Metagenomic approach to the study of halophages: the environmental halophage 1

Abstract

Summary Hypersaline environments, such as crystallizer ponds of solar salterns, show one of the highest concentration of viruses reported for aquatic systems. All the halophages characterized so far are isolates obtained by cultivation from described haloarchaeal species that have only low abundance in the environment. We employed a culture‐independent metagenomic approach to analyse for the first time complete genomes in the halophage community and explored the in situ diversity by transmission electron microscopy and pulsed‐field gel electrophoresis. We report the genomic sequence of a not yet isolated halophage (named as environmental halophage 1 ‘EHP‐1′) whose DNA was obtained from crystallizer samples with a salinity of 31%. The sequenced genome has a size of 35 kb and a G + C content around 51%. The G + C content is lower than that of previously characterized halophages. However, G + C content and codon usage in EHP‐1 are similar to the recently cultivated and sequenced Haloquadratum walsbyi, the major prokaryotic component in solar salterns around the world. Forty open reading frames have been predicted, including genes that putatively code for proteins involved in DNA replication (ribonucleotide reductases, thymidylate kinase) normally found in lytic viruses.

Dynamic Viral Populations in Hypersaline Systems as Revealed by Metagenomic Assembly

“…This has allowed us to uncover previously unrecognized patterns of viral population dynamics on relatively short timescales. We have nearly doubled the number of haloviruses and VLEs with sequenced representatives (15,18,55), and the assembly of genomes from as little as 0.068% of the reads in a sample suggests that comprehensive viral genomic reconstruction may be possible in more complex systems through deep metagenomic sequencing. Importantly, we were able to obtain enough DNA for metagenomic sequencing without amplification, avoiding potential biases associated with multiple-displacement amplification (MDA) and presumably achieving more accurate relative abundance estimates.…”

“…Annotations that could be associated with lysogeny include a putative phage integrase in LTVLE3 and a putative excisionase in LTV6. LTV2 encodes ribonucleotide reductases, which are more often associated with lytic than temperate viruses (55). Of the functions that could be identified, the most prevalent is methyltransferase or DNA methylase, found in LTV1, -2, and -5 and LTVLE3.…”

“…Using gsMapper (Newbler [33]) with a minimum overlap length of 40 and a minimum overlap identity of 90%, unique reads from each of the eight samples were used as queries against the 7 genomes or the 28 genome fragments as references (duplicate reads were removed, though nearly identical mapping counts were obtained when duplicate reads were included). Similarly, reads from three previously described haloviral metagenomes (14,49,57,61) were used as queries against the 35 LT genomes and genome fragments, and 10 previously described haloviral genome sequences (His1 and His2 [10]; HF1 [67]; HF2 [68]; HRPV-1 [44]; HHPV-1 [51]; SH1 [8]; BJ1 [42]; Ch1 [26]; and EHP-1 [55]) were used as references for mapping reads from each of our eight samples. The same fragment recruitment parameters were applied to data from the previously published San Diego halovirome (14, 49), using two sets of references (first, the 10 largest contigs assembled directly from the San Diego samples, and second, the 10 previously sequenced haloviruses described above).…”

Culture-Independent Approaches for Studying Viruses from Hypersaline Environments

“…Soon later, the viruses in the Dead Sea (38) were also observed under TEM. Later, more salterns (20,54) and hypersaline lakes, such as the Retba Lake in Senegal, Great Salt Lake in Utah, and Mono Lake in California, were studied (10,15,58).…”

Evolutionarily Conserved Orthologous Families in Phages Are Relatively Rare in Their Prokaryotic Hosts

“…Most viruses detected in the environment are head-tail phages that infect prokaryotes (mostly bacteria, though archaeophages are being actively studied, and there is no reason to believe that they will be less diverse than bacteriophages [14,44,48]). There are almost 600 fully sequenced genomes of phages in sequence databases as of this writing.…”