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Title: Viral infection of marine picoplankton under nutrient depletion conditions : pseudolysogeny and magic spot nucleotides
Author: Rihtman, Branko
ISNI:       0000 0004 6348 5826
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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Cyanobacteria are major players in marine biogeochemical processes, primarily CO2 fixation via oxygenic photosynthesis, and nitrogen cycling. These phototrophs occupy a variety of oceanic niches, with their distribution and abundance being shaped by a range of abiotic (e.g. temperature, light, nutrients) and biotic factors (e.g. grazing and virus infection). Viruses infecting cyanobacteria are termed cyanophage. During evolution these cyanophages have acquired an arsenal of ‘auxilliary metabolic genes’ (AMGs), often horizontally acquired, which can influence the metabolism of the infected host thereby optimising viral production. Cyanophage infection of their host under sub-optimal growth conditions can lead to deleterious effects on infection success. For example, cyanophage S-PM2 infection of Synechococcus under P-deplete conditions causes a delayed latent phase and decreased burst size – a process termed pseudolysogeny. In this thesis I set out to provide a molecular understanding of pseudolysogeny, at the same time hypothesising that specific cyanophage AMGs help to avoid the negative effects of sub-optimal host growth conditions on the infection process. In support of this, infection by cyanophages that possess putative P-stress related AMGs do not show signs of delay (Chapter 3), and the presence of these genes in cyanophage genomes correlates well with the prevailing P conditions in the temporal and spatial niches from which these cyanophages were isolated (Chapter 4). Meanwhile, in a cyanophage lacking such nutrient stress genes, and thus entering pseudolysogeny during infection under P-deplete conditions, transcriptional profiling showed retardation in the timing of known cyanophage temporal gene expression clusters (Chapter 6). Moreover, a significant increase in expression of several cyanophage early genes involved in DNA replication was also observed under these P stress conditions compared to infection of a P-replete host. Quantitation of intracellular cyanophage DNA showed that while levels were generally lower under P-deplete conditions, the rate of DNA replication between P-replete/deplete conditions was similar. The observed increased expression of cyanophage genes involved in DNA replication during the early stages of infection may thus be an evolved response to compensate for decreased levels of intracellular phosphate experienced under these conditions (Chapter 6). Overlaid on top of specific bacterial nutrient stress responses is the ‘stringent’ response, mediated by the alarmone molecule (p)ppGpp, a process which occurs under prolonged nutrient stress and in late stationary phase. A bacterial gene mazG, encodes a pyrophosphatase which participates in (p)ppGpp homeostasis. Interestingly, a mazG orthologue is found as part of the cyanomyovirus core genome, suggesting that cyanophages attempt to alter intracellular signalling during the course of infection. The stringent response has been shown to have a particularly negative effect on phage replication, with (p)ppGpp levels in a cyanobacterial host being previously shown to be dramatically reduced under phage infection. In this thesis I show that the Synechococcus host mazG is dispensable for growth under normal laboratory conditions. However, this Synechococcus mazG mutant shows a modified cyanophage infection profile, slower and less productive, compared to the WT, under P-deplete conditions (Chapter 5). Furthermore, comparison of enzymatic activity of host and cyanophage MazG showed that the viral orthologue exhibits an increased affinity towards GTP, compared to the host protein and a general preference towards G and C nucleotides (Chapter 5), possibly reflecting the low GC content of cyanophage genomes. Thus, the cyanophage and host MazG may have additional functions in phosphate metabolism and controlling DNA integrity, a hypothesis strengthened by experimental evidence for the cyanophage mazG being over-expressed under P-deplete conditions (Chapter 6). Taken together, data presented in this thesis demonstrates a general strategy by cyanophages to acquire host genes involved in modification of central metabolism or that regulate host signalling. Furthermore, once acquired, cyanophage genes appear to have evolved divergent functions to suit specific differences in genome content, compared with their host, as well as mechanisms to regulate transcription of these genes in response to external nutrient stimuli. Thus, this study expands our view of lytic phages, and suggests sophisticated mechanisms occur for overpowering their hosts under a range of infection conditions. This new information provides a mechanistic understanding of viral infection in a ubiquitious primary producer under environmetally relevant conditions, and will undoubtedly improve our ability to understand and model biogeochemical cycling performed by these key marine phototrophs in a more accurate manner.
Supervisor: Not available Sponsor: University of Warwick
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology