Genomewide comparison and novel ncRNAs of Aquificales
- Marcus Lechner†1,
- Astrid I Nickel†1,
- Stefanie Wehner†2,
- Konstantin Riege2,
- Nicolas Wieseke3,
- Benedikt M Beckmann4,
- Roland K Hartmann1Email author and
- Manja Marz2Email author
© Lechner et al.; licensee BioMed Central Ltd. 2014
Received: 29 November 2013
Accepted: 8 May 2014
Published: 25 June 2014
The Aquificales are a diverse group of thermophilic bacteria that thrive in terrestrial and marine hydrothermal environments. They can be divided into the families Aquificaceae, Desulfurobacteriaceae and Hydrogenothermaceae. Although eleven fully sequenced and assembled genomes are available, only little is known about this taxonomic order in terms of RNA metabolism.
In this work, we compare the available genomes, extend their protein annotation, identify regulatory sequences, annotate non-coding RNAs (ncRNAs) of known function, predict novel ncRNA candidates, show idiosyncrasies of the genetic decoding machinery, present two different types of transfer-messenger RNAs and variations of the CRISPR systems. Furthermore, we performed a phylogenetic analysis of the Aquificales based on entire genome sequences, and extended this by a classification among all bacteria using 16S rRNA sequences and a set of orthologous proteins.
Combining several in silico features (e.g. conserved and stable secondary structures, GC-content, comparison based on multiple genome alignments) with an in vivo dRNA-seq transcriptome analysis of Aquifex aeolicus, we predict roughly 100 novel ncRNA candidates in this bacterium.
We have here re-analyzed the Aquificales, a group of bacteria thriving in extreme environments, sharing the feature of a small, compact genome with a reduced number of protein and ncRNA genes. We present several classical ncRNAs and riboswitch candidates. By combining in silico analysis with dRNA-seq data of A. aeolicus we predict nearly 100 novel ncRNA candidates.
Aquificales are gram-negative, non-sporulating bacteria that are thermophilic to hyperthermophilic [1, 2], living in terrestrial and marine hot springs. They are autotrophs that primarily fix carbon by the tricarboxylic acid (TCA) cycle [3–5]. The hyperthermophile A. aeolicus, living under extreme temperatures of up to 95°C, has been proposed to have adopted 10% of its protein-coding genes by horizontal gene transfer [6, 7] from Archaea. Accumulation of all the special properties of thermophiles (also referred to as accumulation profiles ) are rarely understood. Special protein-protective mechanisms have been analyzed [9, 10], but we are far away from a comprehensive understanding of the molecular biology of extremophilic bacteria. Beyond idiosyncratic features of Aquificales genomes, our interest focussed on their transcriptomes. Experimentally, we performed a deep sequencing analysis on the model hyperthermophile A. aeolicus with the primary goal of identifying novel ncRNAs candidates. NcRNAs are known to have various functions in all domains of life. Apart from their general importance as gene expression regulators [11–13], ncRNAs are involved in processing  and translation  of other genes, in defending genomes from viral invasion , in shaping and maintenance of bacterial chromosome architecture , and they can even be multifunctional [18, 19]. According to 16S rRNA analysis, the Aquificales constitute the most deeply rooted bacterial group . However, protein-based phylogenetic reconstructions are not in line with this model [21–26].
We compared the genomes of the three Aquificales families, i.e. Aquificaceae, Hydrogenothermaceae and Desulfurobacteriaceae. We have extended the protein annotation of the mentioned Aquificales and reconstructed the phylogenetic position of these species based on 16S rRNAs as well as on a set of orthologous proteins. Moreover, we have identified ncRNAs based on known homologs and present a complete set of novel ncRNA candidates based on sequence analyses and deep sequencing data obtained for A. aeolicus. For selected ncRNA loci, we provide independent experimental evidence for their expression.
