Denitrification and the hypoxic response in obligate aerobic methane-oxidizing bacteria By Kerim Dimitri Kits A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Microbiology and Biotechnology Department of Biological Sciences University of Alberta Kerim Dimitri Kits, 2016 Abstract Aerobic methanotrophic bacteria lessen the impact of the greenhouse gas methane (CH4) not only because they are a sink for atmospheric methane but also because they oxidize it before it is emitted to the atmospheric reservoir. Aerobic methanotrophs, unlike anaerobic methane oxidizing archaea, have a dual need for molecular oxygen (O2) for respiration and CH4 oxidation. Nevertheless, methanotrophs are highly abundant and active in environments that are extremely hypoxic and even anaerobic. While the O2 requirement in these organisms for CH4 oxidation is inflexible, recent genome sequencing efforts have uncovered the presence of putative denitrification genes in many aerobic methanotrophs. Being able to use two different terminal electron acceptors – hybrid respiration – would be massively advantageous to aerobic methanotrophs as it would allow them to halve their O2 requirement. But, the function of these genes that hint at an undiscovered respiratory anaerobic metabolism is unknown. Moreover, past work on pure cultures of aerobic methanotrophs ruled out the possibility that these organisms - denitrify. An organism that can couple CH4 oxidation to NO3 respiration so far does not exist in pure culture. So while the role of aerobic methanotrophs in the carbon cycle is appreciated, the hypoxic metabolism and contribution of these specialized microorganisms to the nitrogen cycle is not understood. Here we demonstrate using cultivation dependent approaches, microrespirometry, and whole genome, transcriptome, and proteome analysis that an aerobic - methanotroph – Methylomonas denitrificans FJG1 – couples CH4 oxidation to NO3 respiration - with N2O as the terminal product via the intermediates NO2 and NO. Whole transcriptome and proteome analysis reveals that respiratory nitrate (Nar, Nap), nitrite (Nir), and nitric oxide (Nor) reductases are expressed and upregulated at the transcript and protein levels under denitrifying conditions. Physiological analysis of denitrifying cultures of M. denitrificans FJG1 also confirms ii - that NO3 respiration is bioenergetically advantageous. We also confirm denitrification activity and upregulation of denitrification gene expression in another obligate methanotroph – Methylomicrobium album BG8 – and also show that this activity is supported by a diverse array of energy sources. Formaldehyde fermentation has been identified by others as an important adaptation of aerobic methanotrophs to hypoxia. We additionally illustrate here that M. denitrificans FJG1 can also ferment formaldehyde and that nitrate respiration and fermentation occur simultaneously during hypoxia. The work herein is a significant contribution to our knowledge of how aerobic methanotrophs affect greenhouse gas flux though CH4 oxidation in low O2 environments and through N2O production via denitrification. Preface This thesis is an original work by Kerim Kits and serves as a compendium of original research articles that are either currently published in peer reviewed journals or are currently in preparation for submission. The research objectives and background for these research articles are detailed in the opening chapter. The closing chapter – Chapter 5 – provides a synopsis of the conclusions, contributions, and major findings of this thesis and the articles contained within. The original research articles that contribute to this thesis have been reformatted from the original publication style and are as follows: Chapter 2: Kits, K. D., Klotz, M. G., and Stein, L. Y. (2015). Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1. Environ. Microbiol. 17, 3219–3232. doi:10.1111/1462-2920.12772. (in press) Chapter 3: Kits, K. D., Campbell, D. J., Rosana, A. R., and Stein, L. Y. (2015). Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8. Front. Microbiol. 