Category Archives: mobile DNA

The Transposon/piRNA/Chromatin Nexus

Close observation of chromatin states at piRNA-silenced genomic loci demonstrates the power of transposons to change native gene expression.

As reviewed in an earlier post, the Drosophila Piwi/piRNA transposon silencing pathway can be divided into two facets; a complex pathway operating in the germline centred on the Piwi-family argonautes Aubergine and AGO3 localised in peri-nuclear nuage, and a linear pathway operational in the somatic follicle cells. In this linear pathway, piRNAs derived from uni-directional piRNA clusters such as flamenco target Piwi to mediate silencing of a limited subset of retrotransposons. Unlike Aub and AGO3, Piwi is localised to the nucleus, leading to speculation that rather than silencing transposons post-transcriptionally by ‘slicing’ their transcripts, it may act at the transcriptional level. There are many precedents in other organisms for argonautes mediating transcriptional silencing via interactions with chromatin modification and DNA methylation pathways. However, whether one of these silencing modes is employed by Drosophila Piwi was unresolved. A new paper from the lab of Julius Brennecke, generally analysing the linear piRNA pathway active in a cell line derived from the somatic follicle cells surrounding the oocyte (OSC cells) includes important findings for a number of aspects of Piwi-mediated transposon silencing leading to insights on the wider genomic ecology of transposon insertions.

In the first section of the paper, Sienski et al. demonstrate that Maelstrom (Mael), a protein containing putative RNA and DNA binding domains, expressed in both cytoplasm and nuclei and previously implicated in a number of Piwi-pathway effects, acts downstream of Piwi to effect TE silencing. Silencing requires the nuclear localisation of both Piwi and Mael. Further, mutation of the residues necessary for ‘slicer’ activity in Piwi did not de-repress TEs, suggesting a different mechanism for Piwi-mediated silencing.

Sienski et al. go on to marshal three different high-throughput techniques to show that Piwi mediates gene silencing at the transcriptional level. Knocking down (KD) the expression of Piwi pathway factors (piwi, mael) in OSC cells they determined the set of repressed transposable elements (TEs) by comparing RNA levels (RNA-seq). Changes in the steady-state RNA levels were highly correlated with transcription rate as monitored by RNA polymerase II occupancy (ChIP-seq) and levels of nascent RNAs (GRO-seq). Judging by how closely correlated derepression of TEs was to transcription rate, it seems unlikely that the linear piRNA pathway active in follicle cells acts post-transcriptionally at all.

Reasoning that Piwi-mediated transcriptional gene silencing may involve chromatin modification, Sienski et al. profiled the distribution of the repressive histone mark H3K9me3 in OSCs after piwi or mael knockdown. H3K9me3 levels at transposable elements known to be repressed by the piRNA pathway were significantly reduced in the absence of Piwi (and to a lesser extent Mael). This data was from across the genome irrespective of whether the TE was inserted into heterochromatic or euchromatic regions. To negate general effects associated with heterochromatin, the authors looked more closely at TE insertions within euchromatic regions.

Approximate sketch of the patterns of RNA pol II occupancy (ie Transcription), and H3K9me3 at the mdg1 locus after piwi or mael knockdown and normally in control.

Approximate sketch of the patterns of RNA pol II occupancy (ie Transcription), and H3K9me3 at the mdg1 locus after piwi or mael knockdown and normally in control.

At a specific euchromatic insertion of the retrotransposon mdg1, they observed that upon either piwi KD or mael KD, transcription downstream of the insertion strongly increased. However, although this transcriptional bleeding into the surrounding area was similar upon TE derepression due to either piwi KD or mael KD, the pattern of H3K9me3 was very different. Normally this mdg1 insertion displays H3K9me3 in the surrounding 12kb, peaking at the insertion site. This was strongly reduced in piwi KD cells, but in mael KD, H3K9me3 was moderately reduced at the insertion site but had actually spread further downstream (see figure). Similar patterns were observed at nearly all euchromatic mdg1 insertions, as well as other TEs known to be targeted by the linear piRNA pathway active in OSC cells.

Strikingly, most euchromatic H3K9me3 peaks were sensitive to piwi knockdown, whilst 88% of H3K9me3 peaks were found within 5Kb of TE insertions. Piwi-mediated transposon silencing therefore seems to be the main trigger for H3K9 trimethylation in euchromatin.

This transposon silencing mechanism appears to have a major impact on native genes upon TE insertion in their vicinity. An insertion of the retrotransposon gypsy into the first intron of the expanded (ex) gene serves as paradigm for these effects. In OSC cells, the gypsy insertion triggered H3K9me3 spreading into the surrounding 10-12Kb. In control cells RNA polymerase II occupancy was observable at the ex transcription start site (TSS) but weak. Upon piwi or mael knockdown, transcription from the ex TSS was massively increased. As in the earlier mdg1 example, H3K9me3 levels were greatly reduced upon piwi KD but not in mael KD cells. Sienski et al. observed similar effects on the transcription of 28 more genes with nearby TE insertions in OSC cells.

This data has a number of ramifications speaking of a complex interplay between transcription, the establishment and maintenance of repressive chromatin states and the Piwi pathway. Firstly, H3K9me3 considered a transcriptionally repressive histone mark is compatible with transcription. In fact, based on it’s pattern in mael KD cells, the authors propose that downstream transcriptional bleeding leads to the spread of H3K9me3. Further, although H3K9me3 has an integral role in Piwi-mediated silencing, it is not the final silencing mark. H3K9 trimethylation is downstream of Piwi action, but is either upstream or acts in parallel to Mael, which mediates an unknown silencing step crucial to Piwi transcriptional gene silencing.

