Dissimilatory nitrate reduction in Bacillus Yihua Sun Promotors Dr. Kim Heylen Prof. Dr. em. Paul De Vos Prof. Dr. Anne Willems Dissertation submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) of Science: Biotechnology (Ghent University) Yihua Sun – Dissimilatory nitrate reduction in Bacillus Copyright ©2017, Yihua Sun ISBN-number: 978-94-6197-486-0 All rights are reserved. No part of this thesis protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage or retrieval system without written permission of the author and promotors. Printed by University Press | www.universitypress.be Ph.D. thesis, Faculty of Sciences, Ghent University, Ghent, Belgium. This Ph.D. work was financially supported by Chinese scholar council ( 201206330054) and BOF CSC co- funding from Ghent University (01SC2713) Publicly defended in Ghent, Belgium, January 20 th , 2017 Examination committee Prof. Dr. Savvas Savvides (Chairman) L-Probe: Laboratory for Protein Biochemistry and Biomolecular Engineering Faculty of Sciences, Ghent University, Belgium VIB Inflammation Research Center VIB, Ghent, Belgium Prof. Dr. Anne Willems (Promotor) LM-UGent: Laboratory of Microbiology Faculty of Sciences, Ghent University, Belgium Prof. Dr. em. Paul De Vos (Promotor) LM-UGent: Laboratory of Microbiology Faculty of Sciences, Ghent University, Belgium Dr. Kim Heylen (Promotor) LM-UGent: Laboratory of Microbiology Faculty of Sciences, Ghent University, Belgium Prof. Dr. Sofie Goormachtig (Secretary) PSB: Plant systems Biology Faculty of Sciences, VIB, Ghent University, Belgium Prof. Dr. ir. Nico Boon CMET: Center for Microbial Ecology and Technology Faculty of Bioscience Engineering, Ghent University, Belgium Prof. Dr. Bart Devreese L-Probe: Laboratory for Protein Biochemistry and Biomolecular Engineering Faculty of Sciences, Ghent University, Belgium Dr. Boran Kartal Microbial Physiology Group Max Planck Institute for Marine Microbiology, Bremen, Germany iii iv Merci!!! PhD, Doctor of Philosophy, before I fully understand what it means, I came here far away from home for it. This 4-years exploring journey is both long and short. It is long because it took so much effort to figure out one topic in science even not fully understood it in the end, or let’s say that human beings can only take one step further in the exploring of knowledge with much more left to explore. Too short it is because I just figured out what it means to work in science and how to do it better, and 4 years has passed. Anyway in short, it has been my great great great pleasure to do my PhD in our Lab of microbiology in Ghent University. There are so much that I want to say and to thank for my 4 years life here, but here I have to make it short. Firstly I would like to thank my promoter Dr. Kim Heylen who gave me the opportunity to work on this topic. She is the smartest person I ever met and is so enthusiastic in science. During my study here, her extensive knowledge, and inspiring ideas and critical attitude on science inspired me a lot and she is the one that led me into the field of microbial nitrogen cycle, an amazing world that I would like to work on for the rest of my life. I highly appreciate all her contributions of time, ideas and energy to make my PhD experience productive and stimulating and guide me far away from a ‘silly’ beginner to a doctor! Hope now after all my effort in our research, I did satisfy her. ☺ Secondly I would like to thank my supervisor Prof. Dr. Paul De Vos, who has great passion in science. He guided me step by step to build up the chemostat with equipments and assisted me for this experimental work for one year even after his retirement. It was not easy to work with such big and complicated equipments, but thanks to him, this work became so exciting and I’m so proud to start another life journey of the chemostat. His humor, optimism and passion in research which made the tedious work more enjoyable and productive really have been of a great value to me and influenced my view of working in science. Next, I would like to thank Anne Willems who has been my promoter for the last year. She is a very caring person with a lot of patience; helped me a lot, both scientifically and personally. I still remember she worked so hard even in the weekend to carefully revising my manuscript. Thanks to her guidance, I could finish my last journey to my PhD diploma. v To sum up, without the guidance of Kim, Paul and Anne, I can never make this PhD work successful. And many many many thanks to my colleagues Margo, Bart, Evie, Bram, Helen, Guillaume, Charles, Maarten, Diana, Jessy, Charlotte, Eliza, Jindrich, Cindy, Anandi who has been so helpful for my experimental work, and especially