Diversity of the Formyltetrahydrofolate Synthetase (FTHFS)Gene in the Proximal and Mid Ostrich Colon

Hiroki Matsui • Saori Yoneda • Tomomi Ban-Tokuda •

Masaaki Wakita

Received: 6 December 2009 / Accepted: 26 April 2010 / Published online: 11 May 2010

� Springer Science+Business Media, LLC 2010

Abstract We analysed fragments of the formyltetrahy-

drofolate synthetase (FTHFS) gene, which encodes a key

enzyme in reductive acetogenesis, from the bacterial flora in

the proximal (PC) and mid (MC) colon of three ostriches to

assess and compare bacterial diversity in this organ. Two

clone libraries of FTHFS fragments were constructed from

DNA extracted from digesta of the PC and MC, and a total of

46 cloned sequences were analysed from each library. A

wide variety of FTHFS sequences were recovered. The

coverage of the PC and MC libraries was 90.0% and 83.3%,

respectively. Shannon–Wiener index (H’) and Chao1 of the

MC library were higher than those of PC library. The

sequences from each library were classified into 15 opera-

tional taxonomic units (OTUs) and clusters. Only four OTUs

in cluster I were distantly related to known acetogens from

human feces and rumen, suggesting the presence of the

novel acetogens. Phylogenetic analysis suggests that com-

position of FTHFS sequences differs for the PC and MC.

Introduction

The ostrich (Struthio camelus) has adapted to ingesting

plant materials and has developed a large cecum and

haustrated colon where symbiotic microorganisms digest

and ferment plant cell walls. Many short chain fatty acids

are produced during fermentation in the gut, providing the

ostrich with 76% of its metabolic energy [20]. As a result

of fermentation, gases such as carbon dioxide and hydro-

gen are also produced. Hydrogen must be removed from

the gut environment to allow for reoxidation of electron

carriers essential to the fermentation process [21]. Hydro-

gen-utilizing microbes such as methanogens, acetogens,

and sulfate-reducing bacteria contribute to the removal of

hydrogen in the human colon [15]. Although reductive

acetogenesis and methanogenesis are the main pathways

for hydrogen removal in the cecum and colon of the

ostrich, the ratio of acetogenesis to methanogenesis was

found to differ between regions of the large intestine [3].

Acetogenesis is the principle pathway for hydrogen

removal in the cecum and proximal colon (PC), while

methanogenesis predominates in the mid colon (MC) [3].

Hence it is likely that acetogen diversity differs among

regions of the large intestine of ostrich although there is

not, as yet, any information on this diversity.

Leaphart and Lovell [10] developed specific primers to

amplify a fragment of the formyltetrahydrofolate synthe-

tase (FTHFS) gene, an essential gene in the acetyl CoA

pathway of reductive acetogenesis [12], and demonstrated

that this primer set could be used to assess diversity of

acetogenic Bacteria [10]. Recently, we analyzed the

diversity of FTHFS in the bovine rumen using these

primers, and demonstrated the utility of these primers for

this type of analysis in ruminants [13].

In this study, FTHFS sequences in the ostrich colon

were investigated to compare acetogen diversity in two

different regions (PC and MC) of the ostrich colon.

Materials and Methods

Animal and Sampling

Freshly removed colon was obtained from three ostriches

(estimated mean body weight, *90 kg) slaughtered at a

H. Matsui (&) � S. Yoneda � T. Ban-Tokuda � M. Wakita

Graduate School of Bioresources, Mie University, 1557

Kurimamachiya-cho, Tsu, Mie 514-8507, Japan

e-mail: matsui@bio.mie-u.ac.jp

123

Curr Microbiol (2011) 62:1–6

DOI 10.1007/s00284-010-9661-y

commercial facility (Toyohashi City, Aichi Prefecture,

Japan). The birds were given a commercial diet including

maize, wheat, alfalfa meal, corn gluten meal, soybean

meal, rice bran, animal fat, vitamin/mineral/amino acid

premix, calcium carbonate, calcium phosphate, sodium

chloride, and betaine with a mean chemical composition on

a dry matter basis as follows: 15.5% crude protein, 14.0%

crude fiber, 3.0% crude fat, 2.4% calcium, and 0.5%

phosphorus. The anterior end of the colon and posterior end

of the rectum were ligatured to prevent movement of di-

gesta. The colon was ligatured to divide it into three parts

of equal length. The upper colon was designated the PC,

and the middle colon was designated the MC. The surface

of each part of colon was washed with sterilized physio-

logical salt solution and cut with sterile scissors. Colonic

digesta was taken aseptically from the opening and sus-

pended in acetone to give a final concentration of 70% (vol/

vol) to prevent DNA degradation [4]. The digesta samples

were cooled on ice and brought to our laboratory, where

they were stored at 4�C until analysis.

