GC-rich Genomes Research Articles

Evolution and diversity of nucleotide and dinucleotide composition in poxviruses.

Poxviruses (family Poxviridae) have long dsDNA genomes and infect a wide range of hosts, including insects, birds, reptiles and mammals. These viruses have substantial incidence, prevalence and disease burden in humans and in other animals. Nucleotide and dinucleotide composition, mostly CpG and TpA, have been largely studied in viral genomes because of their evolutionary and functional implications. We analysed here the nucleotide and dinucleotide composition, as well as codon usage bias, of a set of representative poxvirus genomes, with a very diverse host spectrum. After correcting for overall nucleotide composition, entomopoxviruses displayed low overall GC content, no enrichment in TpA and large variation in CpG enrichment, while chordopoxviruses showed large variation in nucleotide composition, no obvious depletion in CpG and a weak trend for TpA depletion in GC-rich genomes. Overall, intergenome variation in dinucleotide composition in poxviruses is largely accounted for by variation in overall genomic GC levels. Nonetheless, using vaccinia virus as a model, we found that genes expressed at the earliest times in infection are more CpG-depleted than genes expressed at later stages. This observation has parallels in betahepesviruses (also large dsDNA viruses) and suggests an antiviral role for the innate immune system (e.g. via the zinc-finger antiviral protein ZAP) in the early phases of poxvirus infection. We also analysed codon usage bias in poxviruses and we observed that it is mostly determined by genomic GC content, and that stratification after host taxonomy does not contribute to explaining codon usage bias diversity. By analysis of within-species diversity, we show that genomic GC content is the result of mutational biases. Poxvirus genomes that encode a DNA ligase are significantly AT-richer than those that do not, suggesting that DNA repair systems shape mutation biases. Our data shed light on the evolution of poxviruses and inform strategies for their genetic manipulation for therapeutic purposes.

INTERPIN: A repository for intrinsic transcription termination hairpins in bacteria

The large-scale detection of putative intrinsic transcription terminators is limited to only a few bacteria currently. We discovered a group of hairpins, called cluster hairpins, present within 15 nucleotides from each other. These are expected to work in tandem to cause intrinsic transcription termination (ITT), while the single hairpin can do the same alone. Therefore, exploring these ITT sites and the hairpins across bacterial genomes becomes highly desirable. INTERPIN is the largest archived collection of in silico inferred ITT hairpins in bacteria, covering 12745 bacterial genomes and encompassing ten bacterial phyla for ∼25 million hairpins. Users can obtain details on operons, individual cluster, and single ITT hairpins that were screened therein. Integrated Genome Viewer (IGV) software interactively visualizes hairpin secondary and tertiary structures in the genomic context. We also discuss statistics for the occurrence of cluster or single hairpins and other termination alternatives while showing the validation of predicted hairpins against in vivo detected hairpins. The database is freely available at http://pallab.cds.iisc.ac.in/INTERPIN/.INTERPIN (database and software) can make predictions for both AT and GC-rich genomes, which has not been achieved by any other program so far. It can also be used to improve genome annotation as well as to get predictions to improve the understanding of the ITT pathway by further analysis.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces hosts for natural product discovery

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as alternative NP hosts. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.

Advances in Vertebrate (Cyto)Genomics Shed New Light on Fish Compositional Genome Evolution.

Cytogenetic and compositional studies considered fish genomes rather poor in guanine-cytosine content (GC%) because of a putative "sharp increase in genic GC% during the evolution of higher vertebrates". However, the available genomic data have not been exploited to confirm this viewpoint. In contrast, further misunderstandings in GC%, mostly of fish genomes, originated from a misapprehension of the current flood of data. Utilizing public databases, we calculated the GC% in animal genomes of three different, technically well-established fractions: DNA (entire genome), cDNA (complementary DNA), and cds (exons). Our results across chordates help set borders of GC% values that are still incorrect in literature and show: (i) fish in their immense diversity possess comparably GC-rich (or even GC-richer) genomes as higher vertebrates, and fish exons are GC-enriched among vertebrates; (ii) animal genomes generally show a GC-enrichment from the DNA, over cDNA, to the cds level (i.e., not only the higher vertebrates); (iii) fish and invertebrates show a broad(er) inter-quartile range in GC%, while avian and mammalian genomes are more constrained in their GC%. These results indicate no sharp increase in the GC% of genes during the transition to higher vertebrates, as stated and numerously repeated before. We present our results in 2D and 3D space to explore the compositional genome landscape and prepared an online platform to explore the AT/GC compositional genome evolution.