Aquificaceae: Aquifex aeolicus VF5 (AAE), Hydrogenivirga sp. 128-5-R1-1 (HVI), Hydrogenobacter thermophilus TK-6 (HTH), Thermocrinis ruber (TRU), Thermocrinis albus DSM 14484 (TAL), Hydrogenobaculum sp. Y04AAS1 (HBA),
Hydrogenothermaceae: Sulfurihydrogenibium sp. YO3AOP1 (SSP), Sulfurihydrogenibium azorense Az-Fu1 (SAZ), Persephonella marina EX-H1 (PMA), and
Desulfurobacteriaceae: Desulfobacterium thermolithotrophum DSM 11699 (DTH), and Thermovibrio ammonificans HB-1 (TAM).
Extension of protein annotation
We used BacProt (publication in progress, see  for details) to complement the present annotation of protein-coding genes for each Aquificales genome. It uses a database of groups of orthologous protein-coding genes present in most bacteria . Matches in the genome of interest are annotated, and species-specific features like codon usage, Shine-Dalgarno sequences, Pribnow box motifs and Rho-independent terminators are used to predict additional protein-coding genes. To actually achieve a de novo annotation, we excluded all Aquificales genes from the reference database. Alternative start codons like ATT and CTG were considered as well [35–37]. Re-annotated and previously annotated proteins (genomic positions and sequences) and statistics (mono-/di-nucleotide distribution, position and occurrence of Shine-Dalgarno sequence motifs and Pribnow boxes) for each species are provided in the Supplemental Material.
Annotation of ncRNAs by homology
We used GORAP (v.1.0, publication in progress) to annotate ncRNAs in the following manner: transfer-RNAs (tRNAs) were detected by tRNAscan-SE (v.1.3.1)  with the option -B for bacteria. Split tRNAs were searched using SPLITS (v.1.1) . By applying ARAGORN (v.1.2), we searched for tRNAs containing introns . Searches for RNase P RNA were conducted with Bcheck (v.1.0) . For the detection of putative CRISPR loci, crt (v1.2)  and CRISPRFinder were used. We searched for cas protein genes by blast (v.2.2.26, E-value ≤10-4)  based on known cas genes (downloaded from UniProt (downloaded Jan. 2013) ).
To find further ncRNAs, we used blast and Infernal (v.1.1rc2) . Seed sequences from the Rfam (v.11.0) database  and European Ribosomal RNA Database were used as query with an E-value ≤0.001 for blast and the Rfam-provided family specific noise cutoffa for Infernal.
NcRNAs expected to escape from detection (e.g. 6S RNA) were searched in a second step with rnabob for short motif search in combination with RNAsubopt, RNAduplex, RNAcofold, RNAalifold and RNAup from the RNA Vienna Package (v.2.0) [50–53]. For verification, we aligned candidates with ClustalW (v.2.0.10)  or Locarnate (v.18.104.22.168) . Stockholm alignments were adjusted by hand in the Emacs Ralee mode.
Resulting Stockholm alignments are supplied in the Supplemental Material in the General Feature Format (gff) as well as in Fasta (fa) and Stockholm (stk) formats.
Protein-based phylogeny was performed based on the official NCBI annotations for 42 bacteria shown in the Supplemental Material. In addition to eleven Aquificales species, we included two Archaea as outgroup and a wide phylogenetic range of 29 bacterial species representing all bacterial clades.
Protein sequences were clustered using Proteinortho in the blastp+-mode, thus performing a pairwise all-against-all comparison of sequences from different species to derive orthologous relationships. Whenever an orthologous group did not have a member in a certain species, we applied tblastn to the respective genome to complement for potentially incomplete annotations. The highest scoring alignment to an ORF above a fairly high E-value ≤ 10-20 was added to the initial protein annotation. Finally, Proteinortho was applied again using the expanded annotation.
For a high resolution phylogeny within the Aquificales, we created a whole genome alignment using Pomago. The alignment was analyzed using RAxML (v.7.4.2)  with a GAMMA model of rate heterogeneity with an estimate on the proportion of invariable sites and 100 rapid bootstraps.
In an additional phylogenetic analysis we used single-copy orthologous proteins present in at least 50% of all species in the set (189 groups in 42 species). Each protein group was aligned separately using dialign-tx. Both ends of the group’s alignments were cropped to remove leading and tailing gaps. The remaining sequences were concatenated resulting in a 57,260 aa long alignment and applied to RAxML using the LG substitution model  as well as the GAMMA model of rate heterogeneity with 100 rapid bootstraps.
