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Anaerobic Bacteria: an Investigation of Metabolic Important Enzymes

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Anaerobic Bacteria: an Investigation of Metabolic Important Enzymes Anaerobic bacteria: an investigation of metabolic important enzymes A novel type of oxygen reductase and enzymes of the tetrapyrrole biosynthesis Susana André Lima Lobo Dissertation presented to obtain the Ph.D. degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Supervisor: Dr. Lígia M. Saraiva Co-supervisor: Prof. Miguel Teixeira Opponents: Prof. Martin Warren Prof. Ilda Sanches Dr. Carlos Salgueiro Oeiras, July 2009 i From left to right: José Martinho Simões, Ilda Sanches, Carlos Salgueiro, Martin Warren, Susana Lobo, Lígia Saraiva, Ana Melo, Célia Romão, Miguel Teixeira 8th of July 2009 Second Edition, July 2009 Molecular Genetics of Microbial Resistance Laboratory Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Av. da República (EAN) 2780-157 Oeiras Portugal ii Acknowledgements I would like to express my gratitude to the people that contributed to this work and beyond: To my supervisor, Dr. Lígia Saraiva, whose presence, determination and friendship were a constant factor. For believing in me and in my work, for the 24h availability whenever I needed to ask a question or communicate the result of an experiment that just could not wait! For always seeing the bright positive side of things, when I was only seeing the dark ones. For giving me the opportunity to evolve as a scientist (and as a person) in this infinite scientific world. To my co-supervisor, Prof. Miguel Teixeira, for all his support and fruitful discussions, for spending so much time in my computer playing with Matlab, and for always paying attention when I started talking and talking and talking about Desulfovibrio genomes. To Prof. Martin Warren, for all his collaboration and welcome into his lab, and to all the people in Prof. Martin Warren’s lab, especially to Amanda Brindley, for the help in the tetrapyrrole work, to Evelyne Deery, for her genial genetic tips and to Susanne Schroeder, for her friendly welcome into her home and for all she has done to integrate me when I was in the UK. Claudia Almeida, for being the excellent person she is, for her patience when I was still learning how to put LA in a Petri dish without solidifying the medium before getting into the plate, for her support and belief in me, and most of all, for her huge friendship. Célia Romão, who helped me in the anaerobic (and aerobic) protein purification quest, for the 1500000 protein assays, for the structure of the most pinkish protein, which I had the delight to see in 3D in the computer using special glasses, without having to switch my eyes into 3D mode! Ana Melo that helped me when I started to give my first steps in science (when I was still finishing my fifth year of Biology!). To my group, the Molecular Genetics of Microbial Resistance, for making the lab such a dynamic and nice place to work. To Lígia Nobre, Vera Gonçalves and Marta Justino, for their friendship and for the “conferences” and ”brain stormings” downstairs or in the lab desk surrounding areas, and Filipa Tavares, Joana Baptista and Mafalda Figueiredo, the three “baby girls” in the lab that were always updating me with things from the “future” (e.g. Hi5), for their friendship, good mood, and for saying things like: “Hemos de porfirinas”. I will never forget this haem description! I would like also to thank our former group members, Vera Mónica, Sofia, Nuno and Nuno Félix, for the good moments. To the Metalloproteins and Bioenergetics group (my first group), where I was always welcome every time I had to purify a protein or do hundreds of enzymatic activities, iii for their friendship and for all their help. To Ana Paula Batista, Ana Patricía Refojo, Ana Filipa Pinto, João Vicente, Andreia Fernandes, Tiago Bandeiras, Manuela Pereira, Smilja Todorovic and to Andreia Veríssimo, Filipa Sousa and João Rodrigues, who were my travel companions the first time I went to a congress outside of Portugal. João Carita, for the endless Desulfovibrio growths (with and without oxygen!), for the chats and for the help when I had to look into the microscope. To all the members (and former members) of our neighbour group, the Microbial Biochemistry group, especially to Sofia Venceslau, for the hydrogen bubbling in gels and for all the great chats! And also to Isabel Pacheco, for the welcoming into her lab to use the anaerobic chamber. To Ricardo Louro for the collaboration on the NMR spectroscopy. To Manuela Regalla for the N-terminal sequencing and HPLC analysis. To everyone in the 3rd floor of ITQB, for the friendly environment. Desulfovibrio for being such an interesting and good cell model which allowed me to study so many fascinating proteins! To all my friends; especially to Andreia (who is the most friendly, strong and determined person I know), André and João, for saying: “oh no, there she goes again….and the lab talking begins!” and for all the fantastic and treasured off-lab moments! A special thanks goes also to Tânia, Ana