Importantly, this paper has demonstrated the impact that TE insertion and subsequent piRNA pathway transcriptional repression can have on native gene expression. There are two different modes in which the inactivation of Piwi-mediated TE silencing can lead to the transcriptional activation of these loci. Firstly, the spreading of repressive chromatin marks at transposons can suppress RNA polymerase II access to the genes promoter. Alleviation of TE repression hence leads to (re-)activation of gene expression. Conversely, as TEs (especially the long terminal repeats of some retrotransposons) can serve as promoters, the loss of their repressed chromatin state upon piRNA pathway loss, can activate transcription of downstream regions. Although both these modes lead to transcriptional activation after Piwi pathway loss, they demonstrate that transposon insertion can either activate or repress transcription within relatively extensive genomic surroundings. This underscores the scope for transposons to act as regulatory elements, or to produce new chimerical transcripts and hence potential new genes.

These experiments were mainly performed in one cell type that only partially reflects the activity of what is already a subset of piwi/piRNA action during Drosophila oogenesis.  Piwi and Mael are also active in the nurse cells and oocyte, and this paper suggests that they have similar roles within the context of the expanded piRNA pathways active in the germline. It will be interesting to integrate this nuclear-localised transcriptional-silencing aspect of piRNA silencing into the context of ping-pong amplification and bi-directional piRNA cluster transcripts. Further, do these Piwi-mediated chromatin effects in the germline impact on the transcriptional status of TEs and genes later in somatic development? And if not, do other systems have equivalent activity?

This paper underlines again the importance of the arms race between mobile genetic elements and genomic immune systems such as the piRNA pathway on the wider genomic regulatory context. This contest is being observed to have shaped so many aspects of genome organisation throughout evolution that it sometimes becomes hard to differentiate parasitism from regulation. It is clear however, that to understand the evolutionary impact of mobile elements we must also understand the import of the various epigenetic mechanisms controlling their spread. The minutiae of these mechanisms with regard to their targets, plasticity, adaptability, heritability – often different from organism to organism – has major evolutionary significance. Evolution works differently depending on these mechanisms.

Sienski, G., Dönertas, D., & Brennecke, J. (2012). Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression Cell, 151 (5), 964-980 DOI: 10.1016/j.cell.2012.10.040

A chimeric fusion of RNA and DNA viruses.

The discovery of a new family of viruses leads to speculations on possible modes recombination between RNA and DNA viruses.

The virosphere can be divided into three major classes; viruses with DNA genomes, retroviruses that reverse-transcribe their RNA genome into DNA during their lifecycle, and RNA-only viruses that don’t require DNA intermediates to replicate. In fact, viruses use all sorts of different permutations of genetic material; double-stranded RNA, single-stranded RNA (either negative or positive strand), dsDNA and ssDNA. Viruses evolve notoriously quickly and lateral gene transfer between them is rampant. However, gene transfer has most commonly occurred between closely related viruses or between those with similar replication mechanisms. A recent paper has reported the discovery of a new family of viruses that appear to have arisen via lateral gene transfer between a (non-retroid) +ve single-stranded RNA virus and a ssDNA virus.

Diemer and Stedman discovered the new virus whilst investigating viral diversity in a geothermal lake in California. Boiling Springs Lake is an acidic, high temperature lake with a purely microbial ecosystem composed of archaea, bacteria, and some single cell eukaryotes. Using a metagenomics approach (ie. large-scale sequencing  of environmental DNA from a virus particle sized fraction), they discovered the strange juxtaposition of a capsid protein (CP) gene related to those from the ssRNA plant-infecting Tombusviridae, with a rolling-circle replicase (Rep) gene most similar to those from the circular ssDNA-containing Circoviridae. Using primers designed against CP they confirmed the genome sequence of this putative virus, finding that it consisted of a single-stranded circular DNA containing 4 ORFs. ORFs 3 and 4 are of unknown function and unrelated to known genes. The virus contains a stem loop structure upstream of the Rep gene similar to those that serve as replication origins in other Circoviruses. Thanks to the chimeric origin of the Rep and CP genes, the authors termed it RNA-DNA hybrid virus (RDHV). This term is slightly open to misinterpretation as it could suggest that both molecules are actually encoding its’ genome, but to be clear this is a circular ssDNA virus whose capsid protein is derived from ssRNA viruses.

Organisation of RDHV. Note that ORFs 3 and 4 are not equivalent to those of Tombusviruses, and RDHV is twice the size of other Circoviruses.

Scanning databases of environmental sequence, the researchers found three other instances of homologous CP and Rep sequences arranged in the same configuration, two from global ocean surveys and one from the Sargasso Sea. This shows that RDHV defines a new family of viruses that are common in marine environments and could be more widespread. As CP and Rep are still highly similar to their sibling genes, it appears that the LGT event underlying the evolution of this new family occurred quite recently.

How did recombination occur between a non-retrovirus ssRNA virus and a DNA virus? A number of genes derived from non-retroid RNA viruses have been found in eukaryotic genomes, so perhaps this type of exchange is not as strange or rare as it may seem. The most likely scenario involves the RNA gene being converted into DNA by reverse transcription, followed by DNA-DNA recombination. As reverse transcriptase is not encoded by either virus, it could have been supplied in trans by retrotransposons, group II introns, or retroviruses within a common host cell. This brings us to the problem of metagenomic studies; they have amazing power to identify novel viruses and organisms, but yield very little information on the biology of what is found. In this case of RDHV and it’s family we do not know what their hosts are, don’t know the morphology of the viruses, and don’t know about the functions of half it’s 4 gene genome. I’m not sure how quickly these questions will be answered. Nevertheless, this study shows that amazing diversity is still out there being found, and yields insight into mechanisms underlying virus evolution – possibly in the deep past as well as more recently.

