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. 2010 Jan 19:11:46.
doi: 10.1186/1471-2164-11-46.

Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

Affiliations

Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

Brian V Jones et al. BMC Genomics. .

Abstract

Background: Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome.

Results: Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90%) to two plasmids (pTRACA10 and pTRACA22) were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA) addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria), but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division.

Conclusions: The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In particular the increased relative abundance and broad phylogenetic distribution was identified for a putative RelBE toxin/antitoxin addiction module, a putative phosphohydrolase/phosphoesterase, and an ORF of unknown function. Our analysis also indicates that some plasmids or plasmid families are present in the gut microbiomes of geographically isolated human hosts with a broad global distribution (America, Japan and Europe), and are potentially unique to the human gut microbiome. Further investigation of the plasmid population associated with the human gut is likely to provide important insights into the development, functioning and evolution of the human gut microbiota.

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Figures

Figure 1
Figure 1
Physical maps of complete nucleotide sequences for pTRACA18, pTRACA20, pTRACA22 and pTRACA30. Maps of complete nucleotide sequences of plasmids pTRACA18, pTRACA20, pTRACA22, and pTRACA30 indicating locations of predicted open reading frames (ORFs). ORFs are colour coded according to predicted function as indicated in the associated key. Predicted products and more detail regarding putative functions of annotated ORFS are provided in Table S1.
Figure 2
Figure 2
Identification of sequences homologous to pTRACA10, pTRACA22 and encoded ORFS in human gut metagenomes. The complete nucleotide sequences of plasmids was used to search 15 human gut metagenomes, the combined gut metagenome of lean and obese mice, Sargasso sea, and soil metagenomes [9-11,30,31]. The central ring shows the physical plasmid map with encoded ORFs, as in Figure 1 and Table S1. Concentric rings represent the nine human gut metagenomes in which homologous sequences were identified, and bars indicate regions of homology between sequences retrieved from human gut metagenomes and corresponding regions of the pTRACA10 or pTRACA22 plasmids. Colours of bars indicate the % identity at the nucleotide level for each metagenomic sequence as detailed in the associated key. Only sequences >100 bp in length are shown, and numerals within bars correspond to detailed information on the relevant metagenomic sequences provided in Table 1. Metagenomes are as follows: Hum7, Hum8 - American metagenomes [9]; In-R, F2-W, F2-V, F2-Y, In-E, In-D, In-M - Japanese metagenomes [10].
Figure 3
Figure 3
Distribution and relative abundance of plasmid encoded functions in the human gut microbiome. A) Relative abundance of pTRACA22 ORFs, expressed as hits/Mb, in the combined human gut metagenomes of 15 individuals [9,10] compared to the combined murine gut metagenome [11], Sargasso sea [30], and soil metagenomes [31]. Symbols above bars indicate significant differences between combined human metagenomes and each non-human metagenome (P = 0.01 or less), and colours correspond to each non-human metagenome: Orange = Significant difference between human and murine metagenomes; Blue = Significant difference between human and marine metagenomes; Brown = significant difference between human and soil metagenomes. B) Distribution of amino acid sequences homologous to ORFs encoded by pTRACA10 and pTRACA22 in 15 individual human gut metagenomes. Bars indicate the number of human metagenomes in which amino acid sequences homologous to each plasmid encoded ORF were detected. Colours within bars indicate the overall % identity of the top hits (based on bit score) in each metagenome to the corresponding plasmid ORF. Letters adjacent to bars indicate human metagenomes in which sequences homologous to plasmid ORFs were identified: A) Human7, B) Human8 [9]; C) InA, D) InB, E) InD, F) InE, G) InM, H) InR, I) F2-V, J) F2-W, K) F2-X, L) F2-Y, M) F1-S, N) F1-T, O) F1-U [10]. * indicates metagenomic sequences that correspond to those represented in Figure 2 and Table 1. C) Relative abundance (as hits/Mb) of the pTRACA22 RelBE TA module in human gut, murine gut and environmental metagenomes, compared to relative abundance of MazEF, ParDE and HigBA TA modules. The observed differences between human and non-human metagenomes were explored using the χ2 distribution. Symbols above bars indicate approximate significance of differences between combined human metagenomes and each non-human metagenomes (P = 0.01 or less), as in Fig 1a.