DNA Extraction

Total microbial cells in the colonic content were physically

disrupted with a FastPrep instrument (Bio 101, Vista, CA),

and DNA was extracted as described by Hattori and Matsui

[5]. The crude DNA was purified with a Genomic-tip 100/

G column (Qiagen GmbH, Hilden, Germany) and dissolved

in TE buffer. The DNA concentration was adjusted to

15 ng ll-1. The DNA solutions from three ostriches were

mixed together to eliminate individual difference. This mix

DNA solution was used as the PCR template.

PCR, Construction of FTHFS Clone Library and DNA

Sequencing

PCR to amplify FTHFS fragments, the construction of

FTHFS clone libraries, and the DNA sequence analysis of

cloned fragments were performed as described previously

[13, 14]. Briefly, PCR was performed using Ex Taq (Ta-

kara, Otsu, Japan) with a TP600 thermal cycler (Takara).

Each 20 ll PCR mixture contained 1.0 ll template DNA,

19 ExTaq reaction buffer, 200 lM each of deoxynucleo-

side triphosphate (dNTP mixture), 0.5 U ExTaq DNA

polymerase, and 0.5 g l-1 bovine serum albumin. Forward

and reverse FTHFS primers (described previously [10])

were used at a concentration of 2.5 lM. A touchdown PCR

protocol was employed as described by Lovell and Leap-

hart [10] with some modification. Initial denaturation was

performed at 94�C for 2 min. Nine touchdown cycles of

94�C for 30 s, 63�C for 30 s (decreased by 1�C per cycle to

55�C), and 72�C for 30 s, were performed. After the

touchdown cycles, 15 additional cycles of 94�C for 30 s,

55�C for 30 s, and 72�C for 30 s were performed, followed

by a final extension at 72�C for 10 min. Amplification of

the PCR products was confirmed by 1.0% agarose gel

electrophoresis. The PCR products were cloned with a TA

Cloning Kit (Invitrogen, Carlsbad, CA) as described in the

manufacturer’s protocol. Positive clones were randomly

selected from each clone library for PC and MC. Sequences

from the cloned DNA fragments were analysed with ABI

PRISM 3100 Genetic Analyzer (Applied Biosystems,

Foster City, CA) using DYEnamic ET Terminator Cycle

Sequencing Kit (Amersham Biosciences, Piscataway, NJ)

as described elsewhere [5].

Homology and Phylogenetic Analysis

Amino acid sequences predicted from DNA sequences

were used as search queries in BlastX [1]. Operational

taxonomic units (OTUs), coverage, the Shannon–Wiener

index (H’), and the Chao1 were calculated with the DO-

TUR program [18]. Sequences were assigned to individual

OTUs based on a 98% amino acid sequence similarity

criterion [7]. The amino acid sequences were aligned with

ClustalX ver. 2.0 [9], and phylogenetic trees were con-

structed using the neighbor-joining method [17]. Boot-

strapping (1,000 resamplings) was used to estimate the

confidence of the branch patterns.

Nucleotide Sequence Accession Numbers

All nucleic acid sequences obtained in this study were

deposited in the DDBJ, EMBL, and GenBank databases

under accession numbers AB531025–AB531070 (for the

PC library) and AB531071–AB531116 (for the MC

library).

Results

A total of 46 sequences for each the PC and MC libraries

were analysed. The cloned sequences from each library

were classified into 15 OTUs. Sequences were composed

of between 352 and 357 predicted amino acid residues.

Sequence of PC-OTU15 (14 sequences) and MC-OTU15

(13 sequences) were composed of 336 predicted amino

acid residues. The deleted region of these two OTUs

included the 12th amino acid residue of signature sequence

of FHTFS (V-[ASV]-[TS]-[IVLA]-[RQ]-[AGS]-[LIM]-

[KER]-x-[HN]-[GAS]-[GLKD] [13]). These OTUs were

therefore omitted from analysis. A total of 32 sequences for

PC library and 33 sequences for MC library were analysed.