6, 1072. doi:10.3389/fmicb.2015.01072. (in press) iii Chapter 4: Kits, K.D., Manuel, K., Liu, D., and Stein, L. Y. (2016). Simultaneous expression of denitrifying and fermentation pathways in the methanotroph, Methylomonas denitrificans FJG1. (in preparation) Additional original research articles in which the thesis author is the primary author but that do not contribute to the individual outlined chapters are included as appendices: Appendix A: Kits, K. D., Kalyuzhnaya, M. G., Klotz, M. G., Jetten, M. S. M., Op den Camp, H. J. M., Vuilleumier, S., et al. (2013). Genome Sequence of the Obligate Gammaproteobacterial Methanotroph Methylomicrobium album Strain BG8. Genome Announc 1, e0017013–e00170– 13. doi:10.1128/genomeA.00170-13. (in press) Publications in which the thesis author was a co-author and not the primary author and are therefore not included in the thesis are: Stein, L. Y., F. Bringel, A. A. DiSpirito, S. Han, M. S. M. Jetten, M. G. Kalyuzhnaya, K. D. Kits, M. G. Klotz, H. J. M. O. den Camp, J. D. Semrau, S. Vuilleumier, D. C. Bruce, J. Cheng, K. W. Davenport, L. Goodwin, S. Han, L. Hauser, A. Lajus, M. L. Land, A. Lapidus, S. Lucas, C. Medigue, S. Pitluck, and T. Woyke. (2011). Genome sequence of the methanotrophic Alphaproteobacterium, Methylocystis sp. Rockwell (ATCC 49242). J. Bacteriol. 193, 2668– 2669. doi:10.1128/JB.00278-11. Boden, R., Cunliffe, M., Scanlan, J., Moussard, H., Kits, K. D., Klotz, M. G., et al. (2011). Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09. J. Bacteriol. 193, 7001–7002. doi:10.1128/JB.06267-11. Hamilton, R., Kits, K. D., Ramonovskaya, V. A., Rozova, O. N., Yurimoto, H., Iguchi, H., et al. (2015). Draft genomes of gammaproteobacterial methanotrophs isolated from terrestrial ecosystems. Genome Announc 3, e00515–15. doi:10.1128/genomeA.00515-15. Kozlowski, J. A., Kits, K. D., and Stein, L. Y. (2016a). Genome Sequence of Nitrosomonas iv communis Strain Nm2, a Mesophilic Ammonia-Oxidizing Bacterium Isolated from Mediterranean Soil. Genome Announc 4, e01541–15. doi:10.1128/genomeA.01541-15. Kozlowski, J. A., Kits, K. D., and Stein, L. Y. (2016b). Complete Genome Sequence of Nitrosomonas ureae Strain Nm10, an Oligotrophic Group 6a Nitrosomonad. Genome Announc 4, e00094–16. doi:10.1128/genomeA.00094-16. The contribution of the thesis author to the aforementioned published journal articles was as follows. For chapter 2, the thesis author performed all of the physiology, genome sequencing, transcriptome sequencing and all of the data analysis while Martin G. Klotz performed the phylogenetic analysis of the M. denitrificans FJG1 genome. The thesis author wrote the manuscript (Chapter 2) with input and guidance from M. G. Klotz and L. Y. Stein. For chapter 3, the thesis author performed all of the physiology, all of the data analysis, and a portion of the expression analysis while Albert R. R. Rosana finished the expression analysis. Chapter 3 was written by the thesis author with input from L. Y. Stein who also helped develop experimental design for this chapter. For chapter 4, the thesis author carried out the transcriptome sequencing and all data analysis, and Manuel Kleiner and Dan Liu performed the proteomics. In all cases, Lisa Y. Stein contributed guidance and editorial comments on written manuscripts. With regard to the publications in which the thesis author was a co-author and not a primary author, the contributions from the thesis author were as follows. The thesis author performed the main bioinformatics role, assembled, and closed genome sequences for Kozlowski et al., (2016a) and Kozlowski et al., (2016b). In all other co-authored original research articles, the author contributed to genome annotation. v Acknowledgements First, I would like to thank my supervisor – Dr. Lisa Stein. When I first started working in your lab, during my third undergraduate year, my CV was not that of a promising future research assistant or graduate student. You recognized something in me and you then played an enormous role in the development of my skills and interests as a then (very) junior scientist. Thank you for accepting me as a graduate student, thank you for providing me with funding and space to do research, and thank you for always giving me the independence to pursue my interests. Also, I have to thank you for always allowing and encouraging me to attend meetings and meet other scientists – these experiences gave me immeasurable maturity and perspective. I also have to levy a massive thank you to the other members of my graduate studies committee – Dr. Yan Boucher and Dr. George Owttrim. Thank you both so much for taking the time to read over my work and assessing my progress during our committee meetings. You were both valuable committee members and I am incredibly grateful for your suggestions, advice, and support. It is also important that I mention Dr. Martin Klotz, although Martin wasn’t officially part of my committee; Martin, your approach to critical thinking massively influenced me and ignited in me a true appreciation for exactitude of meaning, nomenclature, and logic. I have to recognize the contribution that other members of the University of Alberta had to my work. Thank you to the storeroom staff