Diemer, G., & Stedman, K. (2012). A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses Biology Direct, 7 (1) DOI: 10.1186/1745-6150-7-13

Beating a Toxin-Antitoxin System; Evading Suicide

Bacteria have evolved many different systems to evade viral predation. One strategy, abortive infection (Abi), involves altruistic suicide. Mediated by a toxin-antitoxin (TA) system, the suicide of the infected cell protects the clonal bacterial population by preventing the spread of replicated bacteriophage. A new paper in Plos Genetics has discovered a molecular mimicry-based strategy that allows phage to escape abortive infection.

Toxin-antitoxin systems are widespread prokaryotic genetic elements found on both plasmids and bacterial chromosomes. Encoding a relatively long-lived toxin and a more labile antitoxin expressed from a single bi-cistronic operon, they were originally characterised as ‘addiction modules’. In the event that a plasmid expressing a TA system fails to be inherited by a daughter cell, the absence of antitoxin allows the persisting toxin to kill the cell – post-segregational killing. These attributes as ‘selfish elements’ made it slightly surprising that so many TA systems have been found encoded on bacterial chromosomes themselves. I’ve previously written about an example of one such TA system’s activity in mediating a stress response in E. coli, and they’ve also been implicated in the formation of antibiotic resisting ‘persister’ cells.

TA systems have been classified into three different classes defined by the level of the molecular interaction between their two components. In type I systems, translation of the toxin is prevented by an antisense RNA antitoxin binding to its’ transcript, whilst in type II systems both partners are proteins. Most recently, type III TA systems have been characterised in which the toxin is neutralised by binding of an RNA antitoxin. Examples of all three varieties have been found to protect bacteria from phage infection via abortive infection; phage replication disrupts the normal cellular transcriptional program, interrupting antitoxin production and hence leading to cell death.

The first type III TA system to be characterised, ToxIN, was found on plasmids in the phytopathogen, Pectobacterium atrosepticum (Pba), and shown to inhibit the propagation of multiple different bacteriophage. ToxN is an endoribonuclease, whilst the antitoxin ToxI, is a 36nt RNA structured as a ‘pseudoknot’. The partners combine into a hetero-hexameric structure composed of 3 ToxN molecules and 3 ToxI pseudoknots.

Blower et al. have discovered that phage can evade the Abi system by producing molecular mimics of the ToxI RNA. The lytic bacteriophage ΦTE, normally fails to infect Pba carrying a toxIN-containing plasmid. At low frequency however, new phage strains emerge capable of evading the Abi system. Upon sequencing the genomes of these ‘escape strains’, the researchers discovered that they all contained sequence expansions at one specific locus. The toxI locus contains 5.5 repeats of the 36nt RNA pseudoknot-encoding sequence. The ‘escape locus’ from the phage normally encoded 1.5 repeats of a pseudo-ToxI sequence. In all the escape strains this repeat had been expanded so that it contained either 4.5 or 5.5 repeats. These expansions had probably arisen due to strand-slippage during phage replication. In one escape strain homologous recombination had occurred between the phage pseudo-ToxI and the endogenous toxI; the phage had effectively hijacked a normal antitoxin- encoding gene.

The 1.5 repeat pseudo-ToxI could not inhibit Abi (as the sequence was out of phase it did not actually encode a functional psudoknot). However, the repeat expansions had allowed the phage to make an antitoxin mimic that protected them from the TA system and hence Abi.

ΦTE is capable of generalised transduction – the ability to package and transfer chromosomal and plasmid DNA from its’ host and transfer it during infection. Blower et al. showed that one of the ΦTE escape strains is able to transduce the plasmid encoded ToxIN – a case of a bacteriophage horizontally transferring an anti-phage defence mechanism. This brings into focus the complex evolutionary dynamics operating between the three different genetic entities being studied; the bacterial cell, the plasmid encoding the TA system, and the bacteriophage evading it and potentially propagating it. From the selfish viewpoint of the TA module what’s best, preventing the spread of the phage or being disseminated by it? These speculations aren’t about to be easily answered, however, it is an interesting way to analyse further examples of similar systems.

Blower, T., Evans, T., Przybilski, R., Fineran, P., & Salmond, G. (2012). Viral Evasion of a Bacterial Suicide System by RNA–Based Molecular Mimicry Enables Infectious Altruism PLoS Genetics, 8 (10) DOI: 10.1371/journal.pgen.1003023

Chromosomal Flip-Flop

A study describes how phenotypic switching in Staphylococcus aureus is caused by a reversible large-scale genomic inversion.

Clonal bacterial populations often display various phenotypes. This diversity is most obviously identifiable as colony variation. Many different bacterial genera display ‘small colony variants’ (SCVs), the occurrence of which is generally attributable to deficiencies in various metabolic pathways.

Cui et al have characterised an STV strain of Staphylococcus aureus which reverts to a normal colony variant (NCV) at a frequency of 1-3 in a 1000. Interestingly the NCV progeny revert back to SCV in 1-10% of cases. This frequent bi-directional reversion was stably maintained at these ratios; homogeneous colony populations could never be isolated.

The small colony variant displayed some important phenotypic differences to the NCV. As well as slow growth and less pigmentation, it was susceptible to β-lactam antibiotics whilst the NCV was not. The authors identified over a hundred genes were differentially expressed between the two variants, and that their susceptibilities to many chemicals were different.

Diagram showing reversible genomic inversion forms caused by homologous recombination at inverted repeat regions (break points. BPs)

When Cui et al. sequenced the genomes of the two variants, they discovered that nearly half of the genome (1.26 Mb of 2.87 Mb) was differenttly aligned. This ‘X-shaped’ chromosome inversion occurred between two oppositely oriented pathogenicity islands, symmetrically opposite each other on the chromosome with respect to the replication axis. Each pathogenicity island contained two copies of an identical 3,638bp long sequence. It appears that homologous recombination can occur at these sites and generate the genomic inversion. This is in agreement with experiments in which the authors altered levels of the key recombination regulatory protein RecA; finding that they could increase the rate of reversion with higher recA expression.

