Complete genome sequence of Acidaminococcus fermentans type strain (VR4T)

Acidaminococcus fermentans (Rogosa 1969) is the type species of the genus Acidaminococcus, and is of phylogenetic interest because of its isolated placement in a genomically little characterized region of the Firmicutes. A. fermentans is known for its habitation of the gastrointestinal tract and its ability to oxidize trans-aconitate. Its anaerobic fermentation of glutamate has been intensively studied and will now be complemented by the genomic basis. The strain described in this report is a nonsporulating, nonmotile, Gram-negative coccus, originally isolated from a pig alimentary tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Acidaminococcaceae, and the 2,329,769 bp long genome with its 2,101 protein-coding and 81 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain VR4 T (= DSM 20731 = ATCC 25085 = CCUG 9996) is the type strain of the species Acidaminococcus fermentans, and the type species of the genus Acidaminococcus [1,2]. A. fermentans was originally isolated by Fuller from a pig alimentary tract [3] and included in the nomina incertae sedis. It was subsequently characterized and classified in 1969 as type strain of the then-novel genus Acidaminococcus [1]. An emendation of the description of A. fermentans was provided by Cook et al. in 1994 [4]. Originally, the principal physiological and taxonomically distinctive feature of the strain was its ability to use amino acids as sole source of energy for growth anaerobically [1]. Three accompanying strains (VR7, VR11, VR14) from the alimentary tract of the same pig were reported and deposited in the ATCC (American Type Culture Collection) [3]. Closely related strains belonging to the species have been isolated from humans (EF060089-91, >99.9% sequence identity) [5] and from cow rumen [4]. Several uncultured clones were isolated from human fecal samples (DQ904734, DQ904735 and DQ904837, 99.8%) [6], and from rabbit cecum (EF445291, 99.8%) [7]. The type strain of the only other species in the genus, A. intestini [5] shows 95.8% 16S rRNA sequence identity with strain VR4 T , whereas the type species of the other genera of the Acidaminococcaceae are less than 91.7% identical [8]. Of the many publicly available human gut metagenomes only one (BAAV01001815, 96.1%) [9] contained a highly similar 16S rRNA gene sequence, whereas none of the environmental genomic surveys indicated any moderately related phylotypes, shedding doubt on a wide-spread occurrence of members of the species A. fermentans (as of February 2010). Here we present a summary classification and a set of features for A. fermentans VR4 T , together with the description of the complete genomic sequencing and annotation. Figure 1 shows the phylogenetic neighborhood of A. fermentans VR4 T in a 16S rRNA based tree. The sequences of the six copies of the16S rRNA gene in the genome of strain VR4 T differ from each other by up to five nucleotides, and differ by up to seven nucleotides from the previously published 16S rRNA sequences from DSM 20731 (X78017, X77951), which contain two ambiguous base calls. Phylogenetic tree highlighting the position of A. fermentans VR4 T relative to the other type strain within the genus Acidaminococcus and the type strains of the other genera within the family Acidaminococcaceae. The tree was inferred from 1,348 aligned characters [10,11] of the 16S rRNA gene sequence under the maximum likelihood criterion [12] and rooted with the type strain of Anaerococcus prevotii, a member of the neighboring family Peptococcaceae. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates [13] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [14] are shown in blue, published genomes in bold and include. the recently published GEBA genome from A. prevotii [15] and Veillonella parvula [16].

Classification and features
Cells of A. fermentans strain VR4 T often occur as oval or kidney-shaped diplococci that are 0.6 to 1.0 μm in diameter [1] (Table 1 and Figure 2). The organism is anaerobic, nonsporulating, nonmotile, chemoorganotrophic, and Gram-negative [1]. Its optimum growth temperature is 30 to 37°C, over a pH range from 6.2 to 7.5, with an optimum at 7.0 [1]. Weak or no growth occurs at 25 and 45°C. , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [25]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. Standards in Genomic Sciences A. fermentans thrives mainly by glutamate fermentation via the 2-hydroxyglutarate pathway in the intestinal tract of homeothermic animals [1,25], utilizing glutamate, citrate, and trans-aconitate as sole energy sources, in the presence of sodium [4,26,27]. Ammonia, acetate, butyrate, and hydrogen are the main end products of growth [1,4]. Transport and catabolism of these substrates are dependent on a sodium motive force as a method of membrane energetics [27,28]. The fermentation of trans-aconitate in strain VR4 T takes a different pathway, which is via citrate, oxaloacetate, and pyruvate, producing CO2, acetate, butyrate and hydrogen [26]. Unlike the citrate uptake, the aconitate uptake may adopt two mechanisms: a citrate/aconitate carrier with low affinity for sodium and an aconitate carrier with high affinity for sodium [29].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [32], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [33]. The genome project is deposited in the Genomes OnLine Database [14] and the complete genome sequence in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler version 2.0.0-PostRelease-11/04/2008 (Roche). Large Newbler contigs were broken into 2,561 overlapping fragments of 1,000 bp and entered into the final assembly as pseudoreads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [35]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 256 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher). The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 63.3× coverage of the genome. The final assembly contains 22,991 Sanger and 557,705 pyrosequencing reads.

Genome annotation
Genes were identified using Prodigal [36] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [37]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [38].

Genome properties
The genome is 2,329,769 bp long and comprises one circular chromosome with a 55.8% GC content (Table 3 and Figure 3). Of the 2,182 genes predicted, 2,101 were protein-coding and 81 were RNAs; 75 pseudogenes were also identified. The majority of protein-coding genes (75.3%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.

Insights from the genome sequence
Different from most organisms for which the type strain genomes have so far been described in the GEBA series, A. fermentans strain VR4 T 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 [1,4]. Strain VR4 T 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 ATPdependent one-electron reduction [29,39,40]. The extra energy produced is conserved via ΔμNa + generated by the decarboxylation of glutaconyl-CoA [41].
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. Standards in Genomic Sciences

Overview on transport systems
About 310 putative transporter genes are annotated in the genome of strain VR4 T , 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 Table 5. 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 Fe 2+ 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. Figure 5 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 VR4 T and A. intestini strain D21), as well as in the two clostridia, whereas Fusobacterium differ slightly.
The second most abundant ABC transporter group consists of amino acid transporters, followed by multi drug or antimicrobial efflux pumps (Table  5). 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.   The numbers represent the number of the ABC system or the number of the genes involved, respectively. A functional ABC system, containing all necessary subunits, is referred to as complete system, otherwise it is labeled as incomplete.
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 VR4 T . 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, hostmediated 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 VR4 T 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 255.99 T (AF473835) by just three nt, but it shares only 95.86 to 96.05% sequence similarity with A. fermentans VR4 T , 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 (Fig.5). 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 T is able to utilize trans-aconitate and citrate as an energy source [4,28]. 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. Table 6 lists major genes identified from the fermentation pathways discussed above.
Acidaminococcus 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 T and A. sp. D21, there are about 10 βlactamase or β-lactamase related genes (Table 6). This indicates that both A. fermentans VR4 T 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.