The 16S rRNA-based phylogeny was computed with Mafft (v.7.017)  using the L-INS-i method with 1000 iterations. We used different approaches: (1) Neighbor Joining with the Kimura correction model  (1000 bootstraps), (2) Bayesian inference with MrBayes (v.3.1.2)  with default parameters, (3) Maximum likelihood with RAxML (v.7.2.8)  (200 bootstraps) with the base substitution models (3a) GTRGAMMA (most accurate, 1000 steps) and (3b) GTRCAT for the bootstrapping phase. For all previously mentioned methods the Archaea Methanobacterium sp. AL-21 and Archaeoglobus fulgidus were used as outgroup. As state of the art, we have estimated a tree with (4) Sate (v.2.2.5)  (200 iterations). Related sequences were aligned with Mafft and subsequently merged by Muscle (v.3.7) . The tree was computed using RAxML.
dRNA-seq of A. aeolicustotal cellular RNA
Transcriptome analysis of A. aeolicus was based on cDNA libraries from a differential deep sequencing approach (dRNA-seq) [67, 68]. A. aeolicus cells, provided by M. Thomm and R. Huber (Regensburg, Germany), were grown for 1 day (late exponential phase) and harvested as described . For preparation of total cellular RNA, we used the hot phenol method : cell pellets were resuspended in extraction buffer (10 mM sodium acetate pH 4.8, 150 mM sucrose) and incubated for 10 min at room temperature with 0.1 volumes of lysozyme (20 mg/ml, Roth, Karlsruhe, Germany). SDS was added to a final concentration of 1% followed by vigorous vortexing. After addition of 1 volume phenol (preheated to 65°C) and vortexing, the mixture was incubated for 5 min at 65°C, then cooled on ice for 5 min, and centrifuged for 30 min at 4°C and 8200 g. Phenol extraction was repeated, followed by chloroform (1+1) extraction and ethanol precipitation. Finally, the DNA was digested with 10 U Turbo DNase (Ambion, Austin, USA) for 30 min at 37°C, followed by addition of another 10 U DNase and incubation for another 30 min at 37°C. Subsequently, the RNA was subjected to phenol/chloroform extraction and ethanol precipitation. After redissolving the RNA in double-distilled water, its concentration was determined by UV spectroscopy. Before cDNA library construction, the RNA was split into two fractions; one fraction was treated with Terminator 5’ P-dependent exonuclease (Epicentre, Madison, USA) for depletion of transcripts carrying a 5’-monophosphate. Both fractions were treated with Tobacco Acid Phosphatase (TAP) before 5’-linker ligation, poly(A) tailing and conversion into cDNA (vertis Biotechnologie AG, Freising, Germany). The cDNA libraries were then sequenced on a Roche FLX sequencer and resulted in the (-)-library with 25,816 reads and the (+)-library (33,697 reads) containing the enriched primary transcripts.
Detection of novel ncRNAs
We used the IGB (Integrated Genome Browser)  to visualize the following features of A. aeolicus: (1) nucleotide sequence; (2) local GC-content (for each nucleotide 15 nt on both sides were included for the calculation of GC-content); (3) protein genes annotated by NCBI and BacProt; (4) locally stable secondary structures: calculation was performed with RNALfold with options -d2 and -L120 for both strands with a maximum base-pair span of 120 nucleotides. Sequences with local structures of fewer than 50 nt were discarded. For the prediction of thermodynamically stable RNA structures, each sequence was shuffled 1000 times while preserving the dinucleotide frequencies; to classify extraordinarily stable RNA secondary structures, we chose to use a Z-score cutoff of -3.0 (∼ top 5% of stable structures); (5) conserved regions among the Aquificales: with default parameters of TBA and Pomago we aligned 11 genomes; the TBA alignment was projected to each of the reference genomes; coverage, WSoP and gap ratio are given in Figure 1; (6) novel ncRNAs: novel ncRNA candidates were predicted using RNAz. We used rnazWindow.pl –min-seqs=4 and RNAz -n -b -p 0.5 on the alignments of Pomago and TBA. As rnazWindow.pl assumes lower case nucleotides to be masked, the alignments were converted to upper case letters beforehand; (7) dRNA-seq: cDNA libraries were mapped with segemehl (v.0.0.9.3)  applying the parameters -m 12 -D 1 -e 2 -p 4 -X 8 -A 90 -E 5.0.
