# Phylogenetic and Guanine-Cytosine Content Analysis ofSymbiobacterium thermophilum Genes

Authors: Hiromi Nishida; Choong-Soo Yun
Journal: International Journal of Evolutionary Biology
 (2011)

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## Abstract

Although the bacteriumSymbiobacterium thermophilum has a genome with a high guanine-cytosine (GC) content (69%), it belongs to a low GC content bacterial group. We detected only 18 low GC content regions with 5 or more consecutive genes whose GC contents were below 65% in the genome of this organism. S. thermophilum has 66 transposase genes, which are markers of transposable genetic elements, and 38 (58%) of them were located in the low GC content regions, suggesting that Symbiobacterium has a similar gene silencing system as Salmonella. The top hit (best match) analyses for each Symbiobacterium protein showed that putative horizontally transferred genes and vertically inherited genes are scattered across the genome. Approximately 25% of the 3338 Symbiobacterium proteins have the highest similarity with the protein of a phylogenetically distant organism. The putative horizontally transferred genes also have a high GC content, suggesting that Symbiobacterium has gained many DNA fragments from phylogenetically distant organisms during the early stage of Firmicutes evolution. After acquiring genes, Symbiobacterium increased the GC content of the horizontally transferred genes and thereby maintained a genome with a high GC content.


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## Body

## 1. Introduction

Symbiobacterium thermophilum is a syntrophic bacterium that grows effectively when cocultured with a cognate Geobacillus sp. [1]. Because of the lack of carbonic anhydrase in the course of Symbiobacterium evolution [2, 3], the major growth factor for this organism is CO2 generated by the growth of Geobacillus [4]. S. thermophilum has a 3.57 Mbp circular genome that consists of 3338 protein-coding sequences [3]. On the basis of the comparative genomic studies, Symbiobacterium is classified as a member of the class Clostridia [3, 5]. Although Symbiobacterium phylogenetically belongs to Clostridia (low guanine-cytosine (GC) content bacterial group), the species S. thermophilumhas a genome with a high GC content (69%).GC content is commonly used as a marker in bacterial systematics; for example, actinobacteria have a high GC content genome, and clostridia have a low GC content genome. This variation in nucleotide content in bacteria is not clearly understood [6–8]. Analyzing a high GC content genome of a bacterium that belongs to a low GC content group or vice versa is useful and important. Symbiobacterium belongs to the class Clostridia (low GC content group), but its genome has a high GC content (69%). One possibility is that Symbiobacterium has acquired this high GC content from DNA fragments through horizontal gene transfer [9, 10], homologous gene recombination [11, 12], or both. Another possibility is that Symbiobacterium has increased the GC content of the acquired genes and maintained the high GC content during evolution. In this study, we identified GC content of each gene of S. thermophilum. In addition, we identified the horizontally transferred and vertically inherited genes. In order to elucidate why the Symbiobacterium genome has a high GC content, we compared the GC contents of the horizontally transferred genes with those of the vertically inherited genes.


## 2. Materials and Methods

In this study, we classified the 3338 protein-coding sequences ofS. thermophilum on the basis of the amino acid sequence of each coded protein. A BLAST search was conducted for all proteins from 147 eukaryotes, 1047 bacteria, and 84 archaea in the KEGG database (http://www.kegg.jp) considering the parameter values given on the GenomeNet website (http://www.genome.jp). The top hit (best match) for each Symbiobacterium protein was recorded. However, if the top hit was missing or if the E-value of the top hit exceeded 0.1, we considered the Symbiobacterium protein to have no similar protein (category, “No hit”). If the top hit was another protein(s) of Symbiobacterium (category, “Symbiobacterium”), the protein-coding gene was considered to have duplicated during evolution. Top hit analysis at the genome level is a powerful tool for elucidating the phylogenetic lineage of an organism [13, 14].


