Breaking down the microbiology world one bite at a time
What genes make a bacteria?
All organisms possess a wide collection of genes in their genome. Humans have about 20,000 genes; Escherichia coli, the most well-studied bacteria, about 4,500. A great mystery in biology is to find which of these are actually important for the organism. A simple way of finding out is to remove a given gene from the genome: if you can suppress it, then it was not that important. Think of it as if you had never seen a car, and are trying to understand how it works. You could easily remove the radio: you would not have music, but the car would otherwise drive perfectly fine. If on the other hand, you remove the wheels, the car would not move at all. Finally, you could not remove the gas tank from an engine car, but you could remove it from a hybrid car. Electric cars don’t even have one! Similarly, some bacterial genes are essential (required for the organism to grow), while others are dispensable. Like cars, bacteria are very diverse, and a gene essential in one strain can be inutile in a closely related one. Indeed, bacteria from the same species sometimes have very different lifestyles, resulting in a very broad genetic diversity. This is the case for Escherichia coli, which is so diverse that we discover new genes in every newly sequenced genome! Which genes are essential in all strains? Why are some genes essential in some strains and not others?
To find out, F. Rousset and co-workers used a technique called CRISPRi. Instead of removing the gene from the genome, CRISPRi blocks its expression. To continue with the car analogy, it is as if instead of removing the part, you would pour glue on it. They focused on a set of 18 strains representative of the diversity of Escherichia coli, and used CRISPRi to target the set of 3,400 genes present in most of these strains. They performed the experiments using different culture media, the traditionally used LB “rich” medium, with plenty of nutrients, the M9 minimal medium which only has a handful, and a medium (GMM) mimicking the conditions found in our gut, one of the natural environments of E. coli. They found out that only a surprisingly small number of common genes were essential in all 18 strains (266 in LB, 304 in M9 and 248 in GMM). Fascinatingly, many more were essential in at least one strain (about twice as many: 506 in LB, 602 in M9 and 555 in GMM). In fact, most essential genes are essential either in all strains or only in a handful.
They examined whether more closely related strains had more similar sets of essential genes. Even though the profile of gene expression was very similar in related strains, they showed that their essential gene sets could significantly differ. Indeed, the evolutionary distance* was only very weakly correlated to the similarity of essential gene sets. It should be noted that it was only in one of the tested mediums. Thus, factors other than relatedness explain differences in essentiality.
Rousset and co-workers then focused on genes presenting highly contrasted essentiality (genes essential in at least one strain, and not essential in at least one strain). They investigated the genetic basis for these differences. They found 120 such genes (32 in LB, 55 in M9 and 66 in GMM), and explored the mechanisms that could explain differences in essentiality for them. The authors searched for functional redundancy* and realized that it was the case for the gene metG. Additionally, metG is inessential in strains where the gene is duplicated: when targeting one copy, the other can still be expressed and the cell can continue to grow normally. Think of it as an electric car having two motors: if one fails, the other can still drive you home. Similarly, the ycaR-kdsB genes were not essential in strains possessing the homolog (a gene with a shared ancestry) kpsU, which could perform the same function. They found that each strain possessed between 10 and 17 extra genes homologous to essential genes. However, homologs are sometimes not sufficiently expressed to counterbalance the lack of the essential genes. As an example, nrdA and nrdB remain essential in strains APEC O1 and TA447, despite having homologs: these homologs are simply not expressed enough.
In more subtle cases, they observed functional redundancy but could not find any homologous gene. This was the case for the gene dut in strains APEC O1 and TA447. They realized that these strains possessed a plasmid (a small extra-chromosomal piece of DNA) with genes thought to have a related function. They could narrow down to one gene in particular, which they proved to perform the exact same function as dut. As this gene is not evolutionarily related to dut, it is an analog. You can think of the engine and electric motor of hybrid cars: the two motors are very different, yet perform the same function.
Finally, they wanted to know why some genes are non-essential in most strains, but suddenly become essential in others. The scientists observed such genes in 13 of the 18 strains. For most of these “variably essential” genes of strain K-12, they noticed that it was because they possessed a dysfunctional homolog: if your electric car has two motors but one is broken, the remaining one becomes critical. In order to understand the variable essentiality of other genes, they used experimental evolution. That way, they generated mutant bacteria in which the gene of interest was not essential anymore. By sequencing the genomes of the evolved and the ancestral strains, Rousset and co-workers found that the gene of interest is part of a toxin-antitoxin system. Such systems are “addictive” genetic elements, in which one gene forms a toxic product that the second gene neutralizes. If both genes are present, the cell is fine, but the cell dies if the antitoxin is lacking. Toxin-antitoxin systems are used by some bacteria to defend themselves against viruses. Other “variably essential” genes also corresponded to viral defense systems. As for us, it seems that bacteria are always fighting against viruses!
*: number of elementary substitution events (mutations) that occurred during the time of divergence of two sequences (in this case, the genomes of two organisms). It measures how related organisms are.
Link to the original post: Rousset, F., Cabezas-Caballero, J., Piastra-Facon, F. et al. The impact of genetic diversity on gene essentiality within the Escherichia coli species. Nat Microbiol 6, 301–312 (2021).
Written by: Théophile Grébert