Northern blot experiments
Total RNA preparation
Total RNA was prepared from cell pellets using the hot phenol method as described .
Positive and negative controls
The positive and the negative controls for the Northern blot experiments were synthesized by in vitro transcription using the "TranscriptAid T7 High Yield Transcription Kit" (Thermo Scientific, Germany), according to the protocol supplied by the manufacturer. PCR products generated with the "Long PCR Enzyme Mix" (Thermo Scientific) served as templates for in vitro transcription. As positive controls for the antisense tRNA blots, chemically synthesized RNA oligonucleotides from "Integrated DNA Technologies" (IDT, Belgium) were used (for sequences, see Supplemental Material). RNA oligonucleotides were 5’-phosphorylated before gel electrophoresis. The in vitro transcribed full-length sense tRNAs (generated from PCR products) were used as negative controls for the Northern blots of antisense tRNAs.
Digoxigenin and LNA probes
For the Northern blot detection internally digoxigenin-labeled probes were transcribed using the DIG RNA Labeling Mix (Roche Diagnostics, Germany) as described . The antisense tRNA transcripts were detected with chemically synthesized 5’-digoxigenin-labeled DNA/LNA mixmer probes (Exiqon, Denmark; for sequences, see Supplemental Material).
5’-Phosphorylation of RNA oligonucleotides
67 ng/ μl RNA oligonucleotide, 2.5 mM DTT, 2 mM ATP and 10 U T4 polynucleotide kinase (T4 PNK; Thermo Scientific) were incubated in 1 × T4 PNK buffer (Thermo Scientific) in a volume of 15 μl for 1 h at 37°C, followed by transfer to and storage at -20°C.
RNAs were separated on 8% or 10% denaturing (8 M urea) PAA gel with 1 × TBE as electrophoresis buffer .
Blotting, crosslinking, hybridization and detection
RNA blotting, hybridization (EDC crosslinking or baking at 80°C for 40 min) and immunological detection were performed as described , except that RNA blotting was carried out at 0.36 mA/cm 2 overnight. Prehybridization and hybridization were performed at 68°C (except for 50°C in the case of antisense tRNA 44) using 12 ml hybridization solution. 3.5 μl of in vitro transcribed, internally digoxigenin-labeled probe were added for overnight hybridization. 300 pmol of chemically synthesized, 5’-digoxigenin-labeled DNA/LNA mixmer probe were used for Northern detection of antisense tRNAs. Blotted membranes were stored at room temperature.
In vitrotranscripts, probes and primers
Further details on in vitro transcripts, probes and primers are listed in the Supplemental Material.
Results and discussion
Genome analysis – general observations
The genomes of the Aquificales range from 1.50 Mb (T. albus) to 1.98 Mb (P. marina), thus being at the lower limit of bacterial genomes ranging in size from 0.14 to 14.38 Mb with a mean of ∼ 4 Mb . The current annotation file of Hydrogenivirga sp. contains 3.04 Mb, which is considerably larger than the genome size of the other Aquificales, which might be an assembly artefact as discussed later.
Aquificales are known to be AT-rich with a GC-content of about 43% [72, 76]. In Hydrogenobaculum sp., Sulfurihydrogenibium sp. and S. azorense even only one-third of the nucleotides are guanine or cytosine. For T. ammonificans an atypically high GC-content of more than 50% was observed.
Between 6.5% (S. azorense) and 28.5% (Hydrogenobaculum sp.) of the genomes were found to be unique to each member bacterium (Figure 1). The comparatively low coverage of Hydrogenivirga sp. is due to the currently assembled genome being almost twice as long as those of other Aquificales. 10.5% to 13.0% of the Pomago alignment, resp. 8.4% to 9.6% of the TBA alignment, consist of gaps. According to the WSoP each nucleotide from the alignment is conserved on average in slightly less than half of the other 10 species (4.43 to 5.09 out of 11 and 3.81 to 4.91 out of 11, for Pomago and TBA, respectively) indicating that the genomes diverged relatively fast. Genomic rearrangements among the Aquificales, underlining the diversity, can be seen in an overview of the Pomago alignment in the Supplemental Material.