The coverage of the PC and MC libraries was 90.0 and

83.3%, respectively (Table 1). Diversity indices of FTHFS

gene fragment expressed as H’ and Chao1 of the MC

2 H. Matsui et al.: Diversity of FTHFS in Ostrich Colon

123

library was higher than those of PC library (Table 1).

Sequences were further classified into 6 clusters based on

phylogenetic placement (Fig. 1, summarized in Table 2).

This clustering resulted in minimum value of amino acid

sequence distance between two sequences of different

cluster was 0.272 (cluster I vs. cluster II). The similarity of

the predicted amino acid sequences to the nearest FTHFS

sequence from a known bacterial species ranged from 60 to

95% (Table 2).

Five OTUs from the PC library and three from the MC

library were classified into cluster I (Fig. 1); the PC library

included more clones than the MC library (8 vs. 5 clones).

PC-OTU01 and 02 and MC-OTU01 and 02 were grouped

into the same subcluster within cluster I (Fig. 1), and the

nearest relative of these OTUs was a distantly related

FTHFS sequence from Bryantella formatexigens isolated

from human feces [22]. PC-OTU03 showed high similarity

to Ruminococcus obeum (93%) (Table 2) The nearest

FTHFS sequence to PC-OTU04 and MC-OTU03 was from

the cultivated species Eggerthella lenta. PC-OTU05 was

distantly related to FTHFS from cultivated acetogens. PC-

OTU03, 04, and 05 and MC-OTU03 were closely related to

FTHFS clones retrieved from other gut environments such

as the rumen of ruminant (FPD03, R021) [13] or human

feces (OTU1, 9, 10, 31) [15] or FTFHS sequences from

Blautia producta (formerly Ruminococcus productus) [11]

and B. formatexigens [22] (Fig. 1).

Cluster II was the largest cluster with five OTUs (15

clones) from the PC library and six OTUs (22 clones) from

the MC library (Table 2). PC-OTU06 to 09 and MC-

OTU04 to 08 were closely related to each other and formed

a subcluster with the FTHFS sequence from Clostridum

bartlettii isolated from human feces with a high similarity

(93–95%) [19]. No FTHFS sequence from the bovine

rumen formed a cluster with these OTUs. Clostridium

carboxidivorans FTHFS was the nearest sequence to PC-

OTU10 (four clones) and MC-OTU09 (nine clones) with

76% similarity. Cluster III contained only MC-OTU10

which showed 72% similarity to B. formatexigens

(Table 2). MC-OTU10 did not cluster with any other

sequences and branched deeply from clusters I and II.

PC-OTU11 and 12 and MC-OTU11 and 12 were clas-

sified into cluster IV (Fig. 1), with 7 clones from the PC

library and 3 from the MC library (Table 2). PC-OTU12

was almost identical to rumen clone R036 (Fig. 1).

PC-OTU13 and 14, and MC-OTU13 clustered with

Roseburia inulinivorans FTHFS (81–83% similarity)

(Table 2). MC-OTU14 clustered with Bacteroides plebius

FTHFS (75% similarity).

Discussion

We recovered a wide variety of FTHFS sequences from the

PC and MC of ostrich, demonstrating that the primer set

used in this study was applicable to diversity analysis of

FTHFS in the 3 ostriches colon.

The diversity of FTHFS gene fragments calculated as

H’ and Chao1 for the PC library was slightly higher than

those of the MC library. The number of clones from the

PC library classified into clusters I and IV was greater

than from the MC library (Table 2). In contrast, the

number of clones from the MC library classified into

cluster II was greater than from the PC library. These

results suggest that the overall diversity of acetogens was

similar between the PC and MC, but the composition of

acetogenic bacteria differed in these regions. Fievez et al.

[3] showed that the extent of acetogenesis for hydrogen

disposal was higher in the proximal colon than lower part

of the colon. The difference of the composition of FTHFS

sequence in the present study could be reflected this their

observation.

PC-OTU01 and 02 and MC-OTU01 and 02 in cluster I

formed an independent subcluster and are distantly related

to acetogens from human feces and rumen (Fig. 1). This

result suggests that these OTUs are derived from novel

acetogens. PC-OTU03, 04, and 05 and MC-OTU03 in the

cluster I were closely related to FTHFS clones from other

gut environments [13, 15] or FTHFS from acetogens found

in a variety of animal feces [11, 22, 23]. These results

suggest that acetogens related to these OTUs are widely

spread in gut environments.