Stoichiometry versus ecology: the relationships between genome size and guanine-cytosine content, and tissue nitrogen and phosphorus in grassland herbs.

Plant tissue nitrogen (N) and phosphorus (P) and genome traits, such as genome size and guanine-cytosine (GC) content, scale with growth or metabolic rates and are linked to plant ecological strategy spectra. Tissue NP stoichiometry and genome traits are reported to affect plant growth, metabolic rates or ecological strategies in contrasting ways, although the elemental costs for building and maintaining DNA are typically overlooked. We formulated stoichiometry- and ecology-based predictions on the relationship between genome size and GC content to tissue N, P and N : P and tested them on a set of 130 herbaceous species from a temperate grassland using ordinary, phylogenetic and quantile regression. Genome size was only negatively linked to plant N and N : P in species with very small genomes. We found no link between genome size and plant P. GC content was negatively linked to plant N and P but we found these significant links consistently in both GC-rich and GC-poor species. Finally, GC content correlated positively with plant N : P but only in species with GC-rich genomes. Our results provide stronger support for the ecology-based predictions than the stoichiometry-based predictions, and for the links between GC content and plant N and P stoichiometry than for genome size. We argue that the theories of plant metabolic rates and ecological strategies (resource-acquisitive vs. conservative or ruderal vs. stress-tolerator spectra) better explain interspecific genome-NP stoichiometry relationships at the tissue level (although relatively weakly) than the stoichiometric theory based on the elemental costs for building and maintaining DNA.

A positive correlation between GC content and growth temperature in prokaryotes

BackgroundGC pairs are generally more stable than AT pairs; GC-rich genomes were proposed to be more adapted to high temperatures than AT-rich genomes. Previous studies consistently showed positive correlations between growth temperature and the GC contents of structural RNA genes. However, for the whole genome sequences and the silent sites of the codons in protein-coding genes, the relationship between GC content and growth temperature is in a long-lasting debate.ResultsWith a dataset much larger than previous studies (681 bacteria and 155 archaea with completely assembled genomes), our phylogenetic comparative analyses showed positive correlations between optimal growth temperature (Topt) and GC content both in bacterial and archaeal structural RNA genes and in bacterial whole genome sequences, chromosomal sequences, plasmid sequences, core genes, and accessory genes. However, in the 155 archaea, we did not observe a significant positive correlation of Topt with whole-genome GC content (GCw) or GC content at four-fold degenerate sites. We randomly drew 155 samples from the 681 bacteria for 1000 rounds. In most cases (> 95%), the positive correlations between Topt and genomic GC contents became statistically nonsignificant (P > 0.05). This result suggested that the small sample sizes might account for the lack of positive correlations between growth temperature and genomic GC content in the 155 archaea and the bacterial samples of previous studies. Comparing the GC content among four categories (psychrophiles/psychrotrophiles, mesophiles, thermophiles, and hyperthermophiles) also revealed a positive correlation between GCw and growth temperature in bacteria. By including the GCw of incompletely assembled genomes, we expanded the sample size of archaea to 303. Positive correlations between GCw and Topt appear especially after excluding the halophilic archaea whose GC contents might be strongly shaped by intense UV radiation.ConclusionsThis study explains the previous contradictory observations and ends a long debate. Prokaryotes growing in high temperatures have higher GC contents. Thermal adaptation is one possible explanation for the positive association. Meanwhile, we propose that the elevated efficiency of DNA repair in response to heat mutagenesis might have the by-product of increasing GC content like that happens in intracellular symbionts and marine bacterioplankton.