Extended annotation of proteins
We added between 0.7% of H. thermophilus (1352/1343) and 10.6% of A. aeolicus (1002/897) protein-coding genes to the NCBI annotation.
Codon usage of A. aeolicus
Homology search and annotation of known ncRNAs
A search for ncRNA candidates with RNAz predicted a relatively constant fraction of the genome to code for ncRNAs (between 0.36% for S. azorense and 0.91% for A. aeolicus). Besides the well-known and described rRNAs and tRNAs, only a handful of other wide-spread ncRNAs were detected (Figure 1).
Most of the Aquificales genomes have two rRNA operons (Figure 1). H. thermophilus and T. albus appear to harbor only one operon. The genomes of T. ammonificans and Sulfurihydrogenibium sp. contain three operons, whereas Hydrogenivirga sp. appears to have two 16S, two 23S and three 5S rRNA genes.
With the exception of Hydrogenivirga sp. (see below), tRNAscan identified between 39 (S. azorense) and 46 tRNAs (T. ammonificans) per Aquificales species. With SPLITS and ARAGORN no split tRNAs were found.
All possible codons are utilized in the Aquificales (see Table 2 for A. aeolicus, and Supplemental Material for other Aquificales), but the number of tRNA genes is reduced to a minimum in contrast to reference bacteria such as E. coli which encodes multiple copies of many tRNA isoacceptors.
The catalytic RNA subunit of the tRNA processing endoribonuclease RNase P was previously identified in P. marina and S. azorense. Additionally, RNase P RNAs were easily identified here with Bcheck in Sulfurihydrogenibium sp., T. ammonificans and D. thermolithotrophum. In the Aquificaceae, RNase P RNA candidates were neither detected with Bcheck, rnabob nor by manual in silico search methods using cDNA libraries of A. aeolicus. This is consistent with the negative results of previous searches for RNase P RNA in A. aeolicus[85, 86].
All identified RNase P RNAs lack the P18 element, which appears to be a general feature of type A RNase P RNAs in the Hydrogenothermaceae and Desulfurobacteriaceae. The Sulfurihydrogenibium sp., T. ammonificans and D. thermolithotrophum RNAs differ from their P. marina and S. azorense counterparts by a weaker L9-P1 tertiary contact (L9 5’-GYAA tetraloop docking on an A-U/G-C tandem bp instead of a G-C/G-C tandem which is a hallmark of RNase P RNAs from thermophiles [84, 87]). Other differences are: (1) very short P12 stems in T. ammonificans and D. thermolithotrophum, (2) particularly weak P17 stems in Sulfurihydrogenibium sp. and D. thermolithotrophum, (3) a destabilized L8-P4 interaction, a destabilized P14 helix, but a stabilized L14-P8 interaction in T. ammonificans. For details, see RNase P RNA 2D structures in the Supplemental Material.
Bacterial 6S RNAs, about 200 nt in length, form a rod-shaped secondary structure with a central bulge region flanked by largely helical arms on both sides. Their structure is thought to mimic the structure of an open DNA promoter [88, 89]. 6S RNAs bind to the housekeeping RNA polymerase holoenzyme to block transcription at DNA promoters, primarily upon entry of cells into stationary growth phase. When nutrients are resupplied (including NTPs), RNA polymerase massively synthesizes transcripts (so-called product RNAs – pRNAs) on 6S RNA as template, which lead to a structural rearrangement of 6S RNA and release of RNA polymerase. Thus, 6S RNA is a fast riboregulator that makes RNA polymerase instantly available for a new exponential growth when nutrients are resupplied [68, 90–93].
In A. aeolicus the 6S RNA was clearly identified via an experimental RNomics approach . 6S RNA candidates in the other Aquificales were predicted computationally using the Rfam covariance model and, as expected, vary substantially in primary, but less in secondary structure. For Hydrogenivirgia we found two copies. Predicted 6S RNAs for T. ammonificans and D. thermolithotrophum remain candidates since they differ substantially from those of other Aquificales.
