Note Bioscience Microflora Vol. 26 (2), 37–40, 2007 Analysis of Acetogenic in Human Feces with Formyltetrahydrofolate Synthetase Sequences

Yuji OHASHI*, Tomoko IGARASHI, Fumi KUMAZAWA and Tomohiko FUJISAWA

Laboratory of Food Hygiene, Department of Food Science and Technology, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan Received January 23, 2007; Accepted for publication, February 21, 2007

The diversity of in human feces was analyzed with partial formyltetrahydrofolate synthetase (FTHFS) gene sequences. FTHFS sequences affiliated with Ruminococcus productus were predominantly recovered. Several sequences of acetogens that have not previously been identified were also recovered. Analysis of FTHFS sequences is available for the study of acetogenic ecology in the human intestinal tract. Key words: ; formyltetrahydrofolate synthetase; human feces

Numerous bacteria inhabit the human large intestine. colon was poorly understood due to a culture-dependent These microbes ferment undigested food materials and technique that limits the representation of the assemblage endogenous substances, such as mucus. The major end of acetogenic bacteria. A molecular approach allows the products of this are short chain fatty acids, phylogenic identification of microbes including , and hydrogen. Hydrogen, which is an uncultivable bacteria in the gastrointestinal microflora intermediate metabolite in anaerobic fermentation, must (14). With a molecular approach targeting the be removed to allow the reoxidation of electron carriers formyltetrahydrofolate synthetase (FTHFS) gene, which that are essential to the fermentation process (16). is a key enzyme in the CO2-reductive acetogenesis Hydrogen-utilizing bacteria, such as sulfate-reducing pathway catalyzing the ATP-dependent activation of bacteria, methanogenic archaea, and acetogenic bacteria, formate, the population of the acetogenic bacteria in contribute to the disposal of hydrogen in the colon (1, 3). some environmental samples has successfully been Sulfate-reducing bacteria are obligatory anaerobic analyzed (8–10, 13). In this study, we examined the bacteria that utilize hydrogen and sulfate as an electron diversity of acetogenic bacteria in human feces with donor and an electron acceptor, respectively. The major recovery and analysis of the FTHFS gene sequences end product of sulfate reduction is sulfide. from feces. Methanogenic archaea utilize H2 to reduce CO2 and The feces of four healthy male volunteers aged produce methane. Acetogenic bacteria utilize H2 to between 21 and 24 years old were collected. Bacterial reduce CO2 and form . These microbes compete DNA was extracted from 0.5 g of feces according to for H2 in an anaerobic fermentation system. In about 30– Godon et al. (5). The partial FTHFS gene was amplified 50% of the people in Western countries, methanogenesis with a polymerase chain reaction (PCR) using specific is an important H2 disposal pathway (4). In non-methane primers FTHFS-f (5’-TTYACWGGHGAYTTCCATGC- producers, H2 is consumed with sulfate reduction or 3’) and FTHFS-r (5’-GATTTGDGTYTTRGCCATACA- reductive acetogenesis (1, 2, 4, 12). The sulfate-reducing 3’). The PCR conditions were as follows: 10 mmol/l bacteria found in the human colon belong to genera Tris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2, Desulfovibrio, Desulfobacter, Desulfomonas, 160 μmol/l of each deoxynucleoside triphosphate Desulfobulbs, and Desulfotomaculum (4). The (dNTP), 400 μmol/l of each primer FTHFS-f and predominant methanogenic archaea are FTHFS-r, 1U rTaq polymerase (TOYOBO, Tokyo, Methanobrevibacter smithii (11). Although some Japan), and 1 μl of extracted bacterial DNA in a total acetogenic bacteria have been isolated from human feces volume of 25 μl. PCR was performed as described by (1, 17), the diversity of acetogenic bacteria in the human Leaphart and Lovell (9) with minor changes. Briefly, the touchdown thermal cycling protocol used included initial *Corresponding author. Mailing address: Laboratory of Food denaturation at 94°C for 3 min, followed by 9 cycles of Hygiene, Department of Food Science and Technology, Nippon denaturation at 94°C for 30 sec, annealing at 63°C for Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan. Phone: +81-422-51-6121. Fax: 30sec (decreased by 1°C per cycle to 55°C), and +81-422-52-9984. E-mail: [email protected] elongation at 72°C for 75 sec. After the touchdown

