While genetic modification technologies have been expanding rapidly in the biological and medical spheres, there is an obvious limitation in how this sort of research is applied. For the microscopic level and the macro beyond it, only a select number of organisms have proven to be amenable to such modification techniques. Others have either shown resistance or have systems that are entirely unworkable with the tools available to us. Even with new advancements like CRISPR, there remain problems that interfere with broadening the pool of available species that can be used.

For bacteria, even though there are hundreds of thousands of species out there, only around 30,000 have been able to be cultured in a lab setting. And most of those besides have yet to have a significant amount of validation done to prove their ability to be grown consecutively and without issues. Out of all of them, only around 80 species have been shown to be open to genetic modification techniques and many only after a fair amount of domestication efforts. Meaning that new wild strains of the same bacterial species would remain resistant to modern forms of gene editing.

Restriction Modification Systems

One of the most common reasons for this resistance is known as the restriction-modification (RM) system. Almost all known bacteria have at least one and they are similar to CRISPR itself in many ways. But rather than defending against viral genomic material, RM systems are used to tell the difference between one’s own DNA and foreign DNA that has been incorporated. Methylation status is the main way, since if a new piece of DNA was inserted into a methylated region and was itself non-methylated or not methylated in the right way, as it is likely to be, then that would be a clear reason to view it as foreign and excise it from the genome. Thus, potential insertion sites in the genome are heavily restricted thanks to this.

Because the RM system itself acts as another layer of defence against bacteriophages trying to attack the cell, scientists have been employing techniques copied from the bacteriophages themselves that have evolved to get around these system. Thus, they are known as anti-restriction mechanisms. The most basic form of which is mimicry-by-methylation, which manually methylates inserted genes to make them indistinguishable from the host cell genome.

But since not all RM systems work with the same methylation process, such bacteriophage methods only apply to some bacterial species. And that then brings us back to the original problem of having to design individual and personal systems for each species in order to have genetic modification be possible, which is time consuming and expensive to accomplish. Finding a way for there to be a more flexible technique able to tackle multiple types of RM systems would be far more useful.

Methylation Investigation

That’s where a team of researchers from the Forsyth Institute in Cambridge comes into play. They knew they had a challenge, however, as bacteria can contain multiple RM systems at once and the forms are so variable that even different strains of the same species can have different ones. The types of DNA motifs they target to check for host or non-host DNA is also highly variable and there are several hundred known to date with more being found every year.

An avenue of attack they noted is that the restriction endonucleases that are able to recognize methylation status are extremely specific in the DNA sequences they check. Those motifs just mentioned being the sequences in question. But they need to be that specific, as a more general system could start falsely recognizing the host genome sequences as foreign and cause those to be excised, likely killing the cell entirely. That also means though that with the right approach, one could stealth right underneath that endonuclease checking mechanism by tricking the methyltranferases that add the methylated status.

Their plan was rather straightforward. If the DNA one desires to insert doesn’t have any of those recognized motifs, then the defense systems won’t notice the sequence after insertion into the genome. So long as all the motifs can be identified and removed from the sequence, the number and variety of RM systems will be irrelevant, as none of them will notice the inserted sequence. Therefore, the plan was to make an identical, otherwise known as syngenic, piece of DNA that didn’t have the motifs thanks to a single nucleotide change or using a synonymous sequence for the same amino acid. This makes the gene “RM-silent”.  

A MRSA Test

To showcase the effectiveness of their system, they tested it in a commercial strain of Staphylococcus aureus due to it being a currently important pathogen thanks to the spread of methicillin-resistant (MRSA) forms of it. They first identified that the bacteria has two RM systems and noted both the two DNA motif sequences they target and also the type of RM systems they were in regards to the form of methylation they used. In this instance, it was cytosine methylation.

The DNA to insert was chosen to be a plasmid from Escherichia coli that is inactive in S. aureus thanks to the two RM systems. The motif sequences in the plasmid were modified with only six nucleotide changes being needed. Then they inserted both the modified plasmid and the original as a control into the S. aureus strain. They immediately found that their modifications had resulted in a 70,000 fold increase in transformation efficiency and they then made a few more tweaks that pushed the efficiency above a 100,000 fold increase.

The researchers hope that this new method and technique can be used not only for direct genetic modification purposes of intractable bacterial species, but also in synthetic biology for construction of new genetic tools and other mechanisms that can serve biotechnology efforts as a whole. Having a deeper understanding of RM systems and how bacteria network their defenses together can improve our ability to deal with pathogens and create new molecular products at the same time. And this method is yet another piece to deciphering the puzzle of life at a fundamental level.

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Photo CCs: Methicillin-resistant Staphylococcus aureus (MRSA) Bacteria2 from Wikimedia Commons

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