The RNAalifold consensus structure for the 6S RNA candidates from all other Aquificales analyzed here is shown in the Supplement. Individual RNAfold predictions (see Supplemental Material for details) support the notion that they are bona fide 6S RNAs.
SRP RNA was found once per genome being highly conserved in sequence and structure (see Supplemental Material). Additionally, we show some riboswitch candidates: TPP, MOCO, Cobalamin and crcB (see Figure 1). The MOCO riboswitch found in T. ammonificans and the two crcB riboswitches identified in Hydrogenobaculum sp. conform well to the Rfam conservation model (see Supplemental Material). Riboswitches were only found sporadically among the Aquificales.
Novel ncRNAs in A. aeolicus
Besides the annotation of ncRNAs with known functions, we additionally aimed to detect novel ncRNAs, as they often regulate transcription or play an important role as posttranscriptional regulators. Here we combined in silico analysis of the A. aeolicus genome and dRNA-seq data from the same organism to identify novel ncRNA candidates, some of which were subsequently analyzed by Northern blot analysis.
In the in silico search, small ncRNAs (sRNAs) were distinguished from proteins by the following analysis steps: (1) The GC-content of the A. aeolicus genome is 43%. However, the ncRNAs described above show an average GC-content of 66%. We associated each nucleotide with a local GC-value. (2) The function of small ncRNAs, e.g. 6S RNA, is often determined by their stable secondary structure. To each position in the genome, we assigned the minimum free energy of the most stable local secondary structure including this nucleotide, using RNALfold. (3) Most ncRNAs are conserved among closely related organisms. We calculated genomewide multiple sequence alignments (MSA) with TBA and Pomago of all Aquificales genomes, which can be viewed in the Supplemental Material. (4) Based on the MSAs we performed a novel ncRNA prediction with RNAz and displayed their probability.
Selection of highly potential novel ncRNA candidates of A. aeolicus
Structure and Sequence
Downstream of 5S RNA
Putative Novel ncRNAs
Tail to tail Transcripts (T2T)
tRNAs with sense transcripts only
tRNAs with sense and various antisense transcripts
For comparison reasons, we also added tRNAs to our table of ncRNAs, which show the feature of sense-antisense (s/as) expression. To exclude the possibility of mapping or other artefacts, we confirmed the presence of antisense transcripts exemplarily by Northern blots for tmRNA and tRNA 44 (Pro-TGG) (Figure 10).
Furthermore, Northern blots were conducted for the loci encoding candidates n25 and n75, for which the dRNA-seq data indicated sense and antisense transcription each differing between the (+)- and (-)-library (Figure 11). For n25, we found most transcripts on the plus strand in the (+)-library (582), whereas less than half as many transcripts (250) were detected in the (-)-library. Interestingly, an inverse relation was observed for the minus strand (50/361). For n25, Northern blot detection revealed a signal somewhat shorter than the one expected from the cDNA read boundaries, whereas no signal could be detected for antisense transcripts (Figure 11, top). This finding suggests that the sense transcript is the major one. In the case of n75, both sense and antisense transcripts of comparable intensity were detected, the major signals of the Northern blot representing RNAs larger and smaller than anticipated from the read boundaries (Figure 11, bottom). Thus, the polarity of the putative ncRNA gene remains unclear.
Interestingly, very high transcription levels are found in overlapping 5’-upstream regions of two protein-coding genes located on opposing strands (Table 3). Beside these so-called head-to-head (h2h) transcripts we furthermore observed tail-to-tail overlaps (t2t, two 3’-untranslated regions overlapping on opposing strands) that are represented by very high read coverage (Supplemental Material). If these are real transcripts with a certain function or artefacts remains unclear.
With the advent of a growing number of Aquificales genome sequences in public databases, we have re-analyzed this group of bacteria thriving in extreme environments. The Aquificales share the feature of a small, compact genome with a reduced number of protein and ncRNA genes. The genes for tRNAs are reduced to a minimum but retain the capacity to decode all types of codons, and rRNA genes are confined to 2–3 copies each. Several classical ncRNAs are present, such as SRP RNA, tmRNA, 6S RNA, RNase P RNA (except for all Aquificaceae) and riboswitch candidates in some Aquificales. Furthermore, by combining in silico analysis with dRNA-seq data of A. aeolicus, we were able to predict nearly 100 novel ncRNA candidates, some of which might be specific to the Aquificales. Finally, CRISPR systems of bacterial immunity were identified.