37 38 Y. OHASHI, et al. portion protocol had been done, 9 additional cycles, in Clostridium were not detected in this study. The which annealing temperature of 55°C was used, were acetogens with FTHFS genes similar to R. productus performed, and this was followed by a final elongation were predominant in the human colon. Furthermore, step consisting of 72°C for 5 min. After confirmation of some acetogens whose FTHFS genes have not previously the sizes and amounts of PCR products, clone libraries been identified colonized in the human colon. were constructed to analyze the composition of Cluster B included a partial FTHFS sequence from acetogens. PCR products were purified with Wizard SV known FTHFS-containing nonacetogens, such as C. Gel and PCR Clean-Up system (Promega, Madison, WI, acetobutyricum, C. acidurici, and Proteus vulgaris. C. USA). The purified PCR products were ligated and acetobutyricum and C. acidurici are known to utilize transformed into competent E. coli JM109 cells FTHFS in purine fermentation. P. vulgaris has FTHFS (TOYOBO) with the pGEM-T Easy vector system activity (15), and its FTHFS gene has previously been (Promega) according to the manufacturer’s instructions. detected (10). Although 12 OTUs (from OTU-19 to -30) The diversity of the clone library was characterized with belonged to cluster B, they were not affiliated with restriction fragment length polymorphisms (RFLP) using FTHFS sequences of known nonacetogens. The clones HaeIII, RsaI, and HhaI (TOYOBO) using adaptation of a in these OTUs were recovered from all volunteers, and previously described method (6). In this study, the RFLP they accounted for 26.4% of the clone library. group was regarded as a bacterial operational taxonomic Furthermore, 3 OTUs (from OTU-16 to -18) formed unit (OTU). The plasmid of 2 clones from each OTU independent clusters (cluster C) of known FTHFS was subjected to sequencing in the major OTU, which sequences. The percentage of clones in the library consisted of more than 10 clones. Otherwise, the plasmid belonging to these OTUs was 29.4. These results suggest of one clone was subjected to sequencing. The that some FTHFS-containing bacteria of which FTHFS sequencing was done by the Operon Biotechnologies genes have not yet been identified colonize in the human (Tokyo). Obtained and reference sequences were colon. translated and aligned using MEGA 3.1 (7) according to This is the first report to analyze the diversity of Leaphart and Lovell (9). The reference sequences were acetogens in human feces with the functional gene, obtained from GenBank. A phylogenic tree was FTHFS. Although nonacetogens have FTHFS, the constructed using the neighbor-joining method of sequences of FTHFS derived from acetogens are MEGA 3.1. The partial sequence of Thermoplasma phylogenically different from those of nonacetogens. acidophilum was used as an outgroup for rooting the tree. Therefore, the analysis of acetogens with the FTHFS Amplicons of FTHFS of correct size (approximately gene might be a useful method for the study of 1.1 kbp) were detected in samples from all volunteers. In acetogenic ecology. However, the poor analysis of total, 33 OTUs were identified from 163 clones that were FTHFS-producing bacteria in the human intestinal tract generated from four volunteers (41, 44, 34, and 44 clones limited the understanding of acetogens. Accordingly, from volunteers I, II, III, and IV, respectively). further analysis of FTHFS production in the human The phylogenic tree contained four clusters, A, B, C, intestinal bacteria is necessary. and D (Fig. 1). Cluster A consisted of a partial FTHFS Nucleotide sequence accession numbers. The sequence from known acetogens. Eighteen OTUs (from nucleotide sequences determined in this study have been OTU-1 to -15 and from OTU-31 to -33) belonged to deposited in the GenBank database under accession no. cluster A. Fifteen OTUs (from OTU-1 to -15) of those AB291639 to AB291671. groups were affiliated with Ruminococcus productus, and shared more than 87% amino acid identity with the REFERENCES FTHFS from R. productus. Clones belonging to these (1) Bernalier A, Rochet V, Leclerc M, Doré J, Pochart P. OTUs were predominantly recovered from all 1996. Diversity of H2/CO2-utilizing acetogenic volunteers, and their proportion reached 41.7% of the bacteria from feces of non-methane-producing clone library. The other 3 OTUs (from OTU-31 to -33) humans. Curr Microbiol 33: 94–99. (2) Chassard C, Bernalier-Donadille A. 2006. H2 and belonging to cluster A were not affiliated with known acetate transfers during xylan fermentation between a acetogens. The clones belonging to these OTUs were butyrate-producing xylanolytic species and recovered from volunteer II, and they made up 2.5% of hydrogenotrophic microorganisms from the human the clone library. Although acetogens related to the gut. FEMS Microbiol Lett 254: 116–122. genus Clostridium and R. productus have been isolated (3) Gibson GR, Cummings JH, Macfarlane GT, Allison C, from human feces (1), acetogens related to the genus Segal I, Vorster HH, Walker ARP. 1990. Alternative ACETOGENIC BACTERIA IN HUMAN FECES 39

