pSC101 plasmid Construction of biologically functional bacterial plasmids in vitro', PNAS USA, 70/11 (1973), 3240–4. 

 Plasmids, usually, are circular extrachromosomal DNA molecules, this additional genetic information is found free in the cytoplasm, and it contains genes that usually give an evolutionary advantage to the host cell. Different types of cells can host plasmids, the majority are found in bacterial cells, yeasts, and fungi, but some other, exotic types of plasmids, can be found in plants and even in animals.
As the title of this post suggests, I think that plasmids play an important role in our definition of life; they are strange entities that are able to replicate and that are subjected to evolution as other organisms. So what is the problem?
Plasmids, like viruses, are entities unable to replicate on their own, they use the machinery and extra information from their host, and so, are not considered living organisms. I really like the comparison between viruses and plasmids, because they can be seen as two different ways in which a non-living form of genetic information can evolve and survive. If viruses can use the resources of the hosting organism to replicate, and, in doing so, causing the death of the cell, on the other hand, plasmids seem to give an advantage to their hosts, ensuring their “survival”.  As viruses are obligated intracellular parasites, plasmids could be seen as a strange form of mutualism.

Plasmid and host co-existence

As I said before, the majority of plasmids can be found in nature as circular dsDNA molecules (what is double-stranded DNA?), their size can vary quite a bit, from a few kb up to several Mb, as well as their GC content and copy number, the last refers to the number of copies of the same plasmid that can be present in the cell. The copy number is an important factor that regulates the expression of the genes that are carried by the plasmid itself, simply, the higher the copies of the gene in the cell, the higher the number of related proteins synthesized, and so, stronger is the phenotype.

 As the cell can host several copies of the same plasmid (up to 50 in some cases), can the cell host several different plasmids? Yes, it can, but with some restrictions.

The incompatibility of some plasmids is due to the presence of very similar sequences, involved in the replication and partition of plasmids during cell replication. Due to these high similarities, the cell is not able to distinguish among two different plasmids, and so their partition in the next generation is completely random, and the probability of each plasmid to be transferred, is equal.

Let’s imagine that two incompatible plasmids are present in the same cell, at the moment of cell division, some plasmids will be randomly selected to be segregated in the newly forming cell, and as the cell cannot identify the plasmids as different, they are seen as two copies of the same one, and so, separated, and not replicated first. 

At this phenomenon, we can also add that slight differences in the copy number of each plasmid, will cause an uneven representation of the plasmid in the population, obtaining, at the end of several generations, a mixed population with cells of two types, each of which with only one plasmid.

Plasmid classification

As we defined the concept of incompatibility, we can now understand how they have been classified experimentally, in different families: two plasmids belong to the same family if they are not compatible, and so, cells can hosts different plasmids only if they belong to different families. Another type of classification takes into consideration the phenotypes given by the plasmid to the host:

Resistance plasmids: they code for genes that give resistance towards a particular antibiotic or toxin. In these sequences, several genes, arranged in cassettes, are present and can be moved and shuffled, or even transferred to genomic DNA, due to the presence of mobile genetic elements. The mobility of these genes puts in danger the usage of particular therapies that rely on antibiotics, whose resistance could be transferred between different species.

Col plasmids: plasmids that carry the colicin factor; colicin is a bacteriocin produced by Escherichia coli that is able to create resistance towards particular groups of microorganisms. Colocin, and bacteriocin in general, are antibiotic-like proteins that often kill the target organism by affecting negatively the cellular membrane stability, or by acting as harmful nucleases.

Degradative plasmids: they confer the ability to degrade particular substrates that are unusual or recalcitrant. These plasmids give a huge evolutionary advantage to their hosts in environments or conditions in which the related substrate, e.g. toluene, is present. The activity of enzymes that are found in degradative plasmids, is particularly interesting for microbial bioremediation, in which a pollutant needs to be degraded by the activity of the local microbiota.

Virulence plasmids: defined as plasmids that carry genes that allow the microorganism to become a pathogenic species. One, well-known, example is Agrobacterium tumefaciens, which plasmid is used to insert pieces of DNA sequences semi-randomly in the genome of the target plant, leading to the development of tumors. This particular type of plasmid is often engineered and used as a vector for the production of recombinant organisms.

F-plasmids: also known as sex-factor, is a plasmid that contains all the necessary information for the production of a sexual pilus by its host (referred to as F+, or donor cell), this structure is necessary to recognize a second cell, laking the same plasmid (F-, or acceptor cell), when recognition is established, a new copy of the plasmid is created and it’s transferred to the new host, forming a second F+ cell.

Horizontal gene transfer

The transfer of genes in prokaryotic cells is not only vertical (from parent cells to the next generation), but it could also be horizontal. This implies that a cell during its lifespan is able to receive new genetic information from another cell or from the environment, as a result of errors of other biological mechanisms. The 3 ways in which this can happens are transformation, transduction, and conjugation.
The latter is mediated by the above-mentioned  F-plasmid that, during its transfer to another host, is able to carry parts of the original F+ cell genomic information. This is possible because F-plasmids are often integrative (episome), which means that they can insert themselves in the genomic DNA of the hosts in particular positions, through the process of homologous recombination. F’ cell is the term used to refer to an F+ cell in which the plasmid is integrated, they are also called high-frequency recombination cells (Hfr) because they are very efficient in transferring part of their genomic DNA during conjugation. Pieces of the host genome can be inserted inside the plasmid as a result of errors during its detachment, this extra information could then be passed to the acceptor cell during conjugation. The new F+ cell now will have both the original plasmid sequence and extra genomic information. This transfer of DNA can be fairly large in some cases, even if entire bacterial chromosome transfers are rare.

This mechanism of gene transfer cannot be fully recognized as a sexual reproduction system because gametes are not formed, it is a result of errors during another mechanism, which is the “reproduction system” of the F-plasmid.  From the evolutionary point of view, it is difficult to frame this type of mechanism, but, surely, this process gives 2 main advantages to the involved entities (cell and plasmid):
  1. ·        The plasmid ensures its “survival” by being passed to several other cells, which now could create also multiple copies of it, and transfer it to other generations.
  2. ·        The cell has the possibility to increase its genetic diversity, which is an advantage, in general, for the species: by using only asexual reproduction systems, it is difficult to have the variability from which advantageous mutations could arise, so, by shuffling genetic information, it is possible that new genes are inserted, or that multiple copies of preexisting genes are created, or that their expression is changed. It is not a surprise that, in fact, other mechanisms for increasing genetic variability like transposon and other mobile genetic elements, are switched on by the cell itself in stressful conditions.
 
 
Before the conclusion, I’d like to underline some points on the topic: first, plasmids are not living entities, and second, they are not sentient. Cells and plasmids are subjected to the rules of evolution, and what could be interpreted as choices or thoughts, arise from the genetic information that those entities carry with them. To simplify, F-plasmids do not have a choice on being replicated and transferred, it is due to the presence of genes that code for those actions, and also the cell does not decide to increase its genetic variability, but the ancestors who adopted that mechanism, had more probability to survive, and pass their genes to the next generation. What I wanted to underly is that these non-living entities, by living in strong relationships with their hosts, and even by mixing their genomic information with them, sometimes make it harder for us to understand where one individual finish, and where a different start (which is at the base of the definition of “life”).

Plasmids play an important role in lab applications, they are used as vectors to insert particular genes, natural or artificial ones, into a suitable host, they are also applied in recombinant protein synthesis, and genetic studies. Another topic that I’d like to explore in the future is the design of artificial plasmids for those purposes, but for today is enough, hope you enjoyed it.