Mutational and molecular analyses of lactococcal bacteriophages TP901-1 and Tuc2009

Bacteriophages belonging to the Siphoviridae family represent viruses with a noncontractile tail that function as extremely efficient bacterium-infecting nanomachines. The Siphoviridae phages TP901-1 and Tuc2009 infect Lactococcus lactis, and both belong to the so-called P335 species. As P335 phages are typically capable of a lytic and lysogenic life cycle, a number of molecular tools are available to analyse their virions. This doctoral thesis describes mutational and molecular analyses of TP901-1 and Tuc2009, with emphasis on the role of their tail-associated structural proteins. Several novel and intriguing findings discovered during the course of this study on the nature of Siphoviridae phages furthers a basic molecular understanding of their virions, and the role of their virion proteins, during the initial stages of infection. While Siphoviridae virions represent complex quaternary structures of multiple proteins and subunits thereof, mutagenic analysis represents an efficient mechanism to discretely characterize the function of individual proteins, and constituent amino acids, in the assembly of the phage structure and their biological function. However, as always, more research is required to delve deeper into the mechanisms by which phages commence infection. This is important to advance our understanding of this intricate process and to facilitate application of such findings to manipulate phage infections. On the one hand, we may want to prevent phages from infecting starter cultures used in the dairy industry, while on the other hand it may be desirable to optimize viral infection for the application of phages as bacterial parasites and therapeutic agents.

Phaeocystis colony distribution in the North Atlantic Ocean since 1948, and interpretation of long-term changes in the Phaeocystis hotspot in the North Sea

Monitoring of Phaeocystis since 1948 during the Continuous Plankton Recorder survey indicates that over the last 5.5 decades the distribution of its colonies in the North Atlantic Ocean was not restricted to neritic waters: occurrence was also recorded in the open Atlantic regions sampled, most frequently in the spring. Apparently, environmental conditions in open ocean waters, also those far oVshore, are suitable for complete lifecycle development of colonies (the only stage recorded in the survey). In the North Sea the frequency of occurrence was also highest in spring. Its southeastern part was the Phaeocystis abundance hotspot of the whole area covered by the survey. Frequency was especially high before the 1960s and after the 1980s, i.e., in the periods when anthropogenic nutrient enrichment was relatively low. Changes in eutrophication have obviously not been a major cause of long-term Phaeocystis variation in the southeastern North Sea, where total phytoplankton biomass was related signiWcantly to river discharge. Evidence is presented for the suggestion that Phaeocystis abundance in the southern North Sea is to a large extent determined by the amount of Atlantic Ocean water Xushed in through the Dover Strait. Since Phaeocystis plays a key role in element Xuxes relevant to climate the results presented here have implications for biogeochemical models of cycling of carbon and sulphur. Sea-to-air exchange of CO2 and dimethyl sulphide (DMS) has been calculated on the basis of measurements during single-year cruises. The considerable annual variation in phytoplankton and in its Phaeocystis component reported here does not warrant extrapolation of such figures.

Biologging, Remotely-Sensed Oceanography and the Continuous Plankton Recorder Reveal the Environmental Determinants of a Seabird Wintering Hotspot

Marine environments are greatly affected by climate change, and understanding how this perturbation affects marine vertebrates is a major issue. In this context, it is essential to identify the environmental drivers of animal distribution. Here, we focused on the little auk (Alle alle), one of the world’s most numerous seabirds and a major component in Arctic food webs. Using a multidisciplinary approach, we show how little auks adopt specific migratory strategies and balance environmental constraints to optimize their energy budgets. Miniature electronic loggers indicate that after breeding, birds from East Greenland migrate .2000 km to overwinter in a restricted area off Newfoundland. Synoptic data available from the Continuous Plankton Recorder (CPR) indicate that this region harbours some of the highest densities of the copepod Calanus finmarchicus found in the North Atlantic during winter. Examination of large-scale climatic and oceanographic data suggests that little auks favour patches of high copepod abundance in areas where air temperature ranges from 0uC to 5uC. These results greatly advance our understanding of animal responses to extreme environmental constraints, and highlight that information on habitat preference is key to identifying critical areas for marine conservation.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.