Tag Archives: Horizontal Gene Transfer

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

Novel modes of lateral gene transfer in bacteria

Understanding the mechanisms of lateral gene transfer (LGT) between bacteria is crucial to our understanding of microbial evolution. It is also important for human health as LGT facilitates the emergence and spread of bacterial virulence and antibiotic resistance. The three ‘classical’ mechanisms of LGT; transformation (in which naked DNA is taken up from the environment), transduction (by which bacteriophage facilitate gene transfer by packaging host DNA as well as their own) and conjugation (when plasmids encode a pilus by which they can be transferred from cell to cell) have been important in the emergence of molecular biology. Three other mechanisms by which DNA can be transferred between bacteria have come to light, potentially broadening our understanding of the importance of LGT in the microbial biosphere.

Gene Transfer Agents (GTAs)

GTAs are virus-like particles that carry random pieces of the producing cell’s genome. The best characterised GTA was discovered in 1974 from the purple, non-sulphur photosynthetic bacterium Rhodobacter capsulatus, a member of the alpha-proteobacteria. The R. capsulatus GTA (RcGTA) packages 4.5kb of DNA, but is encoded by a 14.1kb cluster of 15 genes on the R. capsulatus chromosome. Many of these genes have homology with bacteriophage structural genes. It is unclear how RcGTA particles are released from the cell, as no recognisable lysis genes have been identified. Transcription of the RcGTA gene cluster has been shown to be under the control of a sensor kinase/response regulatory system that transduces environmental signals. RcGTA-like gene clusters are widespread throughout the alpha-proteobacteria and phlogenetic trees based on RcGTA-like sequence recapitulate phylogenies based on 16s rRNA sequences suggesting that the RcGTA ancestor arose early in the evolution of the alpha-proteobacteria lineage.

Other GTAs (with probable independent origins) have been identified in a diverse range of prokarya including the archaebacterium Methanococcus voltae, the delta-proteobacterium Desulfovibrio desulfuricans and the spirochete Brachyspira hyodysenteriae.  None of them packages more than 14kb of DNA, and all of them take the form of small bacteriophage. It appears most likely that GTAs have been derived from bacteriophage that have lost their ability to self-propagate. Recent data suggests that alpha-proteobacterial GTAs are common in marine environments, and transfer genes at high frequency between diverse classes of alpha-proteobacteria. These ‘generalised transducing machines’, under the control of bacterial populations quorum sensing systems, are probably a major force in microbial evolution and ecology.

DNA transfer by membrane vesicle.

DNA encapsulated by MVs. A rosette-like structure is seen in the centre, a plasmid is in the box, linear DNA molecules - arrowheads.

Membrane vesicles (MVs), from gram –ve bacteria can traffic toxins, signals and other proteins between bacteria. They have also been shown to be able to mediate the transfer of DNA between cells. E.coli 0157:H7 MVs were found to contain linear DNA, circular plasmids and rosette-like DNA structures, that included genes from chromosomal DNA as well as plasmid and phage. The MVs were capable of transforming related enteric bacteria and increasing their cytotoxicity. DNA transfer by membrane vesicles could be a more widespread phenomenon than is currently appreciated, however as yet it is more commonly reported as an aside from other MV studies.

Intercellular nanotubes.

top two images show B. subtilis cells with nanotubes (note more intimate thin connections in circle). Lower three images show inter-specific nanotubes.

A year ago Dubey and Ben-Yehuda showed the existence of tubular conduits forming between Bacillus subtilis cells. These nanotubes were shown to be able to mediate the exchange of proteins and non-conjugative plasmids. Nanotubes were also formed between B. subtilis and Staphylococcus aureus (both gram +ve) and a thinner variety were formed between either of the gram +ve species, and gram –ve E.coli. The authors suggest that the formation of ‘syncytium-like synergistic consortia’ mediated by nanotube connections underlies many of the traits displayed by biofilms.

These three phenomena have a tantalising savour, suggesting the depths of our ignorance of the complexity of microbial ecosystems and prokaryotic evolution. However, I imagine that progress in these fields will accelerate. The explosion of microbial and environmental sequencing will be useful in identifying the prevalence of GTAs. Understanding all six modes of LGT will be crucial to our appreciation of the ecology of natural microbial communities and of bacterial evolution, as well as having important application for human health.

See also: A novel gene transfer agent from Bartonella

Stanton, T. (2007). Prophage-like gene transfer agents—Novel mechanisms of gene exchange for Methanococcus, Desulfovibrio, Brachyspira, and Rhodobacter species Anaerobe, 13 (2), 43-49 DOI: 10.1016/j.anaerobe.2007.03.004

Lang, A., & Beatty, J. (2007). Importance of widespread gene transfer agent genes in α-proteobacteria Trends in Microbiology, 15 (2), 54-62 DOI: 10.1016/j.tim.2006.12.001

McDaniel, L., Young, E., Delaney, J., Ruhnau, F., Ritchie, K., & Paul, J. (2010). High Frequency of Horizontal Gene Transfer in the Oceans Science, 330 (6000), 50-50 DOI: 10.1126/science.1192243

Yaron, S., Kolling, G., Simon, L., & Matthews, K. (2000). Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria Applied and Environmental Microbiology, 66 (10), 4414-4420 DOI: 10.1128/AEM.66.10.4414-4420.2000

Dubey, G., & Ben-Yehuda, S. (2011). Intercellular Nanotubes Mediate Bacterial Communication Cell, 144 (4), 590-600 DOI: 10.1016/j.cell.2011.01.015