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Title: The Genome Organization of Thermotoga maritima Reflects Its Lifestyle}
Article:
Abstract:
The generation of genome-scale data is becoming more routine, yet the subsequent analysis of omics data remains a significant challenge. Here, an approach that integrates multiple omics datasets with bioinformatics tools was developed that produces a detailed annotation of several microbial genomic features. This methodology was used to characterize the genome of Thermotoga maritima—a phylogenetically deep-branching, hyperthermophilic bacterium. Experimental data were generated for whole-genome resequencing, transcription start site( TSS) determination, transcriptome profiling, and proteome profiling. These datasets, analyzed in combination with bioinformatics tools, served as a basis for the improvement of gene annotation, the elucidation of transcription units( TUs), the identification of putative non-coding RNAs( ncRNAs), and the determination of promoters and ribosome binding sites. This revealed many distinctive properties of the T. maritima genome organization relative to other bacteria. This genome has a high number of genes per TU( 3. 3), a paucity of putative ncRNAs( 12), and few TUs with multiple TSSs( 3. 7%). Quantitative analysis of promoters and ribosome binding sites showed increased sequence conservation relative to other bacteria. The 5′UTRs follow an atypical bimodal length distribution comprised of “Short” 5′UTRs( 11–17 nt) and “Common” 5′UTRs( 26–32 nt). Transcriptional regulation is limited by a lack of intergenic space for the majority of TUs. Lastly, a high fraction of annotated genes are expressed independent of growth state and a linear correlation of mRNA/protein is observed( Pearson r = 0. 63, p<2. 2×10−16 t-test). These distinctive properties are hypothesized to be a reflection of this organism's hyperthermophilic lifestyle and could yield novel insights into the evolutionary trajectory of microbial life on earth.
Introduction:
A fundamental step towards obtaining a systems-level understanding of organisms is to obtain an accurate inventory of cellular components and their interconnectivities[1]–[3]. The genome sequence and in silico predictions of gene annotation are the starting points for assembling a network. For prokaryotes, these in silico approaches detect open reading frames and structural RNAs with varying degrees of accuracy[4]. Recently, multi-omic data generation and analysis studies[5]–[11] have revealed an abundance of genomic features that are not detected computationally such as transcription start sites( TSSs), promoters, untranslated regions( UTRs), non-coding RNAs, ribosome binding sites( RBSs) and transcription termination sites[12]. However, the rate at which multi-omic datasets are being generated is substantially outpacing the development of analysis workflows for these inherently dissimilar data types[13]. Here, multi-omic experimental data is generated and analyzed in conjunction with bioinformatics tools to annotate numerous bacterial genomic features that cannot accurately be detected using in silico approaches alone. This methodology was applied to study the genome organization of Thermotoga maritima—a phylogenetically deep-branching, hyperthermophilic bacterium with a compact 1. 86 Mb genome. Originally isolated from geothermally heated marine sediment, T. maritima grows between 60–90°C with an optimal growth temperature of 80°C[14]. This species belongs to the order Thermotogales that have, until recently, been exclusively comprised of thermophilic or hyperthermophilic organisms. Compared to most bacteria, Thermotogales are capable of sustaining growth over a remarkably wide range of temperatures. For instance, Kosmotoga olearia can be cultivated between 20–80 °C[15]. Recently, the existence of mesophilic Thermotogales[16],[17] was confirmed with the description of Mesotoga prima, which grows from 20–50 °C with an optimum at 37 °C[18]. Sequencing of M. prima revealed that it has the largest genome of all the Thermotogales at 2. 97 Mb with ∼15% noncoding DNA[19]. T. maritima, which grows at the upper-limit known for Thermotogales, has one of the smallest genomes in this order and maintains one of the most compact genomes among all sequenced bacterial species( <5% noncoding DNA)[20],[21]. The short intergenic regions in the T. maritima genome( 5 bp median) resemble those in the genome of Pelagibacter ubique, a bacterium that has undergone genome streamlining and has the shortest median intergenic space( 3 bp) among free-living bacteria[20]. Although it remains unclear whether T. maritima has also undergone streamlining, both organisms encode only a few global regulators( four sigma factors in T. maritima versus two in P. ubique) and carry just a single rRNA operon. In contrast with P. ubique, T. maritima displays more metabolic diversity through its ability to ferment numerous mono- and polysaccharides[14],[22]. Thermotogales have been the focus of many evolutionary studies[23]–[25]. Organisms in hydrothermal vent communities, where many Thermotogales have been isolated, are thought to harbor traits of early microorganisms[26]. Phylogenetic analysis of 16S rRNA sequences place the Thermotogae at the base of the bacterial phylogenetic tree[27],[28]; however, Zhaxybayeva et al.