Figure 4
Figure 4
Distribution of RelE genes encoded by sequences from human gut metagenomes. RelE sequences homologous to pTRACA22 ORF22-6 identified in human gut metagenomes during comparative metagenomic analysis (Figure 3) were affiliated with a phylogenetic division based on genus represented in top hits (based on bit score) from BlastP and tBlastn searches of public databases encompassed by the nr dataset. Bars represent the proportion of RelE sequences identified which were affiliated with each phylogenetic division, and colours within bars indicate the % identity of metagenomic sequences to homologous sequences in bacterial genomes or plasmids. Numerals adjacent to bars indicate the genus in which homologous RelE were identified at each corresponding level of % identity: 1) Clostridium 2) Faecalibacter 3) Desulfitobacterium, 4) Blautia, 5) Parabacteroides, 6) Bifidobacteria, 7) Escherichia (pARS3), 8) Yersinia, 9) Photorhabdus, 10)Treponema, 11) Fusobacteria, 12) Parachlamydia, 13) "Candidatus Cloacamonas", (Candidate division WWE1) 14) Uncultured bacterium, fosmid clone 3 originating from organically reared pig gut and encoding tetracycline resistance [43].
Figure 5
Figure 5
Identification of conserved regions of RelE. Plot showing the percentage divergence from the pTRACA22 RelE sequence of homologous RelE sequences. Bars represent the percentage divergence in identical amino acids at each position of the pTRACA22 RelE sequence, based on alignments of RelE sequences from human gut metagenomes, plasmids and bacterial chromosomes. The pTRACA22 sequence is presented with the N terminus at the origin of the plot, and the N terminal M start codon omitted. Symbols indicate conserved residues (stars) and those at which between 90 and 100% of represented sequences exhibit a conserved substitution (circles), as indicated by the associated key.
Figure 6
Figure 6
Phylogenetic distribution of RelE toxin sequences. Amino acid sequences homologous to the pTRACA22 RelE (ORF22-6, Figure 1, Table S1) derived from combined human gut metagenomes, plasmids and bacterial genome sequences, were used for construction of a phylogenetic trees. Symbols preceding sequence names denote the origin of the RelE sequence as detailed in the figure legend. Major clades predominantly populated with sequences of common phylogenetic origin are indicated by brackets: A) Firmicutes B) Bacteroidetes/Chlorobi, C) Plasmid D) Proteobacteria E) Actinobacteria. Numerals indicate nodes with bootstrap values of 40 or over (based on 1000 replicates). Scale bar indicates 0.1 amino acid substitutions per site. Accession numbers for chromosomal and plasmid sequences represented in the phylogenetic tree, along with those for gut metagenomic sequences encoding each RelE "type" are listed in Table S2.
Figure 7
Figure 7
Annotation of the available pARS3 IS26Tn sequence fragment and comparison with pTRACA22. The sequence fragment of the E. coli plasmid pARS3 described by Wachino et al[34] was downloaded from GenBank [AB261016]. Due to the high identity of pTRACA22 ORFs with regions of the pARS3 sequence observed during tBlastn searches, the currently available pARS3 sequence (limited to a 3964 bp region of the 9.1 Kb IS26Tn) was analysed using Glimmer v3, and predicted ORFs annotated. All ORFs described by Wachino et al [34] were predicted along with five ORFs not originally described or annotated in the GenBank record, * denotes ORFs in pARS3 annotated during this study. Nucleotide sequences of pTRACA22 and pARS3 were compared using the Artemis Comparison Tool (ACT) and shaded areas between sequence maps represent regions of high identity at the nucleotide level. Areas shaded pink range from 95-97% identity, and areas shaded orange exhibited 89% identity. ORFs described previously by Wachino et al follow nomenclature used in that study [43], and the following functions were originally assigned to these ORFs: Orf4 - putative replication protein, Orf5: hypothetical protein, Orf6 - NpmA aminoglycoside resistance gene, Orf7 - putative ABC transporter substrate binding protein. ORFs are shown as arrows. Open arrows denote ORFs that do not have a homologue in both sequences. Coloured arrows indicate ORFs with homologues present in both pTRACA22 and pARS3, and "paired" ORFs are connected by dashed lines with colours representing % identity at the amino acid level: Red arrows indicate greater than 90% identity between paired ORFs, Orange arrows indicate greater than 80% identity between paired ORFs. Jagged edges indicate truncated ORFs.

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