## 3. Results and Discussion

On the basis of the phylogenetic lineage of the organism possessing the top hit protein shown in the BLAST result, the 3338S. thermophilum protein-coding genes were classified into 17 categories (Figure 1). The largest category was “Clostridia,” and the second largest category was “Bacilli.” This is consistent with the results of previous phylogenetic analyses [3]. The third and fourth largest categories were “No hit” and “Symbiobacterium,” respectively (Figure 1). Most genes belonging to the category “Symbiobacterium” might share their origin with other genes of the same category because 300 of the 341 genes had a similar protein sequence as that of the other organisms that appeared below the top hit of the BLAST result (Table 1 (see Supplementary matrial available online at doi:10.4061/2011/634505)). For example, most transposable elements belonged to “Symbiobacterium,” indicating that they were duplicated on the Symbiobacterium genome after invasion.Figure 1
Pie chart of the categories of the 3338Symbiobacterium protein-coding genes. A BLAST search was conducted for all proteins from 147 eukaryotes, 1047 bacteria, and 84 archaea in the KEGG database (http://www.kegg.jp) considering the parameter values given on the GenomeNet website (http://www.genome.jp). The query amino acid sequence was each protein of Symbiobacterium thermophilum. The top hit (best match) for each Symbiobacterium protein was recorded. However, if the top hit was absent or if the E-value of the top hit exceeded 0.1, the Symbiobacterium protein was considered to have no similar protein (category, “No hit”). We categorized the 3338 Symbiobacterium proteins into the following 17 categories: “Actinobacteria,” “Aquificae,” “Archaea/Eukaryota,” “Bacilli,” “Bacteroidetes/Chlorobi,” “Chloroflexi,” “Clostridia,” “Cyanobacteria,” “Deinococcus-Thermus,” “Dictyoglomi,” “Fibrobacteres/Acidobacteria,” “No hit,” “Proteobacteria,” “Symbiobacterium,” “Thermobaculum,” “Thermotogae,” and “Other bacteria.” If the top hit was another protein(s) of Symbiobacterium, then the query protein was considered to belong to the category “Symbiobacterium.”When each gene was plotted on the basis of its category, we detected 52 clusters containing 5 or more consecutive genes belonging to “Clostridia” (Figure2, pink regions in Supplementary Table 1). These conserved gene clusters are probably not acquired by horizontal gene transfer and are strongly considered to be vertically inherited. The putative vertically inherited genes were scattered across the genome of S. thermophilum (Figure 2). In addition, we detected 18 low GC content regions containing 5 or more consecutive genes whose GC contents were below 65% (Figure 3, yellow regions in Supplementary Table 1). These low GC content regions do not overlap with the 52 vertically inherited clusters (Supplementary Table 1). On the basis of the KEGG gene cluster database, we found 12 gene clusters in the 18 low GC content regions (Supplementary Table 2).Plots of the location and category of theSymbiobacterium protein-coding genes. X-axis: STH gene number. Y-axis: 0: category “No hit” (Symbiobacterium-specific genes); 1: category “Symbiobacterium” (multiple copied genes); 2: category “Clostridia;” 3: category “Bacilli”; 4: categories “Actinobacteria,” “Aquificae,” “Bacteroidetes/Chlorobi,” “Chloroflexi,” “Cyanobacteria,” “Deinococcus-Thermus,” “Dictyoglomi,” “Fibrobacteres/Acidobacteria,” “Proteobacteria,” “Thermobaculum,” “Thermotogae,” and “Other bacteria”; 5: category “Archaea/Eukaryota.” The italicized numbers indicate 52 clusters (pink) containing 5 or more consecutive genes belonging to the category “Clostridia.”Plots of the location and GC content of theSymbiobacterium protein-coding genes.  X-axis: STH gene number. Y-axis: GC content (%) of gene. Red indicates the putative transposase-coding genes, and blue indicates the group II intron-coding maturase genes. The italicized numbers indicate 18 low GC content regions containing 5 or more consecutive genes whose GC contents are below 65%.Approximately 25% of the 3338Symbiobacterium protein-coding genes belonged to categories consisting of organisms phylogenetically distant from Symbiobacterium (Figure 1), suggesting that Symbiobacterium frequently acquired genes during evolution. The proportion of horizontally transferred genes in the Symbiobacteriumgenome is strongly suggested to be the highest among bacteria [15]. These putative horizontally transferred genes are scattered across the genome of S. thermophilum (Figure 2). In addition, considering the species diversification of Bacilli and Clostridia, it is suggested that the categories “Bacilli” and “Clostridia” include not only vertically inherited genes