Raquel, Raquel, Lena, Leonor, Anna and Lara. To Luísa, Tó, Manel, Luís and Catarina, for their friendship and support. To my godson Rodrigo and to my godparents, Tó and Isabel, for their friendship and for being part of my life since June 1981! To António, for his love, patience, unconditional support, for being my best friend and a huge part of my life. To my family, for all their constant curiosity in my work and in science, for always seeing the artistic and beautiful side of science (even if it was just a dry SDS-PAGE), for always asking me: “How are your proteins going?” and “Are your bacteria being nice to you?”, for listening to me every time I went into a lab mode (“bla bla bla, protein bla bla bla, bacteria bla bla bla”), for their support, encouragement and belief in me. Without them I would not be where I am today. Fundação para a Ciência e Tecnologia is acknowledged for financial support, by awarding a PhD grant (PhD Grant SFRH/BD/19813/2004). This thesis is dedicated to my family iv Thesis Publications This dissertation is based on original publications, listed by chronological order: 1. Lobo, S.A., Melo, A.M., Carita, J.N., Teixeira, M., Saraiva, L.M. (2007) “The anaerobe Desulfovibrio desulfuricans ATCC 27774 grows at nearly atmospheric oxygen levels” FEBS Letters, 581(3):433-436. 2. Lobo, S.A., Brindley, A.A., Romão, C.V., Leech, H.K., Warren, M.J., Saraiva, L.M. (2008) “Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris Hildenborough” Biochemistry, 47(21):5851- 5857. 3. Lobo, S.A., Almeida, C.C., Carita, J.N., Teixeira, M., Saraiva, L.M. (2008) “The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II” Biochimica et Biophysica Acta, 1777(12):1528-1534. 4. Lobo, S.A., Brindley, A.A., Warren, M.J., Saraiva, L.M. (2009) “Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough” Biochemical Journal, 420(2):317-325. v vi Dissertation Abstract Desulfovibrio desulfuricans was the first species of a sulphate- reducing bacterium to be isolated, in 1895. Since that time, many questions were raised in the scientific community regarding the metabolic and ecological aspects of these bacteria. At present, there is still a myriad of open questions remaining to be answered to enlarge our knowledge of the metabolic pathways operative in these bacteria that have implications in the sulfur cycle, in biocorrosion, namely in sewers and in oil and gas systems, and in bioremediation of several toxic metals. The work presented in this dissertation aimed at contributing with new insights of enzymes involved in two different metabolic systems on Desulfovibrio species, namely enzymes that play a role in the response to oxidative stress and that are involved in the haem biosynthetic pathway. Although for many years Desulfovibrio species have been considered strict anaerobes, several Desulfovibrio strains are found in environments where they are exposed to oxygen. In fact, these organisms contain several systems that enable the survival under oxidative conditions, but no aerobic growth was until now reported. In this dissertation it was shown, for the first time, that a Desulfovibrio species, D. desulfuricans ATCC 27774, has the ability to sustain growth when exposed to nearly atmospheric oxygen concentrations. Our studies suggest that D. desulfuricans ATCC 27774 utilizes oxygen for growth maintaining at the same time the nitrate dissimilatory pathway metabolism operative. Furthermore, enzymatic studies performed with protein fractions of D. desulfuricans ATCC 27774 cells exposed to 18 % oxygen, showed that proteins involved in scavenge of reactive oxygen species (ROS), namely the superoxide dismutase and catalase enzymes, have increased expression. vii Analysis of the genomes of Desulfovibrio species, revealed that they contain genes coding for proteins that perform the reduction of oxygen to water, including the haem-copper membrane-bound oxygen reductases. A comprehensive amino acid sequence analysis of the haem-copper oxygen reductases encoded by the majority of Desulfovibrio sp. genomes, predicts the existence of a novel A2-type oxygen reductase, ccaa3, with an extra haem c binding motif (CxxCH) in subunit II. The biochemical characterization of a truncated form of D. vulgaris Hildenborough ccaa3 subunit II confirmed the binding of the two c-type haems, with a His- Met haem-iron coordination. Furthermore, we showed that the D. vulgaris cytochrome c553 donates electrons to the ccaa3 oxygen reductase, revealing the first possible physiological electron donor to the haem- copper oxygen reductase in these organisms. With the objective of clarifying the tetrapyrrole biosynthetic pathway in D. vulgaris Hildenborough, we performed the biochemical study of several enzymes involved in the generation of sirohydrochlorin, sirohaem and cobalt-sirohydrochlorin from aminolaevulinic acid, namely porphobilinogen synthase (HemB), porphobilinogen deaminase (HemC), uroporphyrinogen III synthase (HemD), uroporphyrinogen III methyltransferase (SUMT), precorrin-2 dehydrogenase (CysGB) and sirohydrochlorin ferro- and cobalto-chelatases (CysGB and CbiK, respectively).
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