The chromosomal flip-flopping therefore regulates the maintenance of two different S. aureus phenotypic variants. The two forms have different advantages and disadvantages. The original SCV strain isolated from a patient suffering persistent reinfection of a surgical site. It appears that the SCV may facilitate immune evasion, whilst the NCV has higher antibiotic resistance. Maintaining a balance between the two variants within the S. aureus population therefore functions as an evolutionarily useful bet-hedging strategy.

This type of flexible genome organisation serving as a self-organising regulatory mechanism for the maintenance of a bi-stable heterogeneous cell population may well be a more wide-spread bacterial evolutionary strategy.

Cui L, Neoh HM, Iwamoto A, & Hiramatsu K (2012). Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109 (25) PMID: 22645353

Expanding the Conjugative Realm

A recent paper demonstrates that a broader range of plasmids can be transferred by conjugation than previously thought.

Integrative and conjugative elements (ICEs, aka conjugative transposons) are a variety of bacterial mobile element generally found integrated into the host genome, but capable of excision and transfer to other cells via conjugation. I’ve previously written a short review of some of their key features, which may serve as a helpful introduction to this post. As well as transferring themselves between cells, ICEs and conjugative plasmids can mobilise other DNA elements, such as integrative mobilizable elements (IMEs) and mobilizable plasmids, that aren’t independently capable of self-transfer.

The conjugative transfer of any of these elements generally rests upon the generation of a single-stranded DNA molecule from the circular dsDNA mobile genetic element. The ssDNA is formed by a mobilising relaxase (Mob) nicking the circular DNA at an origin of transfer (oriT) sequence, followed by the unwinding of the strands by a host-derived helicase. Mob covalently binds the nicked end forming the ‘relaxosome’. A coupling protein is responsible for targeting the relaxosome to the conjugative apparatus (a type 4 secretion system, T4SS).

Plasmids that are incapable of self-transfer, but can be mobilised in trans by conjugative elements, generally encode their own mobilising relaxase and a cognate oriT site. These functions are separate from their replication system, which does however consist of similar components; a replication relaxase (Rep) which nicks and binds to an origin of replication (ori).

Lee et al. have discovered that three plasmids, which had been classified as non-mobilisable due to their lack of Mob/oriT functions, can in fact be transferred between Bacillus subtilis cells by the conjugation system of an ICE, ICEBs1. The three plasmids’ (pC194, pBS42, and pHP13) transfer required the conjugation machinery and coupling enzyme (ConQ) of ICEBs1, but was not dependent on it’s relaxase (NicK). Transfer could occur concomitantly with that of ICEBs1 or in it’s absence; showing that transfer did not act in cis due to integration of the plasmid into ICEBs1.

The authors found that, in the case of pBS42, it’s replicative relaxase was necessary for mobilisation. They therefore think it likely that in all three cases the Rep/ori system is also capable of mediating mobilisation functions. This blurring of the distinctions between Rep/ori and Mob/oriT systems has important ramifications. It opens up the possibility that many rolling-circle replicating plasmids that had been classed as non-mobilisable can in fact be transferred between cells via conjugation.

From an evolutionary perspective, these findings are important for understanding the persistence of plasmids in bacterial populations. Non-mobilisable plasmids would only be maintained in the population because benefits conferred on their hosts. If however many more ‘non-mobilisable’ plasmids can be disseminated by horizontal transfer, their persistence can be better explained. This study therefore expands the importance of conjugation in understanding bacterial evolution. Future studies will need to analyse the extent of interactions between coupling proteins and replication relaxases to better gauge the trans-mobilisation of genetic elements, and re-classify the mobility status of many plasmids.

Lee CA, Thomas J, & Grossman AD (2012). The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions. Journal of bacteriology, 194 (12), 3165-72 PMID: 22505685

Trans-Extremophile Para-Sex

Cells of Haloferax volcanii showing their cup-like morphology

More than twenty years ago, an extra-ordinary mode of para-sexual genetic exchange was found to occur between cells of the halophilic archaeon Haloferax volcanii, an extremophile isolated from the Dead Sea. This process involves first, cell fusion producing heterodiploid cells. In this state, parental chromosomes can recombine, hence producing novel hybrid daughter cells after cell separation.

Haloferax mediterranei

Naor et al. have now shown that this mechanism of genetic exchange can also occur between cells of H. volcanii, and those of a related species – Haloferax mediterranei. H. mediterranei was originally isolated from a saltern near Alicante in Spain, and shares on average 86.6% sequence identity in protein-coding genes with H. volcanii. Assaying for chromosomal markers, it was found that interspecies cell fusion occurs at a frequency of 4.2 x 10-5, less than one order of magnitude less frequently than between H. volcanii cells (1.0 x 10-4). Of intraspecies fusions, 62% resulted in genetic recombination, whereas only 8% of interspecies fusions did. Although these figures sound quite small, in comparison to modes of genetic exchange between bacterial species, this inter-species genetic interchange occurred  a few orders of magnitude more efficiently.

Analysing a number of hybrid genomes, Naor et al. found that recombinant fragments ranged in size between 310 kb and 530 kb – huge genomic portions, with respect to most known bacterial horizontal genetic transfer mechanisms. Recombination occurs at areas higher sequence identity, such as transfer RNA genes. As these hybrids were isolated by selection for a specific genetic marker, it is quite conceivable, that higher proportions of the genomes can be transferred by this process.