Fig. 1. Phylogenic analysis of partial FTHFS sequences. The tree was constructed using the neighbor-joining method. The partial FTHFS sequence of Thermoplasma acidophilum was used as an outgroup for rooting the tree. The values at the nodes are the percentages of 1,000 bootstrap replicates. Bootstrap values below 70% are not shown. 40 Y. OHASHI, et al.

pathways for hydrogen disposal during fermentation in Environ Microbiol 57: 2602–2609. the human colon. Gut 31: 679-683. (11) Miller TL, Wolin MJ, de Macario EC, Macario AJ. (4) Gibson GR, Macfarlane GT, Cummings JH. 1993. 1982. Isolation of Methanobrevibacter smithii from Sulphate reducing bacteria and hydrogen human feces. Appl Environ Microbiol 43: 227–232. in the human large intestine. Gut 34: 437–439. (12) Robert C, Del’Homme C, Bernalier-Donadille A. (5) Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta 2001. Interspecies H2 transfer in cellulose degradation R. 1997. Molecular microbial diversity of an anaerobic between fibrolytic bacteria and H2-utilizing digester as determined by small-subunit rDNA microorganisms from the human colon. FEMS sequence analysis. Appl Environ Microbiol 63: 2802– Microbiol Lett 205: 209–214. 2813. (13) Salmassi TM, Leadbetter JR. 2003. Analysis of genes (6) Inoue R, Ushida K. 2003. Vertical and horizontal of tetrahydrofolate-dependent metabolism from transmission of intestinal commensal bacteria in the rat cultivated spirochaetes and the gut community of the model. FEMS Microb Ecol 46: 213–219. termite Zootermopsis angusticollis. Microbiol 149: (7) Kumar S, Tamura K, Nei M. 2004. MEGA3: 2529–2537. Integrated software for molecular evolutionary (14) Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, genetics analysis and sequence alignment. Brief Collins MD, Doré J. 1999. Direct analysis of genes Bioinform 5: 150–163. encoding 16S rRNA from complex communities (8) Leaphart AB, Friez, MJ, Lovell CR. 2003. reveals many novel molecular species within the Formyltetrahydrofolate synthetase sequences from salt human gut. Appl Environ Microbiol 65: 4799-4807. marsh plant root reveal a diversity of acetogenic (15) Whitehead TR, Park M, Rabinowitz JC. 1988. bacteria and other bacterial functional groups. Appl Distribution of 10-formyltetrahydrofolate synthetase Environ Microbiol 69: 693–696. in eubacteria. J Bacteriol 170: 995–997. (9) Leaphart AB, Lovell CR. 2001 Recovery and analysis (16) Wolin MJ, Miller TL. 1983. Interactions of microbial of formyltetrahydrofolate synthetase gene sequences populations in cellulose fermentation. Fed Proc 42: from natural populations of acetogenic bacteria. Appl 109–113. Environ Microbiol 67: 1392–1395. (17) Wolin MJ, Miller TL. 1993. Bacterial strains from (10) Lovell CR, Hui Y. 1991. Design and testing of a human feces that reduce CO2 to . Appl functional group-specific DNA probe for the study of Environ Microbiol 59: 3551–3556. natural populations of acetogenic bacteria. Appl