My postdoctoral research in the laboratory of Professor Wolfram Zillig focussed on the characterization and utilization of extrachromosomal genetic elements of the hyperthermophilic archaeon Sulfolobus solfataricus. Hyperthermophilic archaea, some of which can grow at temperatures up to 113°C, are not only of great interest for biotechnology, but also for study of thermostable macromolecules, hot environments, and microbial physiology at very high temperatures. These organisms may even be models for life on other planets. Complete genome sequencing has confirmed that the Archaea are fundamentally different from all other life forms although they display intriguing similarities to both bacteria and eukaryotes. Particularly important for my research, transcription in archaea is very similar to transcription in eukaryotes but potentially easier to dissect at the molecular level due to the apparently small number of basal transcription factors involved in transcriptional initiation. Replication in archaea, on the other hand, is very poorly understood, despite the wide use of DNA polymerases from hyperthermophilic archaea in molecular biology.

Fundamental understanding of hyperthermophilic archaea has lagged behind biochemical characterization of a few gene products particularly due to the absence of a stable transformation system and methods for specific disruption of genes. Sulfolobus solfataricus is an excellent model archaeon and hyperthermophile for a number of reasons; optimal growth at 80°C, a pH optimum of 3, heterotrophy, ease of plating, and aerobiosis. Many plasmids and viruses, including the virus SSV1, have been found by the Zillig laboratory, and recently by myself and my current laboratory, in field samples from Sulfolobus isolates. The complete genome of S. solfataricus has recently been determined by a Canadian and European consortium.

The virus SSV1 is the first virus of Sulfolobus to be characterized and it has been extensively studied both biochemically and genetically. It is completely different from any known virus in morphology. In addition the complete sequence of its double stranded DNA genome showed that it had no sequence similarity to other viruses either. Critical for the development of a genetic system, strains not containing the virus can be transformed with the viral DNA. Using a novel serial selection technique, I developed a functional, high copy number shuttle vector which replicates in both E. coli and S. solfataricus from this virus (Stedman et al., 1999). For the first time, recombinant DNA can be stably introduced into S. solfataricus using this vector (Stedman et al., 1999). Recently we have shown that proteins from other hyperthermophiles can be stably expressed in S. solfataricus and used as markers (Stedman et al., manuscript in preparation). This makes in vivo analysis in a hyperthermophilic archaeon possible for the first time. I plan to use this system to study the regulation of gene expression in vivo in S. solfataricus and to compare the results to those obtained in in vitro experiments.

Naturally occuring plasmids and viruses of archaea, particularly the virus SSV1, are not only the basis for a stable transformation system, but also excellent candidates for studies of fundamental processes in hyperthermophilic archaea. All of the transcripts of SSV1 have been mapped and their production during induction observed. Replication of the virus is induced by ultraviolet irradiation and this induction appears to take place via transcription of one small transcript in the viral genome. The regulation of this transcript is unknown. One of my first goals is to decipher this link between transcription and replication in SSV1.

Most of the open reading frames in the SSV1 genome have no known homologs, these may function in replication or transcription or in other essential virus functions. Using my selection system, I have shown that many of these ORFs are crucial for virus function. I plan to determine the precise biochemical activities and physiological roles of the products of these ORFs. My doctoral training in Prof. Sydney Kustu’s laboratory in Berkeley and previously at Sandoz Pharma, Ltd. has prepared me very well for purification and in vitro characterization of such proteins.

We have also been analyzing and sequencing novel viruses and plasmids from field samples from solfataric acidic hot springs throughout the world. Collection of field samples and screening them for novel genetic elements is critical to both the understanding of genetic diversity in solfataric environments and as a tool to study the organisms which live in these environments. I have participated in a number of successful collecting expeditions already, the results of these studies and characterization of some of these plasmids has recently been published (Zillig et al., 1998; Stedman et al., in the press). We have been performing biogeographical analysis on these new viruses and plasmids.

The diversity of the new viruses is been surprising. Some viruses have completely new morphologies, and others are similar to viruses characterized by the Zillig group. SSV-like viruses are present in samples from Japan, Iceland, Yellowstone National Park, U.S.A., and I recently found some in Kamchatka. Their sequences are obviously related, but they are not as similar to each other as their hosts are. By contrast, SIRV-like viruses, another novel virus of Sulfolobus, which so far have only been found in Iceland and Yellowstone National Parks, U.S.A., are much more similar to each other. What this means is unclear, but the viruses will be characterized in more detail and more viruses from more diverse geographical locations will be isolated.

The genome of S. solfataricus has been shown to be very plastic, both by the presence of a large number of insertion sequences and ORFs potentially encoding transposases and experimentally by its high basal mutation rate. One of the families of plasmids which I characterized, the pING family of conjugative plasmids, is also highly variable and obviously interacts with the host chromosome (Stedman et al., in the press). I plan to continue to analyze this genomic plasticity by inserting marker genes either into my already established SSV1 based vector or into one of the conjugative plasmids. In the long term I also plan to integrate marker genes specifically into the chromosome. I will follow the transfer of these marker genes both in liquid culture and in biofilm environments in collaboration with the Biofilm Engineering Center at Montana State University. These results should lead to greater understanding of the mechanisms and kinetics of horizontal gene transfer, one of the most potent forces shaping evolution.

With the complete genome sequence of S. solfataricus available, genomic and proteomic analysis will be performed, in the short term in collaboration, but I plan to establish the techniques in my own group in future. Primary experiments will be performed to determine which genes are induced upon viral infection. These genes will be candidate genes for those either directly regulated by the virus or those involved in the response of the host to infection. Mutant forms of the viruses will then be tested for the response of the cell to these mutant forms of the virus. The response to ultraviolet irradiation will be tested, both in the presence and absence of SSV1. Conjugative plasmids will also be used in these experiments to see which cellular genes and proteins are influenced by these plasmids which severely affect their host cells.

In summary, for the short term, I plan to use the in vivo system which I have developed in parallel with in vitro analysis to investigate the function of the archaeal virus SSV1, particularly its transcription and replication. In the long term I plan to extend this research to transcription and replication in SSV1’s host, the hyperthermophilic archaeon S. solfataricus. I also plan on collecting and analysing new plasmids and viruses from acidic hot springs both to study diversity of these extra-chromosomal elements and to use them as tools to study their hosts. I will use these viruses and plasmids to study genomic plasticity in S. solfataricus in order to start to understand some mechanisms involved in horizontal gene transfer. Finally I will use genomic and proteomic techniques to address questions regarding the function and response of Sulfolobus to viral infection and conjugation. All together these studies should lead to insight into the biochemistry, regulation and physiology of both archaea and hyperthermophiles, and into evolution.