Margo, Anique, Diana, Bram, Helen, Guillaume, who are willing to hear me out and help me out when I had problems, frustrated with my work or was depressed in my private life and gave me energy to keep on going. You all mean a lot to me!!! Thanks all people in the Laboratory of Microbiology and BCCM, who have been my company for my valuable and happy life here, I will never forget you! In addition, I would like to specially thank my friends and roommates, Leilei, QuQu, Gaozhen, Tongyan, Zhuo, Anan, Shanshan, Zhongjia, thanks for their company and their taking care of me, made me feel so at home in gent. And thanks my foreign friends in Belgium, David Servillo, Diana Goderich, Diana Angie Aguilar, Bodo Sylvianne, Sofie Thijs, Rema, Xin Li, Ewout, Alex, Ocean, thanks for bringing happiness to me and making my life so colourful and joyful! And other soul mates I met during my stay here, Dan, Jiyuan, Maxime and those nice people that I even forgot their names L! Without their wise advice and support, I wouldn’t have gone so far. And Last but not least, thanks to my parents for their support and their love, and thanks to my boyfriend Pieter Van den Meersch, the important PoKeMan, thanks for inviting me for Chinese food :) and falling in loving with me, you are the present from God. Thanks Van den Meersch Luc and Arijs Annie, and your whole family. Thanks for welcoming me to your family and taking care of me, I’m so touched. Many many thanks! I love you all! Yihua Sun Gent, January, 2017 vi 衷心感谢此时远在中国的关心爱护我的家人和朋友！感谢爸爸妈妈！女儿今天 所有的一切都离不开爸妈三十年来辛勤的养育和照顾，这本博士毕业论文和博士学位 是女儿的也是爸爸妈妈的。 虽然我有点任性，有时冲动伤爸爸妈妈的心，但以后会努 力改正的，永远最爱你们！愿用寸草心，来报三春晖！感谢 221 的朋友们！ 感谢林青 在京都的关心和照顾！感谢根特与我同甘共苦的朋友们 : 蕾蕾，曲曲，高团长，潼雁， 薛清，朱云鹏，朱丹，崔卓，李欣 ! 感谢异国他乡相识的同事朋友 David servillo, Diana Goderich, Diana Angie Aguilar , Bodo Sylvianne, Sofie Thijs, Rema, Xin Li, Ewout, Alex, Ocean! 谢谢你们出现在我的生命中带给我快乐和欣喜，让我成长！感谢 Pieter 一家对 我的关心和照顾，让我在这里体会到家的温暖。特别感谢比利时感谢根特这个让我充 满感恩的城市，塑造了我让我成为更好的人，让我自信的站在这里开启我人生新的篇 章！ 孙艺华 Gent, January 20 th , 2017 vii viii List of Abbreviations A amtB / AmtB ammonium transporter gene / protein Anammox anaerobic ammonium oxidation ANOVA analysis of variance ANI average nucleotide identity ATP adenosine triphosphate B BCCM Belgian Coordinated Collections of Microorganisms BLAST basic local alignment search tool BOF Bijzonder Onderzoeksfonds bp base pair C c cytochrome cbaBA nitric oxide reductase Cu ANor gene CDS coding sequences cNirK copper-dependent nitrite reductase cnor / cNor nitric oxide reductase gene / enzyme accepting electrons from heme c Cu ANor nitric oxide reductase accepting electron from cytochrome c CO 2 carbon dioxide codY / CodY GTP-sensing transcriptional pleiotropic repressor gene / protein D DNA deoxyribonucleic acid DNRA dissimilatory nitrate/nitrite reduction to ammonium dnrN / DnrN nitric oxide dependent regulator gene/ protein F fnr / Fnr fumarate-nitrate reductase regulatory protein gene/ protein FWO Fonds Wetenschappelijk Onderzoek ix G GC gas chromatography gdhA / GDH glutamate dehydrogenase gene / enzyme glnA / GS glutamine synthetase gene / enzyme glnH1 ABC-type glutamine transporter, periplasmic component / domain gene glnK /GlnK PII -type signal-transduction protein gene / protein glnP ABC-type glutamine transporter, permease component gene glnQ1 ABC-type glutamine transporter, ATPase component gene glnR / GlnR glutamine synthetase repressor gene / protein gltAB glutamate synthase genes gltT proton/sodium-glutamate symport protein gene GOGAT glutamate synthase or glutamine 2-oxoglutarate aminotransferase H hmp / HMP flavohaemoglobin gene / flavohaemoglobin I IBM International Business Machines Corporation IPCC International Panel for Climate Change K KEGG Kyoto Encyclopedia of Genes and Genomes L LMG Laboratory of Microbiology – Ghent University N NADH nicotinamide adenine dinucleotide nap / NapA periplasmic nitrate reductase gene / enzyme nar / NarG membrane-bound nitrate reductase gene / enzyme narGHJI the nar operon, coding for membrane-bound nitrate reductase narK/NarK nitrate/nitrite antiporter gene / protein narL nitrate/nitrite response regulatory protein gene narX nitrate/nitrite sensor regulatory protein gene x nasA/ NasA assimilatory nitrate transporter gene / enzyme nasC / NasC assimilatory nitrate reductase gene / enzyme NCBI National Center for Biotechnology Information NH 3 ammonia + NH 4 ammonium nirB /NirB gene/enzyme for assimilatory nitrite reductase (small subunit) nirC / NirC formate/nitrite transporter gene / enzyme nirD/NirD gene/enzyme for assimilatory nitrite reductase (large subunit) nirK / NirK periplasmic copper-dependent nitrite reductase gene / enzyme nirS / NirS periplasmic cytochrome cd1-dependent nitrite reductase gene / enzyme NtrB