This form of genetic exchange has important implications for archaeal speciation and evolution. High rates of recombination can act as a homogenising force – unlinking alleles faster than genes can diversify. The authors therefore suggest that geographical isolation may be the primary force in archaeal speciation. Further experiments testing the efficiency of this parasexual genetic exchange mechanism between other Haloferax species, and more information on halophilic archaeal ecology will help to clarify these issues. It will also be interesting to examine more hybrid genomes derived from other selection experiments to find out just how extensive interspecies recombination can be and whether there are any directional biases in transfer.

Naor A, Lapierre P, Mevarech M, Papke RT, & Gophna U (2012). Low species barriers in halophilic archaea and the formation of recombinant hybrids. Current biology : CB, 22 (15), 1444-8 PMID: 22748314

The Birth of Introns

Eukaryotic genes are composed of exons and introns. Introns are non-coding sequences that separate the coding exons, and are spliced out of the pre-messenger RNA after transcription. This modular structure of eukaryotic genes allows alternative splicing, by which single genes can encode multiple isoforms of proteins, hence widening the diversity of the proteome. Introns also have important roles in genetic regulation; for instance as sites of enhancers, and by encoding microRNAs.

Intron position is often conserved between orthologous eukaryotic genes showing that spliceosomal introns originated early in eukaryotic evolution. However, it has been difficult to explain the mechanisms of intron loss, and especially, gain that have maintained a high number of introns in present day eukaryotic genomes. Current models suggest that introns should be being lost faster than they are gained. However, studies in organisms such as the urochordate, Oikopleura dioca, and the green alga, Micromonas pusilla, have shown extensive recent intron gains. Interestingly, the study of the Micromonas genome discovered a form of intronic repeat sequence that ‘extended nearly to donor and acceptor sites, and lacked known TE (transposable element) characteristics’. These sequences were termed ‘Introner elements’. A new study, forthcoming in Current Biology, has discovered and characterised something similar in various fungal clades.

Burgt et al. found numerous introns with near-identical sequences in the Dothidiomycete fungus Cladosporium fulvum. They then widened their analysis to search for similar introns in the ‘intronomes’ of 23 other species of fungi, and found large sets of near-identical introns in 6 different species. Phylogenetic analyses of these ‘introner-like elements’ (ILEs) showed that they could be grouped into related clusters, and that in turn the clusters were related to each other, indicating that all the ILE clusters were derived from a single ancestral element.

Analysis of the molecular structure of the Introner-like elements showed that they contained all the distinguishing features of normal spliceosomal introns, such as splice acceptor and donor sites, and branch point sequences. ILEs were longer than normal introns, and were found to fold into more stable secondary structures. Burgt et al. suggest that these predicted stable secondary structures are likely to have important functions, as they observed compensatory mutations that conserve secondary structure between related ILEs.

Analysing intron gain in the 6 species of fungi in which they found ILEs, Burgt et al find that ILEs account for the majority of recent gains. In closely related sister species that diverged within the last 22,000 years ILEs account for 90% of intron gains, but this figure rapidly drops off for older divergences. This leads Burgt et al. to consider that most intron gains are due to ILE multiplication, with rapid degeneration meaning that ILE identification becomes progressively more difficult.

Introner-like elements therefore appear to be mobile elements that can in some way transpose to new sites leading to intron gain. Just what mechanism is employed in this process is far from clear. Many different mechanisms for intron gain have been proposed but as yet there is little experimental evidence demonstrating that they occur in vivo. These include Intron transposition, in which an intron transposes to a new position in a transcript, which is then reverse transcribed and recombined into the original gene; Transposon insertion in which a transposon becomes a spliceable intron; Intronisation in which exons are converted into intron by accumulated mutation; and other ideas based on genetic duplications and errors during repair processes. Burgt et al think that the most likely mechanism for ILEs is a process by which introns are reverse spliced directly into the genome and then reverse transcribed. It will be interesting to see whether ILE transposition can be observed in vivo and figure out just what mechanism of intron generation is employed.

Interestingly, introner-like elements differ from the introner elements found in Micromonas in important ways. Introner elements were found within introns rather than being the whole intron, and lacked the interesting secondary structures observed in ILEs. Along with the author’s inability to find ILEs in other clades, this suggests that ILEs may not be a very widespread mechanism of intron multiplication. However Burgt et al. disagree, and reckon that ILEs could potentially be an ancestral mechanism for intron gain.

van der Burgt, A., Severing, E., de Wit, P., & Collemare, J. (2012). Birth of New Spliceosomal Introns in Fungi by Multiplication of Introner-like Elements Current Biology DOI: 10.1016/j.cub.2012.05.011

On ICE: Integrative and Conjugative Elements.

Integrative and conjugative elements are bacterial mobile genetic elements that primarily reside in the host cell’s chromosome, yet have the ability to be transferred between cells by conjugation. ICEs can be considered as mosaic elements, combining features from other mobile elements: the integrative ability of bacteriophage or transposons, and the transfer mechanisms of conjugative plasmids. This mosaicism is reflected in modular structures: genes encoding the core functions of integration/excision, conjugation and regulation are generally found clustered together. As well as these core functions, ICEs often carry accessory genes that can bestow adaptive phenotypes on their hosts. Gene cassettes encoding antibiotic resistance, nitrogen fixation, virulence factors and various other functions have all been documented in ICEs. They are therefore important vectors for the horizontal dissemination of genetic information, facilitating rapid bacterial evolution.