Re-annotation of protein genes using BacProt revealed novel proteins with unknown function, some of which might turn out to be specific to the Aquificales as well. On average, 63 additional proteins were found that were missing in the respective original annotation.
In our cDNA libraries of A. aeolicus, we observed massive amounts of antisense reads with similar patterns (length and amount) at putative ncRNA loci and terminal regions of mRNAs. Examples of transcripts antisense to tmRNA and tRNA are illustrated in Figure 10.
We compared 40 bacterial and 2 archaeal genomes (see Supplemental Material), and the presence or absence of proteins was used to determine their position in the phylogenetic tree of bacteria. Both Archaea form a clear outgroup. Thermodesulfatator indicus branches first in the group of Bacteria, followed immediately by the Aquificales, while other bacterial branches diverge later. In an additional protein-based analysis, we took the sequences of single-copy orthologs that were present in at least 50% of all species (concatenated 57,260 aa) (see Supplemental Material). In contrast to the protein presence/absence tree, neither the Aquificales nor T. indicus were placed at a basal position here. However, the two groups are still in close vicinity to each other. This analysis not necessarily excludes the possibility of the Aquificales being a basal clade. The selection of orthologs being present in at least 50% of the species leads to a lower coverage of orthologs present in Archaea species and therefore may favor long branch attraction . The idea behind selecting frequently occurring single-copy orthologs was to produce phylogenetic trees being less influenced by horizontal gene transfer. However, proteins shared by Archaea and Aquificales only are not part of the selected "50% group" of proteins and are therefore not considered in this analysis.
Both protein-based phylogenetic trees disagree with a previous study  where Desulfobacterium autotrophicum HRM2, a δ-proteobacterium, was added to the Desulfurobacteriaceae family based on 16S rRNA analysis. We assume that this was an artefact of the high GC-content of rRNAs due to the high environmental temperatures. Regarding their proteomes, Aquificales and D. autotrophicum are not significantly related.
The results of the 16S rRNA phylogenetic analysis did not show a clear picture. Depending on the method used for reconstruction, the Aquificales were either placed near the root of the bacterial tree (MrBayes and RAxML with GTRGAMMA substitution model) or not (NJ and RAxML with GTRCAT) (see Supplemental Material). In accordance with the results of , the Aquificales were always placed close to the Thermotogales and Thermales-Deinococcales, Archaea were more closely related to the Aquificales than to the Thermotogales.
We identified two 6S RNA and two tmRNA candidate genes in Hydrogenivirga sp., rather than a single one as in the other Aquificales. Likewise, Hydrogenivirga sp. has a comparatively high amount of tRNA copies and CRISPR loci and its genome is estimated to be of roughly double the size of the other Aquificales genomes. Combined, these observations support the notion that the Hydrogenivirga sp. genome assembly is erroneous or two genomes of related bacteria (one type from Hydrogenothermaceae) have entered the sequencing project, being in agreement with . Based on the tmRNA tag peptides identified in the Hydrogenivirga sp. assembly, the second one (Hydrogenivirga sp.-B: IPEREIAIAA) matches the sequence exclusively found among the Hydrogenothermaceae, although Hydrogenivirga sp. belongs to the Aquificaceae (see Figure 7). This suggests that the Hydrogenivirga sp. assembly is a blend of sequences from a member of the Aquificaceae and a member of the Hydrogenothermaceae.
aNoise cutoff is the highest observed false positive bit score for a potential gene which does not belong to the seed model.
We thank Markus Fricke for tmRNA structure visualization, Brice Felden for tmRNA discussion, J. Sugahara for the SPLITS run in A. aeolicus, Jörg Vogel and Cynthia Sharma from the University of Würzburg for help with differential RNA-Sequencing. MM was funded by the Carl-Zeiss-Stiftung. This work was supported by the DFG-Graduiertenkolleg 1384 "Enzymes and multienzyme complexes acting on nucleic acids" (BMB, ML, MM, RKH, SW), and DFG project MA-5082/1 (MM, SW).
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