Functional Gene Analysis Suggests Different Acetogen Populations in the Bovine Rumen and Tammar Wallaby Forestomach

Abstract

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.Methane is a potent greenhouse gas that is implicated in global warming (35). Of the 600 Tg of methane released into the atmosphere each year, 55 to 70% is anthropogenic (48). Enteric fermentation of ruminant livestock is the largest source of anthropogenic methane, contributing between 20 and 25% (48). During enteric fermentation, archaea in the rumen (methanogens) produce methane mainly through the stepwise reduction of CO 2 (4H 2 ϩ CO 2 3 CH 4 ϩ 2H 2 O) (47). As well as contributing to greenhouse gas emissions, methanogenesis is energetically wasteful representing a loss of between 2 and 12% ingested feed energy (23). Reductive acetogenesis is a hydrogenotrophic pathway (4H 2 ϩ 2CO 2 3 CH 3 COOH ϩ 2H 2 O) that results in an energy gain for ruminant livestock through the production of acetate (22) and could be an alternative hydrogen sink to methanogenesis if methanogenesis is suppressed (16).The bacteria capable of reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway (acetogens) exist in a range of environments, including sediments, wastewater treatment systems, soils, and animal gut systems, and they are likely to be natural microbiota of all ruminants (11,22). Naturally, however, reductive acetog...

The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

“…A physiological difference such as shorter retention time of feed in the foregut partially explains this. The presence of a novel group of acetogenic bacteria may be the reason for this lower CH 4 emission (Gagen et al, 2010). Presence of capnophiles may also be contributing to this observation.…”

“…The Tammar wallaby (Macropus eugenii) is a foregut fermenter but it is a low CH 4 emitter. The homoacetogen population in Tammar wallaby forestomach is different compared with that found in ruminants (Gagen et al, 2010), which can be one of the reasons for lower CH 4 emission. The acetogens in the Tammer Wallaby may be more effective hydrogenotrophs than those in the rumen and possibly better competing with methanogens.…”

“…Information on whether Verrucomicrobia are capable of this function in the rumen is still missing. Also, Klieve et al (2012) identified clones related to CH 4 -oxidising archaea in the rumen of cows. Methane-oxidising archaea have been described as having an important role in aquatic ecosystems (Hallam et al, 2003;Knittel et al, 2005).…”

Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes

“…Primers ACSF1/ ACSR1 (Gagen et al, 2010) and FTHFSf/FTHFSr (Leaphart and Lovell, 2001) targeting functional genes acs (acetyl-CoA synthase involved in the acetyl-CoA pathway) of acetogens were used. Primers ME1/ME2 (Hales et al, 1996) and DSR1fdeg/ DSR4rdeg (Leloup et al, 2007) were used for mcrA (coding for the methyl-coenzyme M reductase catalyzing methane formation) and dsrA (dissimilatory (bi)sulfite reductase catalyzing the anaerobic respiration of sulfite or sulfate) genes.…”

Abundance and diversity of mucosa-associated hydrogenotrophic microbes in the healthy human colon

“…qPCR analysis of hydrogenotrophic functional genes and SRB genera Real-time qPCR was performed on all colonic DNA extracts (20 subjects þ replicated biopsies from the five additional subjects; A-E, Supplementary Table S1) using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Both primers ACSF1/ACSR1 (Gagen et al, 2010) and FTHFSf/ FTHFSr (Leaphart and Lovell, 2001) targeting functional genes acs and fhs of acetogens were used (Supplementary Table S2). Primers ME1/ME2 (Hales et al, 1996) and DSR1fdeg/DSR4rdeg (Leloup et al, 2007) were used for mcrA and dsrA genes, respectively (Supplementary Table S2).…”