StartLink and StartLink+: Prediction of Gene Starts in Prokaryotic Genomes.

State-of-the-art algorithms of ab initio gene prediction for prokaryotic genomes were shown to be sufficiently accurate. A pair of algorithms would agree on predictions of gene 3′ends. Nonetheless, predictions of gene starts would not match for 15–25% of genes in a genome. This discrepancy is a serious issue that is difficult to be resolved due to the absence of sufficiently large sets of genes with experimentally verified starts. We have introduced StartLink that infers gene starts from conservation patterns revealed by multiple alignments of homologous nucleotide sequences. We also have introduced StartLink+ combining both ab initio and alignment-based methods. The ability of StartLink to predict the start of a given gene is restricted by the availability of homologs in a database. We observed that StartLink made predictions for 85% of genes per genome on average. The StartLink+ accuracy was shown to be 98–99% on the sets of genes with experimentally verified starts. In comparison with database annotations, we observed that the annotated gene starts deviated from the StartLink+ predictions for ∼5% of genes in AT-rich genomes and for 10–15% of genes in GC-rich genomes on average. The use of StartLink+ has a potential to significantly improve gene start annotation in genomic databases.

Genome-Guided Discovery of Natural Products through Multiplexed Low-Coverage Whole-Genome Sequencing of Soil Actinomycetes on Oxford Nanopore Flongle.

ABSTRACTGenome mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 kb) biosynthetic gene clusters (BGCs) with high GC (>70%) and high-repeat content, it is necessary to use long-read sequencing methods when sequencing actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-di-tryptophan, two novel lasso peptides, and three known actinomycete-associated siderophores, namely, mirubactin, heterobactin, and salinichelin.IMPORTANCE Short-read sequencing of GC-rich genomes such as those from actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30 to 40 per strain) for sequencing actinomycete strain libraries.

Genome-scale determination of 5\xb4 and 3\xb4 boundaries of RNA transcripts in Streptomyces genomes

Streptomyces species are gram-positive bacteria with GC-rich linear genomes and they serve as dominant reservoirs for producing clinically and industrially important secondary metabolites. Genome mining of Streptomyces revealed that each Streptomyces species typically encodes 20–50 secondary metabolite biosynthetic gene clusters (smBGCs), emphasizing their potential for novel compound discovery. Unfortunately, most of smBGCs are uncharacterized in terms of their products and regulation since they are silent under laboratory culture conditions. To translate the genomic potential of Streptomyces to practical applications, it is essential to understand the complex regulation of smBGC expression and to identify the underlying regulatory elements. To progress towards these goals, we applied two Next-Generation Sequencing methods, dRNA-Seq and Term-Seq, to industrially relevant Streptomyces species to reveal the 5´ and 3´ boundaries of RNA transcripts on a genome scale. This data provides a fundamental resource to aid our understanding of Streptomyces’ regulation of smBGC expression and to enhance their potential for secondary metabolite synthesis.

The Use of the Rare TTA Codon in Streptomyces Genes: Significance of the Codon Context?

Streptomycetes, Gram-positive bacteria with huge and GC-rich genomes provide an ample example of codon usage bias taken to the extreme. Particularly, in all sequenced to date streptomycete genomes leucyl codon TTA is the rarest one. It is present (usually once or twice) in 70-200 out of 7000-8000 coding sequences that make up a typical streptomycete genome. tRNALeu UAA of streptomycetes, encoded by the bldA gene, has been shown to be present in mature form only after the onset of morphological differentiation and activation of secondary metabolism. Consequently, during the early stages of cell growth, the translation of genes carrying the TTA codon can be interrupted due to the absence of tRNALeu UAA. Several reports show that mutations of TTA to synonymous codons in certain genes indeed relieve their expression from bldA dependence. However, the deletion of bldA does not always arrest the expression of TTA-containing genes. The nucleotides T/C downstream of TTA were suggested, in 2002, to favor TTA mistranslation. We tested this hypothesis using sizable datasets derived from individual Streptomyces genome and a subset of TTA+ genes for secondary metabolism known for their active expression. Our results revealed nucleotide biases downstream of NNA codons family, such as the preference for C and the avoidance of A. Yet, none of the observed biases was sufficient to claim a special case for TTA codon. Hence, the issue of codon context and TTA codon mistranslation in Streptomyces deserves further elaboration.