Chromosomal integration and excision of ICEs is mediated by integrase (Int) enzymes. Most commonly integrases are tyrosine recombinases related to the well studied phage λ Int. They mediate site-specific recombination events between identical or near-identical sequences in the host and ICE genomes (termed attB and attP respectively). These integration events normally occur into tRNA genes. No definitive reason for this association of tyrosine recombinase mediated integration with tRNA genes is known, however tRNA genes evolve more slowly than protein coding genes, potentially broadening the possible host range. Other ICEs encode transposase family tyrosine recombinase Ints that have broader target sequence preferences. Members of the DDE transposase and serine recombinase families also serve as integrases in some ICEs. Before ICE transfer occurs, the element is excised and circularised. Excision of ICEs also requires Int activity, however the process is biased towards excision by ‘recombination directionality factors’ (RDFs). If chromosomal replication or cell devision occurs whilst the ICE is in the excised chromosomal state, the element could be lost from the cell. To prevent this ICEs (like plasmids) often encode addiction modules (toxin-antitoxin systems) that kill cells not inheriting the ICE.

Conjugative transfer occurs via the formation of a multiprotein apparatus that connects the donor and recipient cells: a type IV secretion system (T4SS). This consists of a membrane spanning secretion channel and often an extracellular pilus. The extrachromosomal ICE DNA is first nicked at the origin of transfer (oriT) by a relaxase (MOB) enzyme. Rolling circle replication is then initiated. MOB remains bound to the displaced single-stranded DNA and this nucleoprotein complex is targeted to the T4SS by a coupling protein (T4CP). Rather than using a T4SS, some ICEs are transferred between cells using FtsK-like DNA translocase pumps (in this case dsDNA is transferred). After transfer, the ssDNA ICE is replicated into dsDNA and integrated into the recipient cell’s chromosome. The ICE in the donor cell is also converted into dsDNA and re-integrated into the genome.

ICE transmission is under the control of networks responsive to environmental stimuli. For instance, transfer of the SXT-R391 family of ICEs is controlled by SelR, a homologue of the λ repressor CI. Regulation occurs by a similar mechanism as that controlling the λ switch from lysogeny to lysis. As with CI, SetR repression can be relieved by the action of RecA, the main effector of the ‘SOS’ response to DNA damage. Other ICEs transmission have been shown to be under the control of quorum sensing networks.

A recent bioinformatic study of ICE prevalence and diversity identified ICEs by finding clustered conjugative apparatus modules (Guglielmini et al). If these were found on chromosomal locations they were defined as belonging to ICEs, whilst those on extra-chromosomal elements were considered conjugative plasmids. No reference was made to the presence of integrases. Within this definition ICEs were more common than conjugative plasmids: 18% of sequenced prokaryotic genomes contained at least one ICE as opposed to 12% possessing conjugative plasmids. ICEs are generally defined as not being capable of autonomous extra-chromosomal replication and maintenance. This is opposed to conjugative plasmids that include replication origins and systems. However, this definition is not watertight, as there appear to be various exceptions. Likewise, conjugative plasmids can be integrated chromosomally, either by homologous recombination at repeat sequences, or by site-specific recombination events. These elements therefore exist on a spectrum. Phylogenetic analysis of VirB4 genes (an ATPase component of T4SS) shows that ICEs and conjugative plasmids do not segregate as monophyletic clades. Instead they are intermingled throughout the tree, suggesting that conjugative plasmids often become ICEs and vice versa. Guglielmini et al. therefore consider them as two sides of the same coin. If selection pressures are strong enough though, ICEs can be stabilised as chromosomal structures for long periods of time.

A striking example of the potency and evolutionary importance of ICEs is found in the genomes of the obligate intracellular bacterial family Rickettsiales. One third of the genome of Orientia tsutsugamushi (the mite borne causative agent of scrub typhus) is made up of degenerate copies of an ICE named RAGE (Rickettsiales amplified genetic element). Multiple invasions of RAGE have also configured the genome of a Rickettsial endosymbiont of a deer tick (REIS). In this case RAGEs have acted as hotspots for recombination and the insertion of other mobile elements, leading to the insertion of clusters of novel horizontally transferred genes (a process termed piggybacking). These two Rickettsiales species have especially large genomes for obligate intracellular bacteria, but it seems likely that RAGE has been important in the evolution of this entire clade.

See also: Expanding the Conjugative Realm

Wozniak, R., & Waldor, M. (2010). Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow Nature Reviews Microbiology, 8 (8), 552-563 DOI: 10.1038/nrmicro2382

Guglielmini, J., Quintais, L., Garcillán-Barcia, M., de la Cruz, F., & Rocha, E. (2011). The Repertoire of ICE in Prokaryotes Underscores the Unity, Diversity, and Ubiquity of Conjugation PLoS Genetics, 7 (8) DOI: 10.1371/journal.pgen.1002222

Nakayama, K., Yamashita, A., Kurokawa, K., Morimoto, T., Ogawa, M., Fukuhara, M., Urakami, H., Ohnishi, M., Uchiyama, I., Ogura, Y., Ooka, T., Oshima, K., Tamura, A., Hattori, M., & Hayashi, T. (2008). The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution DNA Research, 15 (4), 185-199 DOI: 10.1093/dnares/dsn011

Gillespie, J., Joardar, V., Williams, K., Driscoll, T., Hostetler, J., Nordberg, E., Shukla, M., Walenz, B., Hill, C., Nene, V., Azad, A., Sobral, B., & Caler, E. (2011). A Rickettsia Genome Overrun by Mobile Genetic Elements Provides Insight into the Acquisition of Genes Characteristic of an Obligate Intracellular Lifestyle Journal of Bacteriology, 194 (2), 376-394 DOI: 10.1128/JB.06244-11

Transposons and plasmids combine for bacterial chromosomal transfer.

A mechanism that facilitates the horizontal transfer of large segments of chromosomal DNA has been discovered in natural isolates of the pathogenic bacterium Yersinia pseudotuberculosis.