[25] determined through analysis of 16S rRNA and ribosomal protein genes that Thermotogae and Aquificales( a hyperthermophilic order) are sister taxa. The authors also determined that the majority of Thermotogae proteins align best with those found in the order Firmicutes[25]; therefore, the exact phylogenetic position of Thermotogae is still unresolved. Nevertheless, members of this phylum are among the deepest branching bacterial species and, as such, prime candidates for evolutionary studies. Thermophiles such as T. maritima implement numerous strategies at both the protein and nucleic acid levels to support growth at high temperatures. For instance, intrinsic protein stabilization is achieved by utilizing more charged residues at the protein surface, encoding for a dense hydrophobic core, and increasing disulfide bond usage[29],[30]. DNA is typically kept from denaturing by introducing positive supercoils via reverse gyrase activity while phosphodiester bond degradation is prevented by stabilization through interaction with cations( e. g. K+, Mg2+) and polyamines[31],[32]. However, the impact of temperature on genome features essential to gene expression such as promoters and RBSs remains largely unexplored. Bacterial transcription initiation is governed by recognition of promoter sequences by sigma factors, which load the RNA polymerase holoenzyme upstream of the transcription start site( TSS). Translation initiation is predominantly reliant on base pairing between the anti-Shine-Dalgarno sequence found near the 3′-terminus of the 16S rRNA and the Shine-Dalgarno sequence( i. e. the RBS). Therefore, thermophilic macromolecular synthesis machinery must establish and retain contacts with nucleic acids while facing greater thermodynamic challenges. The integrated approach described here enables an experimentally anchored annotation of several bacterial genomic features including protein-coding genes, functional RNAs, non-coding RNAs, transcription units( TUs), promoters, ribosome binding sites( RBSs) and regulatory sites such as transcription factor( TF) binding sites, 5′ and 3′ untranslated regions( UTRs) and intergenic regions. This is achieved through the simultaneous analysis of genomic, transcriptomic and proteomic experimental datasets with complementary bioinformatics approaches. In addition to providing a valuable resource to the research community, this analysis framework facilitates quantitative and comparative analysis of annotated features across microbial species. For the genome of T. maritima, several distinguishing characteristics were identified and their potential causal factors are discussed.
Discussion:
Genome-scale technologies have provided researchers unprecedented access to large volumes of data detailing the composition of a cell. However, approaches for data analysis and interpretation have lagged behind due to the scope and complexity of these data types. Here, we present a framework for multi-omic data analysis that annotates genomic features involved in transcription, translation and regulation. This methodology integrates genome-scale datasets with bioinformatics predictions to produce 1) an improvement of the gene annotation, 2) an experimentally validated TU architecture and 3) the identification of putative antisense, non-coding transcripts and alternative TSSs. Using these annotated genomic features enabled the genome-wide identification of promoters and RBSs, which are difficult to identify solely using in silico approaches[60],[61]. Furthermore, the relative binding strength of individual promoters and RBSs was quantitatively measured using thermodynamic principles enabling multi-species comparison of these sequence features. The annotated genome organization served as a scaffold for analyzing regulatory features. Transcription factor regulation was examined with respect to promoter containing intergenic regions while the translational impact of the 5′UTR distribution was considered. The multi-omic data generation and analysis demonstrated here is applicable to many microbial species. Applying this methodology to study the genome organization of T. maritima revealed that it has many distinctive properties compared to other organisms. Genome-scale analysis of promoters showed that T. maritima encodes a highly conserved, robust architecture that ensures transcription initiation. Similarly, RBS sequence conservation was shown to be thermodynamically sufficient for translation initiation for almost all T. maritima genes at 80°C compared with only a fraction of E. coli genes. The distinctive properties of the T. maritima genome extend beyond sequence composition and are apparent at the organizational level. The high protein-coding density and minimal intergenic space found in this organism have resulted in a high number of genes per TU, a paucity of putative ncRNAs and few TUs with multiple start sites. Furthermore, transcriptional regulation appears to be limited to a few TUs due to a lack of genomic space in PIRs. Interestingly, the 5′UTR component of the PIR was found to be uncharacteristically bimodal and was comprised of an unusually short grouping of 5′UTRs. Lastly, the constrained genome organization of T. maritima is reflected in the physiological state of the cell. Transcription of the vast majority of genes is