PC-OTU06 to 09 and MC-OTU04 to 08 in cluster II

formed a divergent group subclustered with FTHFS of C.

bartlettii. This group would be major constituent of the

acetogen community in the ostrich colon.

PC-OTU13 included FTHFS clones from horse manure

(clone H1) [10], rumen (R019 and R035) [13], and human

feces (clone OTU25) [15] (data not shown). These

sequences formed a cluster with the enteric, non-acetogenic

Proteus vulgaris; therefore the FTHFS sequences may be

derived from non-acetogenic bacteria. MC-OTU14 clus-

tered with FTHFS sequences from Bacteria belonging to

genus Bacteroides, including B. plebius, B. coprophilus,

and B. thetaiotaomicron. Bacteroides are not known to use

the acetyl-CoA pathway [6, 8, 24], suggesting that the gene

product of this OTU is not involved in reductive aceto-

genesis. Since purine fermenting bacteria also possess

Table 1 Coverage, Shannon–Wiener index, and Chao1 of the PC and

MC library

PC library MC library

Coverage (%) 90.0 83.3

Shannon–Wiener index (H’) 2.119 2.190

Chao1 11.0 14.5

H. Matsui et al.: Diversity of FTHFS in Ostrich Colon 3

123

FTHFS gene and activity [10]. FTHFS genes from these

OTUs may be involved in purine metabolism.

In this study, number of ostriches used and feeding

condition is limited. Therefore, studies using larger number

of ostriches which are fed various feeds, e.g., feed con-

taining different level of fibers, should be done. Fievez

et al. [3] demonstrated that reductive acetogenesis and

methanogenesis compete for hydrogen in the hindgut of

ostrich [3]. Similar competition was observed in pig large

intestine [2] and human colon [16]. The factors affecting

competition between acetogenesis and methanogenesis are

still unclear, requiring quantitative comparison between

I

II

III

IV

V

Thermoplasma acidophilum [AL445067]Bacteroides plebeius [ABQC02000012]Bacteroides coprophilus [ZP_03642410]

Bacteroides thetaiotaomicron [NC_004663]

1000

MC�OTU14 [AB531108]

745

PC�OTU14 [AB531045]Human feces clone OTU25 [AB291663]

MC�OTU13 [AB531073]Roseburia inulinivorans [ACFY01000156]

R035AB282726PC�OTU13 [AB531059]

811

Horse manure clone H1 [AF295711]Rumen clone R019 [AB282710]

Proteus vulgaris [AF295710]

979

Termite gut clone E [AY162296]PC�OTU12 [AB531029]Rumen clone R036 [AB282727]

PC�OTU11 [AB531054]MC�OTU12 [AB531077]Human feces clone OTU16 [AB291654]

1000

1000

1000

Thermosinus carboxydivorans [AAWL01000002]

1000

Moorella thermoacetica [J02911]Mitsuokella multacida [ABWK02000017]

Rumen clone R029 [AB282720]MC�OTU11 [AB531113]

Rumen clone R006 [AB282697]Rumen clone R007 [AB282698]

Sporomusa ovata [AF295708]

1000

935

728

MC�OTU10 [AB531090]PC�OTU10 [AB531026]MC�OTU09 [AB531072]Rumen clone R015 [AB282706]

1000

Clostridium magnum [AF295703]Clostridium carboxidivorans [ACVI01000026]

1000

1000PC�OTU09 [AB531027]MC�OTU08 [AB531089]

PC�OTU08 [AB531028]MC�OTU07 [AB531078]

1000

1000PC�OTU06 [AB531035]MC�OTU06 [AB531076]

PC�OTU07 [AB531031]

1000

MC�OTU05 [AB531074]

814

735

Clostridium bartlettii [ABEZ02000016]

761

MC�OTU04 [AB531110]

711

Eubacterium limosum [AF295706]

1000

Acetobacterium woodii [AF295701]

773

Clostridium formicaceticum [AF295702]

790

942

Treponema primita ZAS2 [AY254548]Horse manure clone H4 [AF295714]

PC�OTU05 [AB531030]Rumen clone R021 [AB282712]PC�OTU04 [AB531038]

Eggerthella lenta [CP001726]Human feces clone OTU31 [AB291669]

1000

MC�OTU03 [AB531081]Rumen clone FPD03 [AB085525]

Horse manure clone H2 [AF295715]