A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in Burkholderia.

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B.cenocepacia, B.multivorans, and B.thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne gRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia's pathogenic arsenal.

Compositional Analysis of Flatworm Genomes Shows Strong Codon Usage Biases Across All Classes

In the present work, we performed a comparative genome-wide analysis of 22 species representative of the main clades and lifestyles of the phylum Platyhelminthes. We selected a set of 700 orthologous genes conserved in all species, measuring changes in GC content, codon, and amino acid usage in orthologous positions. Values of 3rd codon position GC spanned over a wide range, allowing to discriminate two distinctive clusters within freshwater turbellarians, Cestodes and Trematodes respectively. Furthermore, a hierarchical clustering of codon usage data differs remarkably from the phylogenetic tree. Additionally, we detected a synonymous codon usage bias that was more dramatic in extreme GC-poor or GC-rich genomes, i.e., GC-poor Schistosomes preferred to use AT-rich terminated synonymous codons, while GC-rich M. lignano showed the opposite behavior. Interestingly, these biases impacted the amino acidic usage, with preferred amino acids encoded by codons following the GC content trend. These are associated with non-synonymous substitutions at orthologous positions. The detailed analysis of the synonymous and non-synonymous changes provides evidence for a two-hit mechanism where both mutation and selection forces drive the diverse coding strategies of flatworms.

Genome Sequences and Methylation Patterns of Natrinema versiforme BOL5-4 and Natrinema pallidum BOL6-1, Two Extremely Halophilic Archaea from a Bolivian Salt Mine.

Two extremely halophilic archaea, namely, Natrinema versiforme BOL5-4 and Natrinema pallidum BOL6-1, were isolated from a Bolivian salt mine and their genomes sequenced using single-molecule real-time sequencing. The GC-rich genomes of BOL5-4 and BOL6-1 were 4.6 and 3.8 Mbp, respectively, with large chromosomes and multiple megaplasmids. Genome annotation was incorporated into HaloWeb and methylation patterns incorporated into REBASE.

Estimation of AT and GC content distributions of nucleotide substitution rates in bacterial core genomes

BackgroundGenomic GC content varies both within and, substantially, between microbial genomes. While some of this variation can be explained by evolutionary divergence and environmental factors, a notable portion is not understood. To investigate further, we explore a non-linear mathematical model (gcMOD) of single-nucleotide polymorphism (SNP) GC content (sbGC, the GC content of substituted bases) as a function of core genome GC content (cgGC). We estimate the model’s parameters using Bayesian inference on empirical genetic data from the microbial core genomes of 35 bacterial species, each of which contains at least 10 representative strains. We utilize 716 bacterial genomes in total. We also explore some possible implications that result from the mathematical properties of gcMOD.ResultsWe find that the median GC → AT substitution rates (β) are almost always considerably higher than the corresponding AT → GC substitution rates (α) for all 35 core genomes. The distribution of β is also noticeably more concentrated (i.e. thinner) than the corresponding distribution of α for almost all species, excepting the bacteria with the most GC-rich genomes. We also demonstrate that at the singularity point of gcMOD (where α = β), the model is reduced to a linear equation. By analyzing the linear model, we show that due to the constraints on gcMOD, the mutation rates can have profound influence on both cgGC as well as sbGC. Moreover, by examining the mathematical properties of gcMOD’s inverse function, we find that change in cgGC, and hence in genomic GC content, can potentially occur quite rapidly.ConclusionsExamining the distributions of the GC → AT and AT → GC substitution rates for 35 bacterial species, we demonstrate that the former (β) are remarkably similar for all species examined. In addition, GC → AT substitution rate distributions were considerably more concentrated for all species, with the mode consistently peaking at higher rates than for AT → GC substitution rates.

Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome.

Determining transcriptional and translational regulatory elements in GC-rich Streptomyces genomes is essential to elucidating the complex regulatory networks that govern secondary metabolite biosynthetic gene cluster (BGC) expression. However, information about such regulatory elements has been limited for Streptomyces genomes. To address this limitation, a high-quality genome sequence of β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27 064 is completed, which contains 7163 newly annotated genes. This provides a fundamental reference genome sequence to integrate multiple genome-scale data types, including dRNA-Seq, RNA-Seq and ribosome profiling. Data integration results in the precise determination of 2659 transcription start sites which reveal transcriptional and translational regulatory elements, including −10 and −35 promoter components specific to sigma (σ) factors, and 5′-untranslated region as a determinant for translation efficiency regulation. Particularly, sequence analysis of a wide diversity of the −35 components enables us to predict potential σ-factor regulons, along with various spacer lengths between the −10 and −35 elements. At last, the primary transcriptome landscape of the β-lactam biosynthetic pathway is analyzed, suggesting temporal changes in metabolism for the synthesis of secondary metabolites driven by transcriptional regulation. This comprehensive genetic information provides a versatile genetic resource for rational engineering of secondary metabolite BGCs in Streptomyces.

Modeling of the GC content of the substituted bases in bacterial core genomes

BackgroundThe purpose of the present study was to examine the GC content of substituted bases (sbGC) in the core genomes of 35 bacterial species. Each species, or core genome, constituted genomes from at least 10 strains. We also wanted to explore whether sbGC for each strain was associated with the corresponding species’ core genome GC content (cgGC). We present a simple mathematical model that estimates sbGC from cgGC. The model assumes only that the estimated sbGC is a function of cgGC proportional to fixed AT→GC (α) and GC → AT (β) mutation rates. Non-linear regression was used to estimate parameters α and β from the empirical data described above.ResultsWe found that sbGC for each strain showed a non-linear association with the corresponding cgGC with a bias towards higher GC content for most core genomes (66.3% of the strains), assuming as a null-hypothesis that sbGC should be approximately equal to cgGC. The most GC rich core genomes (i.e. approximately %GC > 60), on the other hand, exhibited slightly less GC-biased sbGC than expected. The best fitted regression model indicates that GC → AT mutation rates β = (1.91 ± 0.13) p < 0.001 are approximately (1.91/0.79) = 2.42 times as high, on average, as AT→GC α = (− 0.79 ± 0.25) p < 0.001 mutation rates. Whether the observed sbGC GC-bias for all but the most GC-rich prokaryotic species is due to selection, compensating for the GC → AT mutation bias, and/or selective neutral processes is currently debated. Residual standard error was found to be σ = 0.076 indicating estimated errors of sbGC to be approximately within ±15.2% GC (95% confidence interval) for the strains of all species in the study.ConclusionNot only did our mathematical model give reasonable estimates of sbGC it also provides further support to previous observations that mutation rates in prokaryotes exhibit a universal GC → AT bias that appears to be remarkably consistent between taxa.

G4PromFinder: an algorithm for predicting transcription promoters in GC-rich bacterial genomes based on AT-rich elements and G-quadruplex motifs

