An Arms Race at the Tiniest Scale

Defense and anti-defense against genetic attack by plasmids.

Bacteria have a pretty active sex life. And like for us, this involves defense and offense, in a complicated tango of genetic exchange. Only, for bacteria, conjugation mechanisms are overwhelmingly used for attack, even though they are also the conduit of great innovations like horizontal gene transfer from different species, and antibiotic resistance. The top priority of most bacteria most of the time is to defend against alien DNA, which most of the time are selfish genetic elements and viruses, and they have several mechanisms to do so.

Plasmids are a very common feature of bacteria, and fundamental to genetic engineering, as the primary form of manipulated DNA. Plasmids can be amplified to high copy number in bacteria, can be cut, altered, and ligated back together- the very essence of engineering. This dates me, but I remember preparing plasmids from bacterial cultures by cesium chloride isopycnic (high speed) centrifugation, after which the plasmid (marked by the poison ethidium) would end up swimming in the middle of the gradient, and have to be sucked out with a syringe, before further steps to clean up the purified DNA. It was a rather messy, expensive, uncertain, slow, and unsafe process. 

Summary of the current paper, outlining plasmid transfer, plasmid genome structure, and some components (genes and promoters) of the leading strand as it is transferred to target cells.

Anyhow, plasmids in the wild are typically aggressive genetic elements that carry (encode) some or all of the components needed to form an injection attack complex (i.e. type IV secretion system) that conjugates with other bacteria. Plasmids can also carry many other things, like antibiotic resistance, or stray genes from prior bacterial hosts, or transposing genetic elements. So most of the time, bacteria want to defend themselves against this kind of invasion, even though some of the time the gifts they bring can end up being highly beneficial.

One of their defenses is the restriction system. Another foundation of genetic engineering was and remains the restriction enzyme. These are enzymes that cut DNA at a particular sequence. One can imagine how useful it can be to have such specific scissors for these infinitesimal molecules, and most labs would have a freezer filled with a large library of such enzymes that could be used for breaking and reconstructing new (plasmid) molecules, and also for analyzing them by their pattern of "restriction" sites. In the wild, these enzymes are paired with DNA methylation enzymes that make the host genome invisible to the restriction enzyme, leaving only newly arrived alien DNA susceptible to cleavage, and thus destruction, by these enzymes. 

Another defense is the now-famous CRISPR system. Bacteria capture small bits of invading genomes, and, assuming they survive, knit them into special genetic modules in their own chromosomes. Then they express these small modules as RNAs that latch onto an enzyme called Cas9, (or related enzyme plus RNA systems), which are nucleases that are guided by these RNAs to cut and inactivate invading DNA that matches the RNA sequence. This system is noted as a sort of adaptive immune system that learns from experience, and passes its knowledge down genetically to future generations.

More detailed maps of three example plasmid genomes, showing the distribution of anti-defense and other genes at the leading edge of the genome (left end). Red and yellow colored genes are anti-defense genes of various kinds. The tiny arrows are promoters, each of the early-start kind that can operate on single-stranded DNA.

A recent paper discussed specialized anti-defenses that plasmids have against these and other bacterial defenses, including toxin/suicide systems and inducible stress responses. For it is naturally an arms race with innovation on both sides. Plasmids have an origin of replication where new DNA strands start, and this new strand is what is injected into the target cell. So there is a linear order of DNA and thus genes going into the target cell, head to tail. These researchers find that the anti-defense genes of plasmids tend to be bunched up at the head of the genome, where they get into the target cell first. These regions also have a special way to fold up their single-stranded DNA (into a cruciform shape) that forms immediate promoters that allow these "early" genes to be transcribed by the host RNA polymerase, before replication in the host cell has regenerated normal double-stranded DNA. 

The authors do a very wide survey of species and plasmids, and find that there is a wide variety of anti-defense genes, many of which have unknown functions. Among the known ones are: anti-restriction inhibitors, which directly bind and inactivate restriction enzymes, methyltransferases (MTase) that methylate restriction sites to make them look like host DNA, single strand binding proteins (SSB), which coat the single stranded DNA and protect it from detection as single stranded DNA, and may assist in repair after Cas9 or restriction cleavage, and antitoxins that inhibit the toxin systems or the SOS system. There are also anti-CRISPR proteins, which degrade the Cas9 enzyme, or inhibit its binding to target DNA.

Experiment to demonstrate the importance of being first... into the target cell. Targeting means that the bactierial cell has a CRISPR system targeting the incoming plasmid. acrIIA4 is an anti-CRISPR protein that effectively blocks cleavage of plasmid DNA by the CRISPR/Cas9 enzyme. The petri plate exhibits bacteria that can grow only if plasmid transfer was successful. See text for further details.

They finish with an elegant experiment that asks how important it is for the anti-defense gene to be at the front of the plasmid. The gene they chose was acrIIA4, which is an anti-CRISPR that very efficiently inhibits Cas9 cleavage of targeted DNA. The petri plate at top shows the growth of infected bacteria after infection by the experimental plasmid, selected for plasmid presence on antibiotic medium. The grey bar, in both diagrams is the control, which are cells whose CRISPR system does not target this plasmid. The plasmid transfers fine, and the cells grow fine. In contrast, if the cell's CRISPR system does target the plasmid, lack of acrIIA is fatal, (top), decreasing plasmid transfer by about three logs, or a thousand fold. Putting the acrIIA gene at the tail end of the plasmid (middle experiments) helps a little, and transfer is knocked down only a hundred fold. Putting the defense gene at the front of the plasmid, (leading), though, corrects plasmid defense almost fully, and transfer is down a few fold only. Lastly, if acrIIIA is placed in opposite orientation, (inverted), such that plasmid replication is needed before this gene can be expressed (it can't use the cruciform single-stranded promoter), it is virtually useless. So indeed, being first off the block when invading a target cell is critically important, since the host cell makes its defenses all the time- they are ready and waiting.

While most of these transfers are unwanted, sometimes plasmids integrate into bacterial genomes, and then when they start transferring themselves into other cells, they can bring along huge amounts of their host genomes. That starts to look like serious genetic exchange that begins to approximate sex in eukaryotes. So, some balance of defense, offense, and beneficial exchange is the lifeblood of ecology and evolution at this most ancient scale of life.

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