Although horizontal gene transfer (HGT) is recognised as a major force in bacterial evolution, and many mobile genetic elements underlying genomic plasticity have been characterised, the mechanisms by which genetic exchange of chromosomal DNA occurs in natural populations have remained largely hypothetical.

In experiments analysing the mobility of a pathogenicity island (HPI) in Yersinia pseudotuberculosis, Lesic and Carniel (2005) discovered that HPI could be transferred between natural isolates in a process that occurred optimally at 4˚C. However, this process didn’t require integration/excision machinery encoded in the HPI, and also transferred adjacent sequences (>46kb). To test whether this process was specific to the HPI region, Lesic et al inserted various antibiotic resistance markers equidistantly within the Y. pseudotuberculosis chromosome. When this strain was co-incubated with a naïve recipient strain, transfer of these markers was observed in ‘transconjugants’. None of the transconjugants acquired more than one of the antibiotic resistance loci, showing that the transferred chromosomal fragments were less that 1.5Mb in size. The transfer mechanism was also capable of mediating the transmission of a non-mobilisable plasmid (pUC4K). Together these results showed the existence of a mechanism for generalised DNA transfer that functioned at low temperature (termed GDT4).

Lesic et al. found that the GDT4 performing strain contained a very high molecular weight plasmid (≥100Mb). When this plasmid was removed (‘cured’) from the strain, GDT4 was abolished. They therefore termed this plasmid pGDT4. pGDT4 was transferred during transconjugation experiments and was able to confer the ability to transfer chromosomal DNA.

Electron micrograph of aggregating Y. pseudotuberculosis. White arrows point to bridge-like structures.

Sequencing of pGDT4 showed that it contained genes involved in conjugation (the process by which a pilus acts as a conduit for transfer of DNA between cells). When part of this conjugative machinery was deleted GDT4 ability was lost. Interestingly though, pili could not be observed by electron microscopy, and strong shaking of cultures (predicted to disrupt pilus-mediated interactions) had no effect. Instead, the bacteria were seen to tightly aggregate, and seemed to be connected by ‘bridges’.

Organisation of pUC4K plasmids from transconjugants, showing novel IS insertions and IS mediated novel organisations.

pGDT4 also contains a number of Insertion Sequences (IS, short bacterial transposable elements). None of these IS were present on the bacterial chromosome or pUC4K (the non-mobilisable plasmid). After GDT4 transfer of pUC4K, it was observed the size of pUC4K was often altered. Of 10 transconjugant pUC4Ks measured, 3 were the original size, but (pGDT4 derived) IS insertions had lengthened the others. In 5 cases a single copy of ISYps1 had been acquired by pUC4K, whilst in 1 case a large section of pGDT4 had been inserted between two ISYps3 elements.

ISYps1 and ISYps3 (members of the IS6 and Tn3 families respectively) transpose by a mechanism termed replicative transposition. An intermediate stage during this mode of transposition is the formation of a ‘co-integrate’ in which the donor and target replicons (in this case pGDT4 and pUC4K) are fused by 2 copies of the transposon. The cointegrate is then resolved by homologous recombination, leaving a new copy of the transposon on the target and the original still in place on the donor.

Model describing ISYps1 transposition mediated mobilisation of pUC4K via cointegrate formation.

These observations suggested that GDT4 occurs by cointegrates of pGDT4 and other replicons (bacterial chromosome or other plasmids) being formed during IS transposition and then being transferred to recipient cells by conjugation. In further experiments, Lesic et al. confirmed that replicative transposition of ISYps1 was capable of  driving cointegrate formation between different plasmids in E.coli, that could also be transferred by conjugation.

This mode of chromosomal transfer is very similar to the ‘Hfr‘ system characterised in E. coli. In Hfr strains, transfer of chromosomal DNA occurs by conjugation because the conjugative plasmid ‘F’ becomes integrated into the chromosome. Integration of F occurs by homologous recombination at IS sequences shared between the plasmid and the bacterial chromosome. The difference in GDT4 is that no sequence identity between the two replicons is necessary. ISYps1 transposition does not seem to target any particular sequence. This makes this mode of chromosomal conjugative transfer less constrained than Hfr and potentially more powerful. It is notable that plasmids often carry a large number of IS. For instance, a plasmid in Shigella carries 93 copies of IS of 21 different types. The ability to confer chromosomal DNA transfer may be a selective advantage underlying plasmid/IS symbioses.

GDT4 was optimal at low temperature and low iron concentration. However, pGDT4 did not encode any known thermoregulators. This suggests that temperature sensitive pGDT4 conjugation is under the control host genes. GDT4 could therefore be a natural response to challenging growth conditions.

Lesic, B., Zouine, M., Ducos-Galand, M., Huon, C., Rosso, M., Prévost, M., Mazel, D., & Carniel, E. (2012). A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis PLoS Genetics, 8 (3) DOI: 10.1371/journal.pgen.1002529

small silencing RNAs. I: Piwi-interacting RNAs.

Three major classes of small RNAs involved in gene silencing have been found in animals: microRNAs (miRNAs), small-interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). miRNAs are involved in the regulation of mRNA stability and translation, whilst the main purpose of the siRNA and piRNA pathways appears to be the defense of the cell and genome from viruses and transposable elements. Unlike the other two systems that are ubiquitously active, the piRNA pathway is generally only active in germline cells, the most important locus of defense against transposons.

