The ISME Journal (2014) 8, 1323–1335 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Phylogenetic distribution of three pathways for propionate production within the human gut microbiota This article has been corrected since Advance Online Publication and a corrigendum is also printed in this issue Nicole Reichardt1,2, Sylvia H Duncan1, Pauline Young1, Alvaro Belenguer1,3, Carol McWilliam Leitch1,4, Karen P Scott1, Harry J Flint1 and Petra Louis1 1Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, UK and 2Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl- CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota. The ISME Journal (2014) 8, 1323–1335; doi:10.1038/ismej.2014.14; published online 20 February 2014 Subject Category: Microbial ecology and functional diversity of natural habitats Keywords: acrylate pathway; gut microbiota; propanediol pathway; propionate; succinate pathway Introduction breakdown of non-digestible carbohydrates originating from the diet leads to the formation of fermenta- The human large intestine is inhabited by a diverse tion acids, mainly the short-chain fatty acids microbial community that influences host health (SCFAs) acetate, propionate and butyrate. SCFAs are through a number of mechanisms, including the absorbed by the host and used as an energy source production of metabolites, a barrier function against but also have a variety of distinct physiological pathogens, and interactions with the host’s immune effects. Butyrate, in particular, is believed to system and physiology (Flint et al., 2012b). The counteract colorectal cancer and inflammation (Hamer et al., 2008; Berni Canani et al., 2012). Propionate also has potential health-promoting Correspondence: Petra Louis, Rowett Institute of Nutrition and effects that include anti-lipogenic, cholesterol- Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK. lowering, anti-inflammatory and anti-carcinogenic E-mail: [email protected] action (Hosseini et al., 2011; Vinolo et al., 2011). 3Present address: Instituto de Ganaderı´a de Montan˜ a, Finca Furthermore, the potential role of propionate in Marzanas, s/n, 24346 Grulleros, Leo´n, Spain. enhancing satiety (Arora et al., 2011) is of increasing 4Present address: MRC—University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK. interest given the rising incidence of obesity across Received 17 July 2013; revised 20 November 2013; accepted 9 the world. Recent proteomic work suggests that January 2014; published online 20 February 2014 some of the effects of propionate at the cellular level Propionate formation in human gut bacteria N Reichardt et al 1324 differ from the action of butyrate (Kilner et al., gains additional energy from succinate in the 2012). The recently deorphanised SCFA receptors presence of lactate as the main growth substrate FFA2 and FFA3 are likely to mediate some of the (Janssen, 1992), whereas Phascolarctobacterium actions of propionate; however, the exact mechan- succinatutens, isolated from human faeces, can isms remain to be established (Ulven, 2012). grow on succinate alone (Watanabe et al., 2012). Enhancing propionate production in the colon Selenomonas ruminantium strains isolated from the through dietary intervention with non-digestible rumen produce lactate, acetate and propionate from carbohydrates is an attractive approach for increasing carbohydrates, and some are also able to utilise satiety and maintaining health; however, in-depth lactate for growth (Bryant, 1956; Gilmour et al., knowledge of propionate producers within the gut 1994). This class of bacteria remains understudied microbiota is required for the development of diet- in the human gut and it remains to be established ary strategies. which genera dominate in this ecosystem. The In order to understand and manipulate SCFA acrylate pathway for propionate formation has been formation by the human colonic microbiota, we characterised in detail in the soil bacterium Clos- need to know which phylogenetic groups, substrates tridium propionicum (Hetzel et al., 2003), and the and pathways have major roles in the formation of corresponding genes have recently been described each acid. Relevant information is now available for (Kandasamy et al., 2013). This pathway is also butyrate formation (Louis and Flint, 2009, Louis present in the rumen bacterium Megasphaera et al., 2010) but is currently lacking for propionate. elsdenii within the Negativicutes (Hino and Three different biochemical pathways for propio- Kuroda, 1993), which produces butyrate during nate production are known to be present in the growth on glucose, but propionate during growth microbiota (Figure 1). Bacteroidetes utilise the on lactate (Hino and Kuroda, 1993). It is possible to succinate pathway via methylmalonyl-CoA (Macy distinguish the succinate pathway from the acrylate and Probst, 1979), which is also present in several pathway by incubation with stable isotope-labelled Firmicutes bacteria belonging to the recently substrates (Bourriaud et al., 2005; Morrison et al., proposed new class of Negativicutes (formerly 2006). Finally, several different bacteria are known classed as Veillonellaceae or Clostridial cluster IX to produce 1,2-propanediol from deoxy sugars such (Marchandin et al., 2010)). Bacteroidetes mainly as fucose and rhamnose, or via different pathways utilise polysaccharides and peptides for growth from dihydroxyacetonephosphate or lactate (Saxena (Macy and Probst, 1979, Flint et al., 2012a), whereas et al., 2010). In some bacteria, including Salmonella in Firmicutes propionate formation has been enterica serovar Typhimurium, 1,2-propanediol can reported from organic acids as well (Seeliger et al., be further metabolised to propionate or propanol 2002; Watanabe et al., 2012). Veillonella parvula (Bobik et al., 1999). Propionate formation from many hexoses, pentoses fucose, rhamnose oxaloacetate PEP DHAP + L-lactaldehyde malate pyruvate CO2 fumarate succinate lactate succinyl-CoA propane-1,2-diol lactoyl-CoA R-methylmalonyl-CoA lcdA propionaldehyde acryloyl-CoA S-methylmalonyl-CoA pduP pduQ mmdA propionyl-CoA propionyl-CoA propionyl-CoA propanol propionate propionate propionate P1 P2 P3 Figure 1 Known pathways for propionate formation in human gut bacteria. (P1), Succinate pathway; (P2), acrylate pathway; (P3), propanediol pathway. Substrates utilised are shown in boxes. Genes targeted as molecular markers for the specific pathways are indicated. DHAP, dihydroxyacetonephosphate; PEP, phosphoenolpyruvate. The ISME Journal Propionate formation in human gut bacteria N Reichardt et al 1325 fucose via propanediol has also been described in 13C-labelled L-lactate at 10 molar % excess were the human gut anaerobe Roseburia inulinivorans, prepared for estimation of concentrations by isotope which produces butyrate rather than propionate dilution and for determination of enrichments of when grown on glucose (Scott et al., 2006). SCFA and lactate by gas chromatography coupled Our primary aim here was to explore the distribu- with mass spectrometry. They were measured by tion of the three known pathways for propionate analysis of the tert-butyldimethylsilyl derivatives. production within the human microbiota with Procedures were as described previously (Belenguer genomic and metagenomic approaches. In addition, et al., 2007). The mass spectrometer was operated we demonstrate the operation of the acrylate path- under electron impact ionisation conditions. For the way for propionate production for the first time in concentration determinations, appropriate correc- an isolated human colonic bacterium by means of tions were applied for the enrichments