Original citation: Jameson, Eleanor, Fu, Tiantian, Brown, I. R., Paszkiewicz, K., Purdy, K. J., Frank, S. and Chen, Yin. (2015) Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis. Environmental Microbiology. DOI: 10.1111/1462-2920.13059 Permanent WRAP url: http://wrap.warwick.ac.uk/72391 Copyright and reuse: The Warwick Research Archive Portal (WRAP) makes this work of researchers of the University of Warwick available open access under the following conditions. This article is made available under the Creative Commons Attribution- 3.0 Unported (CC BY 3.0) license and may be reused according to the conditions of the license. For more details see http://creativecommons.org/licenses/by/3.0/ A note on versions: The version presented in WRAP is the published version, or, version of record, and may be cited as it appears here. For more information, please contact the WRAP Team at: [email protected] http://wrap.warwick.ac.uk/ bs_bs_banner Environmental Microbiology (2015) doi:10.1111/1462-2920.13059 Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis Eleanor Jameson,1† Tiantian Fu,1† Ian R. Brown,2 Introduction Konrad Paszkiewicz,3 Kevin J. Purdy,1 Bacteroidetes, Firmicutes and Proteobacteria are the Stefanie Frank2* and Yin Chen1** dominant microbes in bacterial communities of the human 1School of Life Sciences, University of Warwick, gut, with the former two accounting for > 90% of microbial Coventry, CV4 7AL, UK. biomass in a healthy gut (Arumugam et al., 2011; 2School of Biosciences, University of Kent, Canterbury, Yatsunenko et al., 2012). Bacteroidetes and Firmicutes Kent CT2 7NJ, UK. are generally strict anaerobes fermenting dietary and 3College of Life and Environmental Sciences, University host-derived proteins and carbohydrates, whereas many of Exeter, Exeter, EX4 4QD, UK. Proteobacteria, particularly those Gammaproteobacteria of the Enterobacteriaceae family are facultative anaer- Summary obes, which live in close proximity to the mucosa of the gut lumen (Eckburg et al., 2005; El Kaoutari et al., 2013; Gammaproteobacteria are important gut microbes Winter et al., 2013b). An imbalance in gut microbiota, but only persist at low levels in the healthy gut. The known as dysbiosis, is usually associated with a sudden ecology of Gammaproteobacteria in the gut environ- increase in the abundance of facultative anaerobic ment is poorly understood. Here, we demonstrate that Gammaproteobacteria, particularly Enterobacteriaceae, choline is an important growth substrate for repre- a characteristic of gut malfunction and intestinal sentatives of Gammaproteobacteria. Using Proteus inflammation (Winter et al., 2013b). Understanding the mirabilis as a model, we investigate the role of choline mechanisms underpinning the sudden increase of metabolism and demonstrate that the cutC gene, Gammaproteobacteria during gut microbiota dysbiosis is encoding a choline-trimethylamine lyase, is essential essential to gain better knowledge of the association for choline degradation to trimethylamine by targeted between gut dysbiosis and a number of bowel diseases, mutagenesis of cutC and subsequent complementa- e.g. Crohn’s disease (Baumgart et al., 2007), irritable tion experiments. Proteus mirabilis can rapidly bowel syndrome (Krogius-Kurikka et al., 2009) and utilize choline to enhance growth rate and cell yield in necrotizing enterocolitis (Normann et al., 2013). broth culture. Importantly, choline also enhances A growing body of evidence has emerged over the past swarming-associated colony expansion of P. decade which supports the interplay between the metabo- mirabilis under anaerobic conditions on a solid lism of gut microbiota and the host response leading surface. Comparative transcriptomics demonstrated to an increase in facultative Gammaproteobacteria during that choline not only induces choline-trimethylamine gut dysbiosis (reviewed in Winter et al., 2013a). For lyase but also genes encoding shell proteins for example, many Gammaproteobacteria can utilize the formation of bacterial microcompartments. tetrathionate as an alternative electron acceptor (Winter Subsequent analyses by transmission electron et al., 2010). Tetrathionate is produced during gut microscopy confirmed the presence of such novel inflammation by the oxidation of hydrogen sulfide, a microcompartments in cells cultivated in liquid broth metabolite abundant in a normal gut through the action of and hyper-flagellated swarmer cells from solid fermentation by Firmicutes and Bacteroidetes with subse- medium. Together, our study reveals choline metabo- quent reduction of sulfate by