A common feature of all three pathways is the formation of RNA-induced silencing complexes (RISCs), composed of a small RNA bound to an Argonaute family protein. The small RNA guides RISC to specific target RNAs, resulting in target silencing (generally by the Argonaute protein ‘slicing’ the cognate RNA). A key stage in the miRNA and siRNA silencing pathways is the recognition of double stranded RNAs, and their cleavage by Dicer proteins. This is not a feature of the piRNA system. Another difference is that piRNAs range from 22nt to 30nt in length, whilst siRNAs and miRNAs are 21 or 22-24nt long respectively. When piRNAs were first discovered they were called repeat-associated small-interfering RNAs (rasiRNAs). However, as they are not always associated with repeat sequences and as they bind a specific clade of Argonaute proteins, the PIWI family, they were subsequently renamed.

The piRNA system in Drosophila

A Drosophila melanogaster egg chamber. The large nurse cell nuclei are visible in the upper half, whilst the follicle cells cover the oocyte in the lower half.

The piRNA transposon silencing system has been most comprehensively analysed during oogenesis in the fruitfly, Drosophila melanogaster. Within a Drosophila egg chamber, the germline cells (fifteen nurse cells and the oocyte) share a common syncytial cytoplasm. They are surrounded by a layer of somatic follicle cells, which exchange developmental signals and nutrients with the germline cells. The Drosophila genome harbours over a hundred transposon families, including representatives of all three major classes (LTR and non-LTR retrotransposons, and DNA elements). Some retrotransposons, such as the gypsy family, form viral particles that have been shown to be able to invade the germline from the follicle cells via cellular transport vesicles. Therefore the germline is under threat from transposable elements primarily from within, but also from the somatic follicle cells. Two different variants of the piRNA system function in the germline and the somatic follicle cells: a more complicated system involving three PIWI family Argonaute proteins and a piRNA amplification system functions in the germline, whilst a simpler system involving only one PIWI protein works in the follicle cells to silence a more limited repertoire of retrotransposons.

The piRNA pathway in somatic follicle cells

Approximately 70% of somatic piRNAs map to transposons or transposon fragments. Of these 90% are antisense to active transposons. Mapping piRNAs to genomic sequence has yielded a great insight into genomic structure and the piRNA system of transposon control: piRNAs are derived from large clusters of densely packed, inactive transposon copies and fragments. piRNA clusters are a conserved feature of piRNA biology. They generally span dozens to hundreds of kilobases and are located in the heterochromatin associated with centromeres or telomeres. In the case of Drosophila somatic follicle cells two piRNA-clusters dominate: The flamenco locus and cluster 20A. Follicle cell piRNAs from these clusters are derived from one DNA strand, meaning that transcription is unidirectional. In flamenco and cluster 20A, the transposon fragments are generally oriented antisense to the direction of transcription, explaining the strong antisense bias of somatic follicle cell piRNAs. A P-element insertion at the beginning of the flamenco cluster blocks piRNA production from the whole 180kb cluster, suggesting that the formation of long single stranded transcripts of piRNA clusters is a necessary stage of piRNA biogenesis. However, the mechanisms of piRNA generation are not clear. It appears likely that the long piRNA precursor transcripts are stochastically cut into smaller fragments. Piwi then selectively binds fragments with a 5′ uridine (75% of Piwi-bound piRNAs have a 5′ uridine residue), and the pre-piRNAs are then 3′ trimmed to generate the final piRNA.

The germline piRNA pathway and ping-pong amplification.

In addition to Piwi, Drosophila ovarian germline cells express two related PIWI family Argonaute proteins: Aubergine (Aub) and AGO3. Unlike Piwi, which is localised to the nucleus, Aub and AGO3 are associated with an electron-dense peri-nuclear region of cytoplasm called nuage. Most importantly, they act together in a sophisticated piRNA amplification loop that is dependent on target expression, termed the ping-pong cycle. In a simplified version: Aub complexed with an antisense piRNA targets and slices a sense transcript of an active transposon, resulting in the production of novel sense piRNA species which are loaded onto AGO3. The AGO3-piRNA complexes then cleave complementary piRNA cluster transcripts, resulting in the production of novel antisense piRNA to be complexed with Aub. The ping-pong cycle results in the amplification of sets of antisense and sense piRNAs that are 10nt out of register with each other, suggesting the site of Aub slicer activity and providing a useful signal that shows that ping-pong amplification has occurred.

In the germline, more piRNA clusters are active, representing a larger spectrum of transposons. They are also expressed bi-directionally. An outstanding question is why this doesn’t trigger ping-pong amplification? The most likely reason is that the processes of piRNA biogenesis and transposon silencing are tightly localised and regulated. The roles of other proteins in these processes are starting to be understood. Proteins containing Tudor domains appear to be very important in the localisation and function of Aub and AGO3 in the nuage.

Many other intriguing aspects of piRNA biology are yet to be understood. Although the bulk of piRNAs are directed against transposons, some are involved in the regulation of cellular mRNAs. These piRNAs are derived from mRNAs rather than cluster transcripts: Are these transcripts marked in some way to be processed into piRNAs? The links between the primary piRNA biogenesis pathway and the ping-pong amplification system are also poorly understood. An interesting aspect of the piRNA system active in mouse spermatogenesis, is that the nucleus localised mouse PIWI family protein MIWI2 has been implicated in guiding de novo DNA methylation at transposon loci. Is this a more widespread phenomenon?

The piRNA system has been likened to an acquired immune response and works together with the (more acute response) siRNA pathway in transposon silencing. Future posts will discuss the other small RNA systems, and go further into piRNA biology.

Senti, K., & Brennecke, J. (2010). The piRNA pathway: a fly’s perspective on the guardian of the genome Trends in Genetics, 26 (12), 499-509 DOI: 10.1016/j.tig.2010.08.007

Khurana, J., & Theurkauf, W. (2010). piRNAs, transposon silencing, and Drosophila germline development The Journal of Cell Biology, 191 (5), 905-913 DOI: 10.1083/jcb.201006034

of further interest: piRNAs in the brain: epigenetics and memory