BackgroundOver the last few decades, computational genomics has tremendously contributed to decipher biology from genome sequences and related data. Considerable effort has been devoted to the prediction of transcription promoter and terminator sites that represent the essential “punctuation marks” for DNA transcription. Computational prediction of promoters in prokaryotes is a problem whose solution is far from being determined in computational genomics. The majority of published bacterial promoter prediction tools are based on a consensus-sequences search and they were designed specifically for vegetative σ70 promoters and, therefore, not suitable for promoter prediction in bacteria encoding a lot of σ factors, like actinomycetes.ResultsIn this study we investigated the possibility to identify putative promoters in prokaryotes based on evolutionarily conserved motifs, and focused our attention on GC-rich bacteria in which promoter prediction with conventional, consensus-based algorithms is often not-exhaustive. Here, we introduce G4PromFinder, a novel algorithm that predicts putative promoters based on AT-rich elements and G-quadruplex DNA motifs. We tested its performances by using available genomic and transcriptomic data of the model microorganisms Streptomyces coelicolor A3(2) and Pseudomonas aeruginosa PA14. We compared our results with those obtained by three currently available promoter predicting algorithms: the σ70consensus-based PePPER, the σ factors consensus-based bTSSfinder, and PromPredict which is based on double-helix DNA stability. Our results demonstrated that G4PromFinder is more suitable than the three reference tools for both the genomes. In fact our algorithm achieved the higher accuracy (F1-scores 0.61 and 0.53 in the two genomes) as compared to the next best tool that is PromPredict (F1-scores 0.46 and 0.48). Consensus-based algorithms produced lower performances with the analyzed GC-rich genomes.ConclusionsOur analysis shows that G4PromFinder is a powerful tool for promoter search in GC-rich bacteria, especially for bacteria coding for a lot of σ factors, such as the model microorganism S. coelicolor A3(2). Moreover consensus-based tools and, in general, tools that are based on specific features of bacterial σ factors seem to be less performing for promoter prediction in these types of bacterial genomes.

Transcriptional regulation of a horizontally transferred gene from bacterium to chordate.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event.

The Frequency of Internal Shine-Dalgarno-like Motifs in Prokaryotes.

In prokaryotes, translation initiation typically depends on complementary binding between a G-rich Shine–Dalgarno (SD) motif in the 5′ untranslated region of mRNAs, and the 3′ tail of the 16S ribosomal RNA (the anti-SD sequence). In some cases, internal SD-like motifs in the coding region generate “programmed” ribosomal pauses that are beneficial for protein folding or accurate targeting. On the other hand, such pauses can also reduce protein production, generating purifying selection against internal SD-like motifs. This selection should be stronger in GC-rich genomes that are more likely to harbor the G-rich SD motif. However, the nature and consequences of selection acting on internal SD-like motifs within genomes and across species remains unclear. We analyzed the frequency of SD-like hexamers in the coding regions of 284 prokaryotes (277 with known anti-SD sequences and 7 without a typical SD mechanism). After accounting for GC content, we found that internal SD-like hexamers are avoided in 230 species, including three without a typical SD mechanism. The degree of avoidance was higher in GC-rich genomes, mesophiles, and N-terminal regions of genes. In contrast, 54 species either showed no signature of avoidance or were enriched in internal SD-like motifs. C-terminal gene regions were relatively enriched in SD-like hexamers, particularly for genes in operons or those followed closely by downstream genes. Together, our results suggest that the frequency of internal SD-like hexamers is governed by multiple factors including GC content and genome organization, and further empirical work is necessary to understand the evolution and functional roles of these motifs.

Mutational bias of Turnip Yellow Mosaic Virus in the context of host anti-viral gene silencing

Plant Dicer-like (DCL) enzymes exhibit a GC-preference during anti-viral post-transcriptional gene silencing (PTGS), delivering an evolutionary selection pressure resulting in plant viruses with GC-poor genomes. However, some viruses, e.g. Turnip Yellow Mosaic Virus (TYMV, genus Tymovirus) have GC-rich genomes, raising the question as to whether or not DCL derived selection pressure affects these viruses. In this study we analyzed the virus-derived small interfering RNAs from TYMV-infected leaves of Brassica juncea showed that the TYMV population accumulated a mutational bias with AU replacing GC (GC–AU), demonstrating PTGS pressure. Interestingly, at the highly polymorphic sites the GC–AU bias was no longer observed. This suggests the presence of an unknown mechanism preventing mutational drift of the viral population and maintaining viral genome stability, despite the host PTGS pressure.