Francisco F. Roberto

Comparative Genomic Analysis of Hyperthermophilic Archaeal Fuselloviridae Viruses

ABSTRACT The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of ∼15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone Hot Springs

ABSTRACT There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.

Heterotrophic Acidophiles and Their Roles in the Bioleaching of Sulfide Minerals

The most familiar and well-studied microorganisms indigenous to acidic mineral leaching environments are autotrophic sulfur- and iron-oxidizing bacteria such as Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans. Some photoautotrophs, such as the thermophilic rhodophyte Cyanidium caldarium, may also be present in extremely acidic environments that receive light. Other microorganisms which require pre-fixed (organic) carbon have been isolated from mineral leach dumps and acid mine drainage (AMD) waters. These heterotrophic microorganisms include eukaryotes, such as some fungi and yeasts1 and protozoa,2 as well as prokaryotic bacteria and archaea. It is somewhat paradoxical, given that heterotrophy is the most widespread form of metabolism among bacteria, that the first acidophilic heterotrophic bacterium which is indigenous and active in mineral leaching environments was isolated and characterized some 40 years after the iron/sulfur-oxidizing chemolithotroph T. ferrooxidans and 70 years after the sulfur-oxidizing acidophile T. thiooxidans.

Virus movement maintains local virus population diversity

Viruses are the largest reservoir of genetic material on the planet, yet little is known about the population dynamics of any virus within its natural environment. Over a 2-year period, we monitored the diversity of two archaeal viruses found in hot springs within Yellowstone National Park (YNP). Both temporal phylogeny and neutral biodiversity models reveal that virus diversity in these local environments is not being maintained by mutation but rather by high rates of immigration from a globally distributed metacommunity. These results indicate that geographically isolated hot springs are readily able to exchange viruses. The importance of virus movement is supported by the detection of virus particles in air samples collected over YNP hot springs and by their detection in metacommunity sequencing projects conducted in the Sargasso Sea. Rapid rates of virus movement are not expected to be unique to these archaeal viruses but rather a common feature among virus metacommunities. The finding that virus immigration rather than mutation can dominate community structure has significant implications for understanding virus circulation and the role that viruses play in ecology and evolution by providing a reservoir of mobile genetic material.

Phylogenetic and functional analysis of metagenome sequence from high-temperature archaeal habitats demonstrate linkages between metabolic potential and geochemistry

Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40–45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G + C content) and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport, and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH). These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high-temperature systems of YNP.

Effects of Culturing on the Population Structure of a Hyperthermophilic Virus

The existence of a culturing bias has long been known when sampling organisms from the environment. This bias underestimates microbial diversity and does not accurately reflect the most ecologically relevant species. Until now no study has examined the effects of culture bias on viral populations. We have employed culture-independent methods to assess the diversity of Sulfolobus spindle–shaped viruses (SSVs) from extremely hyperthermal environments. This diversity is then compared to the viral diversity of cultured samples. We detected a clear culturing bias between environmental samples and cultured isolates. This is the first study identifying a culture bias in a viral population.

Biodiversity of Acidophilic Moderate Thermophiles Isolated from Two Sites in Yellowstone National Park and Their Roles in the Dissimilatory Oxido-Reduction of Iron

Bacteria that bring about dissimilatory transformations of iron are important from both biogeochemical and industrial perspectives (Ehrlich and Brierley, 1990; Johnson, 1995). The oxido-reduction of iron in extremely acidic (pH > 3) environments is particularly interesting because of the greater solubility of ionic (particularly ferric) iron and the relative stability of soluble ferrous iron under these conditions. Acidophilic iron-oxidizing bacteria are generally considered the most significant microorganisms in the biological processing of sulfide ores (“biomining”) in which the accelerated oxidative dissolution of sulfidic minerals (e.g., pyrite, arsenopyrite, and chalcopyrite) solubilizes (e.g., copper) or releases (refractory gold) metals, thereby facilitating their recovery (Rawlings and Silver, 1995). Most research into bacterial iron transformations at low pH has focused on mesophilic chemolithotrophs, particularly Thiobacillus ferrooxidans, though a number of physiologically and phenotypically diverse mesophilic acidophiles, it is now known, are involved in the dissimilatory oxido-reduction of iron (Johnson, 1995; Norris and Johnson, 1997; Pronk and Johnson, 1992).

Reply to “Codon Usage Frequency of RNA Virus Genomes from High-Temperature Acidic-Environment Metagenomes”

Mark Young, Benjamin Bolduc, Daniel P. Shaughnessy, Francisco F. Roberto, Yuri I. Wolf, Eugene V. Koonin Thermal Biology Institute and Departments of Microbiology, Plant Sciences and Plant Pathology, and Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA; National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA; Idaho National Laboratory, Idaho Falls, Idaho, USA