detected independent of culture condition and the correlation between protein and mRNA is stronger than previously observed in other bacteria. We hypothesize that the hyperthermophilic lifestyle of T. maritima could potentially explain the distinctive characteristics of this organism's genome organization. For instance, the increased sequence conservation of promoter elements and RBSs throughout the T. maritima genome may be attributed to the need to maintain gene expression under extreme temperature conditions. Macromolecular interactions( e. g. protein/protein, protein/DNA and RNA/RNA) are intrinsically harder to maintain at higher temperatures. In the case of TF binding sites, it has been shown that each nucleotide deviation from consensus results in a ∼2kbT penalty to the maximum binding free energy for a given TF( where kb is Boltzmann's constant and T is temperature)[62]. Increasing the temperature amplifies the binding free energy penalty for every non-conserved base pair. Therefore at 80°C, mismatches between the Shine-Dalgarno and anti-Shine-Dalgarno sequence are especially severe. Thus, T. maritima must overcome the intrinsic challenge of recognizing and retaining contact at the initiation site for both transcription and translation. Our data suggests that high sequence conservation of promoter and RBS sequences is one of the mechanisms used by T. maritima to ensure sufficient gene expression. This sequence-level adaptation could be analogous to many others observed in thermophilic organisms such as the amino acid composition of proteins[29],[30] and the GC content of structural RNAs[63]. The minimal intergenic space found in the T. maritima genome is reminiscent of a streamlined genome, which could explain the limited regulatory capacity observed in this organism. Inflexibility of metabolic regulons has been previously alluded to for other Thermotogales[64]. Here it is demonstrated that, for most TUs, a lack of physical space exists for transcriptional regulation by TFs. Furthermore, the Short 5′UTR group carries the minimum number of nucleotides needed to recruit the ribosome based on Shine-Dalgarno/anti-Shine-Dalgarno interactions[54]. Further reduction in 5′UTR length would abolish translation. Short 5′UTRs also reduce the capacity to regulate by limiting 5′UTR interactions[65],[66]. Though thermodynamics and physical space are hypothesized to contribute to the characteristic features of the T. maritima genome, the phylogenetic contribution cannot be dismissed. These potential causal factors are difficult to decouple. For RBSs, we were able to determine the impact of phylogeny and optimal growth temperature on RBS binding strength. By analyzing RBSs from 109 bacterial species spanning many phyla and having a diverse range of optimal growth temperatures we were able to demonstrate that both phylogeny and optimal growth temperature were significant determinants of RBSs sequence composition. However, a recent analysis of genome size among species of the order Thermotogales could not resolve the impact of phylogeny from optimal growth temperature[19]. The authors found that a negative correlation between genome size and optimal growth temperature exists within this order but the correlation did not hold when phylogeny was accounted for in the analysis. Interestingly, this study also found that the number of predicted transcriptional regulators and intergenic space is higher in Mesotoga prima, a mesophilic member of the Thermotogales. Thus, the relationship between phylogeny and the genome organization is difficult to elucidate without the generation of more datasets similar to the one presented here. Thermotogae are an ideal phylum for future investigations on the causal impact of factors such as temperature, intergenic space and phylogeny on genome organization. This phylum contains organisms that are found in many diverse environments with a wide range of optimal growth temperatures. Generating multi-omic datasets and analyzing them using an integrated, quantitative workflow for numerous Thermotogae species would enable assessment of various environmental factors in the context of phylogenetic distance. Furthermore, given their phylogenetic depth, characterization of the Thermotogae will also provide insights in the evolutionary trajectory of microbial life on earth.
Genomic studies have greatly benefited from the advent of high-throughput technologies and bioinformatics tools . Here , a methodology integrating genome-scale data and bioinformatics tools is developed to characterize the genome organization of the hyperthermophilic , phylogenetically deep-branching bacterium Thermotoga maritima . This approach elucidates several features of the genome organization and enables comparative analysis of these features across diverse taxa . Our results suggest that the genome of T . maritima is reflective of its hyperthermophilic lifestyle . Ultimately , constraints imposed on the genome have negative impacts on regulatory complexity and phenotypic diversity . Investigating the genome organization of Thermotogae species will help resolve various causal factors contributing to the genome organization such as phylogeny and environment . Applying a similar analysis of the genome organization to numerous taxa will likely provide insights into microbial evolution .