Tag Archives: Bacteriophage

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

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A novel gene transfer agent from Bartonella

A gene transfer agent (GTA) that preferentially packages host adaptability genes has been discovered in the pathogenic bacterial genus Bartonella.

Bartonella are vector borne, facultative intracellular bacteria that infect erythrocytes and endothelial cells in various mammalian species. Berglund et al. sequenced the genome of the rodent infecting species Bartonella grahamii, and compared it with various other sequenced Bartonella genomes. Rodent associated Bartonella species had a larger amount of imported genes than other species. Most of these horizontally transferred genes were either bacteriophage derived, or encoded proteins involved in secretion or transport systems. A large proportion of the imported genes were located in a 461kb segment of the genome that the authors termed the chromosomal high plasticity zone.

The chromosomal high plasticity zone was present in all Bartonella species, but was most expanded in the rodent associated species. It showed similarities to auxiliary replicons (ie large extra-chromosomal elements) from related species.  Genes encoding type IV and type V secretion systems, that mediate bacterial invasion of erythrocytes and endothelial cells, were ancestrally located in the chromosomal high plasticity zone.

Bacteriophage particles were observed in Bartonella cultures. When these phage were isolated it was found that they contained proteins encoded at a cluster of phage genes in the high plasticity zone (phage cluster II), and packaged 14kb strands of DNA. By hybridisation of the phage encapsulated DNA against microarrays covering the bacterial chromosome, it was found that the phage contained DNA from the entire bacterial genome but that DNA from the high plasticity zone was overrepresented. The peak of the hybridisation signal was located at another cluster of phage genes within the high plasticity zone (phage cluster III).

It appears that auxiliary DNA replication from a phage derived origin of replication within the high plasticity zone is amplifying the Bartonella genome. The products of this ‘run-off’ replication are then being packaged into phage-like gene transfer agents. This is the first time a coupling of run-off replication and GTAs has been demonstrated. Previously discovered GTAs have packaged chromosomal DNA randomly, but in this instance the packaged DNA is biased towards DNA from the high plasticity zone, which is enriched for host adaptability genes.

The genes of the Bartonella GTA didn’t show any similarity to the RcGTA-like agents that are found in many other alpha-proteobacteria. Interestingly, Bartonella GTA-like clusters were identified in some other Rhizobiales species that also possessed RcGTA-like clusters. It would be interesting to enquire as to whether the GTAs in these species are also associated with run-off replication.

The authors suggest that the linkage of run-off replication and the GTA is selectively advantageous, in that not only is the rapid spread of new genes facilitated, but so is gene diversification by recombination.

 Berglund, E., Frank, A., Calteau, A., Vinnere Pettersson, O., Granberg, F., Eriksson, A., Näslund, K., Holmberg, M., Lindroos, H., & Andersson, S. (2009). Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii PLoS Genetics, 5 (7) DOI: 10.1371/journal.pgen.1000546

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