Different from most organisms for which the type strain genomes have so far been described in the GEBA series, A. fermentans strain VR4T is biochemically well described. The strain has been intensively studied for many years. Here, we describe the genomic location of the genes for the biochemically characterized enzymes, as well as the annotation of the genome using bioinformatic approaches, which may reveal additional physiological properties of the organism.
Glutmate fermentation via 2-hydroxyglutarate
The ability of A. fermentans
to use amino acids as the sole energy source for growth is a well known characteristic, with glutamic acid being the most important amino acid for the organism [1
]. Strain VR4T
ferments glutamate via
the 2-hydroxyglutarate pathway, in which, glutamate is converted to a key intermediate – (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA, then to ammonia, CO2
, acetate, butyrate and hydrogen. An unusual dehydratase contains an [4Fe–4S]2+
cluster – acting as an activator or initiator of dehydration, is activated by an ATP-dependent one-electron reduction [29
]. The extra energy produced is conserved via ΔμNa+
generated by the decarboxylation of glutaconyl-CoA [41
The dehydratase system of strain VR4T
consists of two oxygen-sensitive protein components: component A – the activator (HgdC
) and component D – the actual dehydratase (HgdAB
]. Component A has been crystallized and its structure has been determined by X-ray crystallography at 3 Å resolution [42
The glutaconyl-CoA decarboxylase of A. fermentans
is a biotin-dependent sodium pump, consisting of three major polypeptide subunits: biotin carrier (alpha, gcdA), carboxytransferase (beta, gcdB) and carboxylase, the actual sodium pump (gamma, gcdC) [43
]. There is additional small subunit (delta, gcdC), whose function is unclear [43
]. Glutaconate CoA-transferase consists of two different polypeptide chains and is necessary for the decarboxylation of glutaconate [44
The hydroxyglutarate operon has been experimentally studied [45
] and all encoding genes are annotated in the genome ()
Figure 4 MetCyc pathway  along with the gene locus names, representing the enzymes identified in the pathway.
The entire 7.9 kb long gene cluster, consists of 2-hydroxyglutaryl-CoA dehydratase D-components hgdAB
(Acfer_1814 and 1815), CoA-substrate-specific enzyme activase (Acfer_1816), glutaconyl-CoA decarboxylase α subunit gcdA
(Acfer_1817), glutaconate CoA-transferase α− and β−subunits (gctAB
, Acfer_1819 and 1818). The glutaconyl-CoA decarboxylase β−, γ− and δ-subunits: gcdB
(Acfer_1834, Acfer_1835 and Acfer_1836) are encoded nearly 15kb upstream from this operon, forming a second operon [43
In addition to the above-mentioned protein complexes, the gene encoding glutamate dehydrogenase (NAD(P)(+)) (Acfer_1756) is encoded at the beginning of the gene cluster. Three acyl-CoA dehydrogenase genes (Acfer_1477, Acfer_1575 and Acfer_1583) were annotated at various locations, completing the pathway. Nevertheless, genes encoding 2-hydroxyglutarate dehydrogenase and Butyl-CoA:acetate CoA transferase have not yet been identified. Possibly these enzymes have additional functions in other pathways and have been annotated distinctly.
shows a comparison of the hydroxyglutarate operon among various organisms. The positional gene cluster is conserved in the two strains belonging to the genus Acidaminococcus (A. fermentans strain VR4T and A. intestini strain D21), as well as in the two clostridia, whereas Fusobacterium differ slightly.
Figure 5 Gene ortholog neighborhoods of hydroxyglutarate operons Colors indicate ortholog groups. The size of the bar approximately corresponds to gene size. Date are taken from 
Overview on transport systems
About 310 putative transporter genes are annotated in the genome of strain VR4T, which constitute roughly 15% of the coding genes. The majority of these transporters belong to two groups: secondary carriers and the ATP-binding cassette ABC-type carriers. The most frequent class of ABC-type transport proteins are for importing iron/metal ions and amino acids, as shown in .
