression occurred in three main phases (early, middle, and late), and by 4.5 hr p.i. the majority of genes were actively transcribed, including those encoding the major structural proteins. Eighty host genes were differentially regulated ≥twofold postinfection, with most of them predicted to be involved in transport, translation, and metabolism. Differentially expressed host genes could also be grouped into early-, middle-, and late-expressed genes based on the timing of their up- and downregulation postinfection. The altered host transcriptional pattern suggests regulation by His2 infection, which may reprogram host metabolism to facilitate its own DNA replication and propagation. This study enhances the characterization of many hypothetical viral genes and provides insights into the interaction between His2 and its host.

The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules.

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.