969

1000

Ruminococcus obeum [AAVO02000015]Human feces clone OTU9 [AB291647]PC�OTU03 [AB531041]

925

Human feces clone OTU1 [AB291639]

952

Bryantella formatexigens [ACCL02000005]Blautia producta [AF295707]Human feces clone OTU10 [AB291648]993PC�OTU02 [AB531056]MC�OTU02 [AB531092]PC�OTU01 [AB531057]

MC�OTU01 [AB531115]

1000

1000

1000

781

959

840

1000

836

0.05

VI

Fig. 1 Phylogenetic tree of the

predicted amino acid sequence

of formyltetrahydrofolate

synthetase (FTHFS) gene

fragments recovered from

colonic digesta of ostrich.

Operational taxonomic unit

(OTU) names include ‘‘PC’’ or

‘‘MC’’ designation, representing

the proximal or mid colon,

respectively. Accession number

of each sequence is shown in

brackets, along with the

accession number of the

representative clone of the

OTU. Roman numerals indicate

cluster designations

4 H. Matsui et al.: Diversity of FTHFS in Ostrich Colon

123

acetogenic and methanogenic environments in the future.

Isolation and phenotypic characterization of acetogens and

methanogens from the ostrich colon may provide further

information on acetogenesis in the ostrich colon.

Acknowledgments The authors thank Professor Kazunari Ushida

and his colleagues (Kyoto Prefectural University, Kyoto, Japan) for

their instruction on DNA extraction. Funding for this study was

provided by a Grant-in-Aid for Scientific Research, Japan Society for

the Promotion of Science (17380157). Nucleotide sequencing was

carried out at Life Science Research Center (Center for Molecular

Biology and Genetics), Mie University (Tsu, Japan).

References

1. Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped

BLAST and PSI-BLAST: a new generation of protein database

search programs. Nucleic Acids Res 25:3389–3402

2. De Graeve KG, Grivet JP, Durand M et al (1994) Competition

between reductive acetogenesis and methanogenesis in the pig

large-intestinal flora. J Appl Bacteriol 76:55–61

3. Fievez V, Mbanzamihigo L, Piatton F et al (2001) Evidence for

reductive acetogenesis and its nutritional significance in ostrich

hindgut as estimated from in vitro incubactions. J Anim Physiol

A Anim Nutr 85:271–280

4. Fukatsu T (1999) Acetone preservation: a practical technique for

molecular analysis. Mol Ecol 8:1935–1945

5. Hattori K, Matsui H (2008) Diversity of fumarate reducing bac-

teria in the bovine rumen revealed by culture dependent and

independent approaches. Anaerobe 14:87–93

6. Hayashi H, Shibata K, Bakir MA et al (2007) Bacteroides co-prophilus sp. nov., isolated from human faeces. Int J Syst Evol

Microbiol 57:1323–1326

7. Juottonen H, Galand PE, Yrjala K (2006) Detection of metha-

nogenic Archaea in peat: comparison of PCR primers targeting

the mcrA gene. Res Microbiol 157:914–921

8. Kitahara M, Sakamoto M, Ike M et al (2005) Bacteroides ple-beius sp. nov. and Bacteroides coprocola sp. nov., isolated from

human faeces. Int J Syst Evol Microbiol 55:2143–2147

9. Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W

and Clustal X version 2.0. Bioinformatics 23:2947–2948

10. Leaphart AB, Lovell CR (2001) Recovery and analysis of

formyltetrahydro-folate synthetase gene sequences from natural

populations of acetogenic bacteria. Appl Environ Microbiol

67:1392–1395

Table 2 Distribution, nearest

sequence, identity, and number

of clones in operational

taxonomic units (OTUs)

Cluster OTU Nearest sequence of cultivated

species (identity %)

