Methane (CH4) is the most abundant organic compound in the atmosphere, largely originating from anthropogenic and natural biogenic sources1. Traditionally, biogenic CH4 has been regarded as the nal product of the anoxic decomposition of organic matter by methanogenic Archaea. However, plants2-4, fungi5, algae6,7 and cyanobacteria8 have recently been shown to produce CH4 in the presence of oxygen. While methanogens produce CH4 enzymatically during anaerobic energy metabolism9, the requirements and pathways for CH4 production by "non-methanogenic" cells are poorly understood. Here we demonstrate that CH4 formation by Bacillus subtilis is triggered by free iron species, enhanced by oxidative stress and restricted to metabolically active life-cycle stages. We also show that other model organisms from Bacteria and Eukarya including a human cell line release CH4 and respond to inducers of oxidative stress by enhanced CH4 formation. Our results imply that all living cells possess a common mechanism of CH4 formation without the need for speci c enzymes. We propose that CH4 formation is a conserved feature of living systems which is coupled to metabolic activity and the concomitant generation of reactive oxygen species. Our ndings open new perspectives for our understanding of environmental CH4 cycling, oxidative stress responses and the search for extraterrestrial life. BackgroundMethane is a highly potent greenhouse gas that affects Earth's climate. Around 70 % of all emissions to the atmosphere derive from biogenic sources 10 . Biological CH 4 formation has long been considered to occur only under strictly anoxic conditions in organisms belonging to the domain Archaea. To generate the cellular fuel ATP, methanogenic archaea convert simple compounds, such as CO 2 , H 2 or acetate, into CH 4 . This process of methanogenesis depends on reactions that are catalysed by unique sets of enzymes and co-enzymes 9 . In addition, small amounts of CH 4 can be formed via 'mini-methanogenesis' in several sulphate-reducing bacteria which contain the enzyme carbon monoxide dehydrogenase 11,12 . However, during the past 15 years, evidence has been accumulating that other organisms produce CH 4 under aerobic conditions. These include both multicellular organisms, such as plants 2-4 and saprotrophic fungi 5 , and unicellular organisms, including marine and freshwater algae 6,13 and cyanobacteria 8 . These organisms generate energy via photosynthesis and/or respiration, and it is unclear why and how they release CH 4 . Multiple marine and freshwater bacteria harbouring the C-P lyase pathway have been reported to generate CH 4 from methylphosphonate [14][15][16][17][18] . Several bacteria and archaea have also been shown to possess alternative nitrogenases 19,20 or nitrogenase-like reductases 21 , which can produce CH 4 and other hydrocarbons. In addition -as we will demonstrate here -living systems can form CH 4 without the need for speci c enzymes, and such pathways could drive CH 4 formation in all cells.In plants, CH 4 formation is enha...

All the people I worked with gave me incredible support during these years, technical and emotional. From the first day, with my shirt decorated with bananas, to the lasts, when they had to bear my nervous and stressed mood. I thank Anna, Linda and Pierre, for the funny moments, the laughs and the many beers together. And for the precious scientific discussions, of course! Thank you also to Eethan, the living scientific encyclopedia, with (almost) always the right answer to my problems in the lab. Thank you, Enoch, for the discussions, especially those in the microscopy room. Emir, thank you for your help with the cloning, microscopy and the bacteriographs. I like your enthusiasm toward science, do not lose it! Thank you, Mehmet, for the support to the protein function prediction of my project, I appreciated the point of view from someone with a "dry biology" background and the comments on my thesis. Jeremy, Mirko, Giada, and Jara, thank you for the daily chats and jokes together. The daily routine was lighter with you around. Thank you, Dagmar, oh Dagmar, for your patience and your smiles. Lastly, thanks to the grumpy Daniel, my dear deskmate and benchmate, fellow of complains.Vorrei anche ringraziare i miei amici rimasti in Italia. Anche se ci vediamo poco, quando ci troviamo è sempre una festa. Sono fortunato a non averli persi dopo più di 5 anni passati all'estero: è anche grazie a loro che ho mantenuto la tenacia per arrivare fino alla fine. In particolare, Claudio, Stefano e Vito, grazie per la vostra vicinanza nonostante la lontananza. Grazie anche a Laura, la mia supervisor durante il mio tirocinio di tesi magistrale in Unimi, che mi ha insegnato come pensare e come muovermi nell'ambito di un progetto di ricerca: quello che ho imparato in quell'anno mi è stato molto utile durante il dottorato.Un grazie immenso alla mia famiglia e al nonno, senza i quali non sarei potuto arrivare dove sono ora, incoraggiandomi e sostenendomi sempre. E infine, Margherita, grazie per essere sempre stata al mio fianco in questi anni, anche quando non avevo occhi che per la tesi, risollevandomi dai momenti di sconforto e spazzando via i cattivi pensieri. ContributionsLeonard Ernst, a former bachelor student of the Di Ventura lab, cloned a 2-plasmid-based library composed of 5 VVD-AraC fusion proteins under IPTG induction and performed the first explorative experiments to demonstrate the light-responsiveness of the system. Emir Bora Akmeriç, technical assistant in our laboratory, helped me during the last year of my doctorate in the cloning process of some constructs and with the induction and imaging of the genes with poor or unknown function. Moreover, he took care of the Blade Runner bacteriograph, repeating it until he made it flawless.Mehmet Ali Öztürk, postdoc in our laboratory, contributed with structural biology analysis of the I1-I2 sequences rearrangements and collected, interpreted and catalogued the function and localization prediction outputs of the genes with poor or unknown function. He also provided valuable sug...

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

Recently it has been proposed that methane might be produced by all living organisms via a mechanism driven by reactive oxygen species that arise through the metabolic activity of cells. Here, we summarise details of this novel reaction pathway and discuss its potential significance for clinical and health sciences. In particular, we highlight the role of oxidative stress in cellular methane formation. As several recent studies also demonstrated the anti‐inflammatory potential for exogenous methane‐based approaches in mammalians, this article addresses the intriguing question if ROS‐driven methane formation has a general physiological role and associated diagnostic potential.