but also horizontally transferred genes.Transposase genes are generally used as markers of transposable genetic elements [16]. Most transposase-coding genes flank horizontally transferred genes [13]. S. thermophilum has 66 putative transposase-coding genes, of which 38 (58%) are located in the low GC content regions (P-value=1.3×10-99; Pearson’s chi-square test) (Figure 3), suggesting that Symbiobacterium has a similar silencing system as that of Salmonella [17, 18]. In the silencing system, a histone-like nucleoid structuring (H-NS) protein binds to the region with a low GC content. Similar functional (H-NS) proteins were reported in Mycobacterium and Pseudomonas [19, 20]. If Symbiobacterium also has such proteins that bind the low GC content regions, the expression of the transposable elements located in these regions might be inhibited. As mentioned above, most transposable elements belong to the category “Symbiobacterium,” which is consistent with the fact that the genes of this category have lower GC contents than those of the other categories (Supplementary Figure 1). The regions consisting of low GC content genes cannot be explained by the directional mutation pressure or amelioration of bacterial genomes [21, 22]. Interestingly, although H-NS proteins bind the low GC content regions in Mycobacterium, Pseudomonas, and Salmonella [17–20], the H-NS protein of Escherichia coli does not specifically bind only these regions [23].In addition,S. thermophilum has 30 group II intron-encoding maturase genes. Group II introns are transposable elements [24] that encode maturase as an intron-specific splicing factor [25]. The GC content of each maturase gene is approximately 65% (Figure 3). These maturase genes are classified in “Symbiobacterium,” on the basis of amino acid sequence similarity. In contrast to the transposase genes, the group II intron-encoding maturase genes are not located in the 18 low GC content regions (Figure 3). If Symbiobacterium has both an H-NS protein binding the low GC content regions and a gene silencing system similar to Mycobacterium, Pseudomonas, and Salmonella, these maturases could be activated and the group II introns could be transposed to the Symbiobacterium genome. Of course, it is also possible that this transposition of the group II introns is inhibited by another gene silencing system.It is suggested thatSymbiobacterium has gained many DNA fragments from phylogenetically distant organisms during the early stage of evolution in the Firmicutes (consisting of Bacilli and Clostridia). As the Symbiobacterium genes of all categories have a high GC content (Supplementary Figure 1), it can be concluded that, after acquiring genes, Symbiobacterium increased the GC content of the horizontally transferred genes and thereby maintained a genome with a high GC content.In contrast to theSymbiobacterium genome, the Fusobacterium (phylogenetically closely related to Firmicutes) genome has a low GC content (27%) [13]. It is suggested that Fusobacterium has gained many genes from phylogenetically distant organisms [13]. In the course of evolution,Fusobacterium has probably decreased the GC content of the horizontally acquired genes and maintained a genome with a low GC content.DoesSymbiobacterium benefit from maintaining a genome with a high GC content? Considering that CO2 is the major growth factor of Symbiobacterium, its symbiotic partners may not be limited to Geobacillus. Symbiobacterium is widespread in different natural environments [26, 27]. The difference in the genome base compositions between Symbiobacterium and its symbiotic partners may lead to a decrease in the frequency of a homologous recombination between the 2 genomes. For example, the 5 sequenced chromosomal genomes of Geobacillus have a GC content ranging from 42.8% to 52.5% (http://insilico.ehu.es/oligoweb/).In addition, homologous recombination is generally effective for adaptive evolution [11]. However, if the population density is low or the recombining population is rare in the environment, adaptive evolution is hampered [11]. Considering the wide distribution of Symbiobacterium in natural environments, the population size of Symbiobacterium may be adequately large, suggesting that homologous recombination between the Symbiobacterium strains and different symbiotic partners may be effective for adaptive evolution. Thus, it is hypothesized that Symbiobacterium has maintained its extreme genome composition to avoid homologous recombination between its genome and the genomes of different species and to promote homologous recombination between its genome and the genomes of the same species (or genus).



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*Source: 634505-2010-12-12.xml*