No. of clones

I PC-OTU01 Bryantella formatexigens (81) 1

PC-OTU02 B. formatexigens (81) 2

PC-OTU03 Ruminococcus obeum (93) 1

PC-OTU04 Eggerthella lenta (85) 3

PC-OTU05 B. formatexigens (74) 1

MC-OTU01 B. formatexigens (82) 1

MC-OTU02 B. formatexigens (81) 1

MC-OTU03 E. lenta (80) 2

II PC-OTU06 Clostridium bartlettii (95) 4

PC-OTU07 C. bartlettii (95) 1

PC-OTU08 C. bartlettii (93) 4

PC-OTU09 C. bartlettii (95) 2

PC-OTU10 Clostridium carboxidivorans (76) 4

MC-OTU04 C. bartlettii (93) 1

MC-OTU05 C. bartlettii (93) 2

MC-OTU06 C. bartlettii (95) 5

MC-OTU07 C. bartlettii (93) 3

MC-OTU08 C. bartlettii (95) 2

MC-OTU09 C. carboxidivorans (76) 9

III MC-OTU10 B. formatexigens (72) 1

IV PC-OTU11 Moorella thermoacetica (68) 2

PC-OTU12 Thermosinus carboxydivorans (69) 5

MC-OTU11 Mitsuokella multacida (86) 1

MC-OTU12 M. thermoacetica (68) 2

V PC-OTU13 Roseburia inulinivorans (82) 1

PC-OTU14 R. inulinivorans (83) 1

MC-OTU13 R. inulinivorans (81) 2

VI MC-OTU14 Bacterooides plebeius (75) 1

H. Matsui et al.: Diversity of FTHFS in Ostrich Colon 5

123

11. Liu C, Finegold SM, Song Y et al (2008) Reclassification of

Clostridium coccoides, Ruminococcus hansenii, Ruminococcushydrogenotrophicus, Ruminococcus luti, Ruminococcus produc-tus and Ruminococcus schinkii as Blautia coccoides gen. nov.,

comb. nov., Blautia hansenii comb. nov., Blautia hydrogeno-trophica comb. nov., Blautia luti comb. nov., Blautia productacomb. nov., Blautia schinkii comb. nov. and description of

Blautia wexlerae sp. nov., isolated from human faeces. Int J Syst

Evol Microbiol 58:1896–1902

12. Ljungdahl LG (1986) The autotrophic pathway of acetate syn-

thesis in acetogenic bacteria. Annu Rev Microbiol 40:415–450

13. Matsui H, Kojima N, Tajima K (2008) Diversity of formyltetra-

hydrofolate synthetase gene (fhs), a key enzyme for reductive

acetogenesis, in the bovine rumen. Biosci Biotechnol Biochem

72:3273–3276

14. Matsui H, Kato Y, Chikaraishi T, Moritani M, Ban-Tokuda T,

Wakita M (2010) Microbial diversity in ostrich ceca as revealed

by 16S ribosomal RNA gene clone library and detection of novel

Fibrobacter species. Anaerobe 16:83–93

15. Ohashi Y, Igarashi T, Kumazawa F et al (2007) Analysis of

acetogenic bacteria in human feces with formyltetrahydrofolate

synthetase sequences. Biosci Microflora 26:37–40

16. Ohashi Y, Andou A, Kanaya M et al (2009) Acetogenic bacteria

mainly contribute to the disposal of hydrogen in the colon of

healthy Japanese. Biosci Microflora 28:17–19

17. Saitou N, Nei M (1987) The neighbor-joining method: a new

method for reconstructing phylogenetic trees. Mol Biol Evol

4:406–425

18. Schloss PD, Handelsman J (2005) Introducing DOTUR, a com-

puter program for defining operational taxonomic units and

estimating species richness. Appl Environ Microbiol 71:1501–

1506

19. Song YL, Liu CX, McTeague M et al (2004) Clostridium bart-lettii sp. nov., isolated from human faeces. Anaerobe 10:179–184

20. Swart D, Mackie RI, Hayes JP (1993) Fermentative digestion in

the ostrich (Struthio camelus var. domesticus), a large avian

species that utilizes cellulose. S Afr J Anim Sci 23:127–135

21. Wolin MJ, Miller TL (1983) Interactions of microbial popula-

tions in cellulose fermentation. Fed Proc 42:109–113

22. Wolin MJ, Miller TL, Collins MD et al (2003) Formate-depen-

dent growth and homoacetogenic fermentation by a bacterium

from human feces: description of Bryantella formatexigens gen.

no., sp.nov. Appl Environ Microbiol 69:6321–6326

23. Wang RF, Cao WW, Cerniglia CE (1996) PCR detection and

quantification of predominant anaerobic bacteria in human and

animal fecal samples. Appl Environ Microbiol 62:1242–1247

24. Xu J, Bjursell MK, Himrod J et al (2003) A genomic view of

the human Bacteroides thetaiotaomicron symbiosis. Science

299:2074–2076

6 H. Matsui et al.: Diversity of FTHFS in Ostrich Colon

123