sulfate-reducing bacteria lism as an adaptation strategy for P. mirabilis and (Winter et al., 2010). Gut inflammation also induces contributes to better understand the ecology of this the formation of a number of other electron acceptors bacterium in health and disease. such as trimethylamine N-oxide (TMAO) and nitrate, which can support anaerobic respiration of these faculta- Received 9 June, 2015; revised 14 September, 2015; accepted 15 tive Gammaproteobacteria but not obligate anaerobic September, 2015. For correspondence. *E-mail [email protected]; Firmicutes or Bacteroidetes (Winter et al., 2013b). Nitrate Tel. +44 1227 824692; Fax 00 44 12 27824034. **E-mail Y.chen.25@ warwick.ac.uk; Tel. +44 24 76528976; Fax 00 44 24 76523701. is generated through the oxidation of reactive nitrogen †These authors contributed equally to this work. species released from neutrophils (Szabo et al., 2007), © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2 E. Jameson et al. and TMAO was postulated to be formed from the oxida- growth of this bacterium in liquid culture. Furthermore, we tion of trimethylamine (TMA) as a result of gut microbiota demonstrate, for the first time, true anaerobic swarming metabolism (Winter et al., 2013b). behaviour of this bacterium and reveal that choline deg- Not only can Gammaproteobacteria utilize these alter- radation promotes the swarming-associated colony native host-derived electron acceptors, but they can expansion rates on solid agar surface. Together, our study also acquire carbon and energy from host-derived mol- contributes a potential explanation to the success and ecules. For example, Salmonella spp. can utilize competitiveness of the cut-containing P. mirabilis in gut ethanolamine anaerobically as a carbon and energy dysbiosis (Garrett et al., 2010). source. Ethanolamine is derived from the phospholipid phosphatidylethanolamine, a constituent of the mem- Results branes of gut epithelial cells, which is constantly produced Identification of a functional choline-TMA lyase in during luminal tissue sloughing (Price-Carter et al., 2001; P. mirabilis Thiennimitr et al., 2011). Metabolism of ethanolamine by Salmonella is carried out in a unique bacterial organelle, The enzyme responsible for the conversion of choline to namely a bacterial microcompartment (Chen et al., 1994; TMA has recently been identified from Desulfovibrio Penrod and Roth, 2006). It is thought that these microbial desulfuricans (Craciun and Balskus, 2012). This enzyme organelles optimize pathways with toxic or volatile inter- belongs to the glycyl radical enzyme family. We have mediates (Havemann et al., 2002; Penrod and Roth, analysed the phylogeny of CutC, a choline-TMA lyase, in 2006). The protein shell of the ethanolamine utilization genome-sequenced bacteria (Fig. 1). The resultant (Eut) microcompartments acts as a selective barrier that phylogenetic analysis shows two distinct clusters of CutC helps to retain volatile aldehyde intermediates and proteins, as previously reported by Martínez-del Campo channel intermediates to the next pathway enzyme by and colleagues (2015). The type I cluster is primarily colocalization of critical enzymes (Chowdhury et al., comprised of obligate anaerobes including the character- 2014). ized CutC from D. desulfuricans (Craciun and Balskus, Another major phospholipid in the gut epithelium is 2012), whereas the type II cluster contains facultative choline-containing phosphatidylcholine (Kawai et al., anaerobes, including P. mirabilis and many isolates of the 1974). It is known that choline can be a precursor for TMA Enterobacteriaceae family. The type II CutC cluster is and the enzyme catalyses this reaction, CutC, has further split in to II.a containing Gammaproteobacteria recently been identified as a glycyl radical-containing and II.b containing Firmicutes and Deltaproteobacteria.A protein (Craciun and Balskus, 2012). Interestingly, the striking difference of these cutC genes appears to be in cutC gene cluster also contains a set of proteins that are their length: type I and II.b cutC are approximately 2.5 kb likely to form microcompartments (Craciun and Balskus, in length, while type II.a are longer, with approximately 2012). Although the cut gene cluster was originally 3.4 kb. The type II.a CutC enzymes have an elongated characterized from a sulfate reducer, Desulfovibrio N-terminus compared with those in cluster I and II.b, while desulfuricans (Craciun and Balskus, 2012), subsequent the rest of the sequence is highly conserved (see analyses have shown that cutC homologues and the shell sequence alignment