Abstract:
Methanogens are a group of archaea that impact the environment through methane emission, harbor potential in biotechnological applications to mitigate carbon dioxide emissions, and are present in human microbiomes with an as of yet unknown impact on human health. Understanding these microbes more deeply will be beneficial in many areas, however, while there is a plethora of research on their physiology and biochemistry, techniques to manipulate methanogens are limited. The role of viruses in controlling environmental microbial populations has been demonstrated and they are being utilized in the medical field as an alternative to antibiotics in so-called phage therapy. Furthermore, microbial viruses can serve as vectors that transmit genetic information into microbes to modify their genomes. I propose to establish a methanogen virus-host model system which can serve as the starting point to investigate methanogen head-tailed viruses in relation to their hosts and their evolutionary relation to head-tailed viruses infecting other kingdoms of life, and also be utilized to generate methanogen manipulation techniques that will promote this important field of research.
The head-tailed virus ΨM2 is highly suitable as a model system for methanogen viruses. It is an isolated virus with a fully sequenced genome and is capable of lysing its host, Methanothermobacter marburgensis, which is beneficial for virus propagation and isolation. To establish ΨM2 as a model for methanogen viruses and monitor the virus-host interaction throughout the infection, growth curves of infected M. marburgensis cultures were performed. Interestingly, ΨM2 did not lyse its host when it was cultivated in the minimal growth medium for Methanothermobacter spp. (MS medium). In a complex medium, meant for cultivation of a broad spectrum of methanogen species (SAB medium), the infected M. marburgensis culture was lysed around fourteen hours post infection. This was an unexpected result, since initial investigations of the virus from the literature had shown reliable lysis of the infected host cell cultures. Furthermore, the cultures in MS medium, which did not exhibit lysis, still produced progeny viruses. It was assumed that an alternative viral lifecycle to the lytic one must be employed by ΨM2. The virus-host interaction was further analyzed through more refined growth experiments that focus on the initial phase of virus infection, adsorption of the virus particles to their host cells. The rate at which this adsorption happens was found to be comparable to other, relatively fast-adsorbing viruses. Additionally, electron micrographs of adsorbing virus particles confirmed the expected orientation of head-tailed virus particles during adsorption with the tips of their tails attaching to the host cell.
As a next step, the ΨM2 lifecycle was investigated further. Beside the lytic lifecycle, in which infected cells lyse and release the newly produced progeny viruses, the lysogenic lifecycle is well-studied in microbial viruses. In the lysogenic lifecycle, viruses integrate their genome into the genome of their host, so that it gets replicated and spreads together with the growing host cells without the need for virus particle production and lysis. The integrated virus is then called a provirus and the carrying host a lysogen. ΨM2 has the genetic prerequisite to assume the lysogenic lifecycle in form of an integrase gene, which is required for provirus integration. However, whole genome sequencing of ΨM2-infected M. marburgensis in MS medium and SAB medium revealed no integration of the ΨM2 viral genome. This result can be explained because the integrase gene of ΨM2 is modified in a way that leads to loss of function in other integrases from the same protein family.
With this, the alternative lifecycle that ΨM2 assumes during infections where no lysis happens, remains to be identified. What will help with this aim is the continued resolution of ΨM2 viral characteristics. For example, to employ a closely related methanogen species, Methanothermobacter thermautotrophicus, which is resistant to ΨM2 infection. One hypothesis is that there is an intracellular defense system of this species, which recognizes and destroys intruding viral genomes in the resistant cell. To further support this hypothesis, the aforementioned adsorption assays were performed with ΨM2-infected M. thermautotrophicus. The results confirmed that virus adsorption still happens in these infected cultures and that the M. thermautotrophicus resistance to ΨM2 infection likely stems from an intracellular defense mechanism.
Overall, this dissertation serves as a stepping stone in advances to fully characterize the methanogen virus ΨM2. With a deeper understanding of its lifecycle and other characteristics, it will be possible to utilize this virus for control and manipulation of methanogens and to gain an advanced understanding of these important microbes.
archaeal virus