Overview of the ABC-type transporters within the genome of A. fermentans VR4T
Among all the ABC-type transport systems, 41% are related to the transport of iron or other metal ions, comprising the largest ABC transporter group annotated. Additionally, two ferrous iron uptake (FeoB) systems (5 genes; TC#9.A.8) were annotated, which are involved in G protein coupled Fe2+ transport. At least 15 other gene products are involved in iron or magnesium transport and heavy metal transport and detoxification. Presumably, Acidaminococcus has adapted the ability to sequester iron from the host as a survival strategy. The abundance of this particular group of transporter genes might suggest diverse mechanisms evolved in order to compete for the limited iron supply in the gastrointestinal environment.
The second most abundant ABC transporter group consists of amino acid transporters, followed by multi drug or antimicrobial efflux pumps (). This trend is visible in secondary carrier proteins; more than 18 and 26 genes encoded for amino acid transport and multi drug or antimicrobial efflux pumps, respectively. At least six genes are annotated as encoding a Na+:glutamate symporter (TC# 2.A.27.1). This corroborates the most prominent physiological characteristic of the organism, namely that glutamate is the most important energy and carbon source. No functional sugar transport protein was identified, indicating that this organism does not utilize sugar.
Transporters for carboxylate are also noticeable. For example, the tripartite ATP-independent periplasmic transporter (TRAP-T) family (TC# 2.A.56) is involved in the uptake of widely divergent compounds, mostly carboxylate derivatives [48
]. Five TRAP systems are found in the genome, including 12 genes, one TAXI type system and four DctPQM systems. The abundance of the TRAP-T proteins is indicative of the capability of this organism to import carboxylate derivatives such as those produced by the host metabolism or fermentation by rumen microbiota, thus constituting a recycled food web and a beneficial nutritional cycle.
The Bile Acid:Na+
symporter (TC# 2.A.28) previously identified in intestinal, liver and kidney tissues of animals is identified at various locations within the genome (Acfer_0208, Acfer_0775 and Acfer_1270). This might be indicate horizontal gene transfer (HGT) between the host and A. fermentans
. It has been shown that the acquisition of eukaryotic genes in bacteria is frequently the result of a transfer from the host [49
]. Given the environmental niche of A. fermentans
, host-mediated HGT might well have occurred.
No genes for flagellar machinery (TC#3.A.6) are encoded in the genome, which is consistent with the observation of non-motility. A. fermentans VR4T probably uses a type II secretion (Sec system) for protein secretion, as all components of the Sec protein export system are present (SecA, SecYEG, SecDF), except for SecB, which may be functionally replaced by a different chaperone.
Comparison with the genome of Acidaminococcus intestini D21
The genome sequence of another member of the genus Acidaminococcus (A. intestini) which was isolated from an human gastrointestinal tract D21, has been partially deciphered by the Broad Institute. The unfinished yet annotated genome sequence is deposited at NCBI (ACGB00000000) and IMG-GEBA (object-ID 643886056). The 16S rRNA sequence from A. sp. D21 differs from the one obtained from A. intestini type strain ADV 255.99T (AF473835) by just three nt, but it shares only 95.86 to 96.05% sequence similarity with A. fermentans VR4, indicating a considerable evolutionary distance between the two species. Despite these discrepancies, the annotated genomes indicated quite a few common physiological traits.
For instance, the 2-hydroxyglutarate operon was well conserved between the two genomes, including position, structure and individual genes (). This suggests that both species have adopted the same glutamate fermentation pathway.
The citrate fermentation via oxaloacetate and pyruvate is another important pathway by which A. fermentans
VR4 is able to utilize trans-aconitate and citrate as an energy source [4
]. Genes responsible for this processing tend to cluster in both genomes. Unlike the case of glutamate fermentation, genes within the trans-aconitate and citrate fermentation pathway exhibit a distinct organization in the two genomes (data not shown). This might imply differences in gene regulation or in substrate uptake. lists major genes identified from the fermentation pathways discussed above.
A list of genes discussed, reflecting the organism’s physiological insights
has been considered highly susceptible to β-lactam antibiotics until Galán et al. [50
] discovered the first β-lactamase in this species. Throughout the genomes of A. fermentans
VR4 and A. sp
. D21, there are about 10 β-lactamase or β-lactamase related genes (). This indicates that both A. fermentans
VR4 and A. sp
. D21 can be resistant to β-lactam antibiotics. The organism might thus contribute, via
HGT, to the origin or spread of resistance genes in one of the most complex microbial ecosystems known.