Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Evolution of Bordetella pertussis post vaccination

Evolution of Bordetella pertussis post vaccination Whooping cough or pertussis is caused by the gram-negative bacterium Bordetella pertussis. It is a highly contiguous disease in the human respiratory tract. Characteristic of pertussis is a paroxysmal cough with whooping sound during gasps of breath after coughing episodes. It is potentially fatal to unvaccinated infants. The best approach to fight pertussis is to vaccinate. Vaccinations against pertussis have been available from the 1940s. Traditionally vaccines were whole-cell pertussis (wP) preparations as part of the combined diphtheria-tetanus-pertussis (DTP) vaccines. More recently acellular pertussis (aP) vaccines have replaced the wP vaccines in many countries. The aP vaccines are less reactogenic and can also be administered to school children and adults. There are several publications reporting variation in the i>B. pertussis virulence factors that are also aP vaccine antigens. This has occurred in the genes coding for pertussis toxin and pertactin about 15 to 30 years after the introduction of pertussis vaccines to immunisation programs. Resurgence of pertussis has also been reported in many countries with high vaccination coverage. In this study the evolution of B. pertussis was investigated in Finland, the United Kingdom, Poland, Serbia, China, Senegal and Kenya. These represent countries with a long history of high vaccination coverage with stable vaccines or changes in the vaccine formulation; countries which established high vaccination coverage late; and countries where vaccinations against pertussis were started late. With bacterial cytotoxicity and cytokine measurements, comparative genomic hybridisation, pulsed-field gel electrophoresis (PFGE), genotyping and serotyping it was found that changes in the vaccine composition can postpone the emergence of antigenic variants. It seems that the change in PFGE profiles and the loss of genetic material in the genome of B. pertussis are similar in most countries and the vaccine-induced immunity is selecting non-vaccine type strains. However, the differences in the formulation of the vaccines, the vaccination programs and in the coverage of pertussis vaccination have affected the speed and timing of these changes.

Carbon dioxide evolution from snow-covered agricultural ecosystems in Finland

Selostus: Hiilidioksidin kulku lumipeitteisessä ja paljaassa maassa