Alternate pathways to account for reduced abundance of methanogenic archaea in mi




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Supplementary text S1

Alternate pathways to account for reduced abundance of methanogenic archaea in MI





Sulfate-reducing bacteria.

Sulfate reducing bacteria (SRB) in the mammalian gut include the genera Desulfovibrio, Desulfobacter, Desulfobulbus and Desulfotomaculum (MacFarlane and Gibson, 1997). The predominant H2-utilizing SRB in the human colon belong to the genus Desulfovibrio (70-90% of the total SRB counts). However, previous attempts to cultivate these bacteria from the gut of marine iguanas on Postgate’s medium B (1984) containing lactate as carbon source were unsuccessful. In order to verify the presence of SRB in the gut population in the iguana colon, we have used a PCR approach that targets the APS reductase (apsA) gene which is a useful phylogenetic marker for sulfate reducing bacteria (Friedrich, 2002).

DNA was extracted from marine iguana (n=4) and land iguana (n=5) samples. PCR amplification of apsA gene fragment was carried out using primers APS-FW and APS-RV that amplify a ~390bp fragment, and APS7-F and APS8-R that amplify a ~900bp fragment of the -subunit of the APS reductase gene (Friedrich, 2002):


  • APS-FW (5’-TGG CAG ATM ATG ATY MAC GG-3’)

  • APS-RV (5’-GGG CCG TAA CCG TCC TTG AA-3’)

  • APS7-F (5’-GGG YCT KTC CGC YAT CAA YAC-3’)

  • APS8-R (5’-GCA CAT GTC GAG GAA GTC TTC-3’)

PCR products were cloned into the pGEM-T Easy vector and sequenced. Analysis of the BLAST reports of the sequenced clones from the APS-FW/RW library (~390bp) and APS-7F/8R library (~900bp) are summarized in Table S1A. All sequences recovered thus far are associated with classical gram-negative, lactate-utilizing sulfate reducing bacteria. Although Desulfomonas pigra and Desulfovibrio desulfuricans differ in phenotypic traits (shape and motility), recent molecular analysis suggests that D. pigra should be reclassified within the genus Desulfovibrio (Loubinoux et al., 2002). Desulfovibrio termitidis is a carbohydrate-degrading, sulfate reducing bacterium isolated from the hindgut of termites (Trinkerl et al., 1990). This species utilizes a wide variety of carbohydrates in contrast to other Desulfovibrio species.


Table S1A. Summary of BLAST analysis for PCR amplification of apsA gene fragments from marine and land iguanas. Values are reported as percentage amino acid sequence identity (number of clones in parenthesis).





PCR product obtained from:

Species

Marine iguana

Land iguana




~ 390 bp

~ 900 bp

~ 390 bp

~ 900 bp

Desulfovibrio termitidis

93-95% (12)

94-95% (4)

-

-

Desulfovibrio vulgaris

-

95-96% (12)

-

68% (1)

Desulfovibrio desulfuricans

-

-

-

86% (1)

Desulfomonas pigra

-

91% (1)

-

91% (1)

Acetogen

We also aimed to provide a preliminary identification of bacterial targets producing acetate (i.e., acetogens) in the iguana feces. Past studies had demonstrated that the formyltetrahydrofolate synthetase (fthfs) gene encodes a key enzyme in reductive acetogenesis, and can be used as a functional representation for acetogens. Therefore, genomic DNA from an individual land and marine iguana was PCR amplified with primer pairs (Leaphart and Lovell, 2001):



  • FTHFS-F (5-TTY ACW GGH GAY TTC CAT GC-3), and

  • FTHFS-R (5-GTA TTG DGT YTT RGC CAT ACA-3)

After amplification, PCR products were purified and cloned into competent E. coli JM109 by using the pGEM-T Easy vector system, and plasmids were sequenced.
Table S1B revealed that the production of acetate is in part due to the presence of acetogens related to phylum Firmicutes and a group of uncultured bacterium that are present in the land and marine iguana feces.
Table S1B. Summary of BLAST results from FTHFS gene fragments (~1060 bp). Values are reported as percentage amino acid sequence similarity (number of clones in parenthesis).

Nearest matches

PCR product obtained from:

Marine iguana

Land iguana

Uncultured bacterium

62-68 % (3)

59-78 % (5)

Ruminococcus productus

-

51-69 % (4)

Clostridium thermaceticum

-

61 % (1)



References in this supplementary text
Friedrich MW. (2002). Phylogenetic analysis reveals multiple lateral transfers of adenosine-5'-phosphosulfate reductase genes among sulfate-reducing microorganisms. Journal of bacteriology 184: 278-289.

Leaphart AB, Lovell CR. (2001). Recovery and analysis of formyltetrahydrofolate synthetase gene sequences from natural populations of acetogenic bacteria. Applied and environmental microbiology 67: 1392-1395.

Loubinoux J, Valente FM, Pereira IA, Costa A, Grimont PA, Le Faou AE. (2002). Reclassification of the only species of the genus Desulfomonas, Desulfomonas pigra, as Desulfovibrio piger comb. nov. International journal of systematic and evolutionary microbiology 52: 1305-1308.

MacFarlane GT, Gibson GR. (1997). Carbohydrate fermentation, energy transduction and gas metabolism in the human large intestine. In: Mackie RI, White BA (ed.). Gastrointestinal Microbiology. Chapman & Hall: New York, pp 269-318.



Trinkerl M, Breunig A, Schauder R, König H. (1990). Desulfovibrio termitidis sp. nov., a carbohydrate-degrading sulfate-reducing bacterium from the hindgut of a termite. Systematic and Applied Microbiology 13: 372-377.





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