by Anna Dragoš
Bacteria live in microbial jungles where they constantly must cope with siblings exploiting their food, predators trying to eat them, or hostile strains shooting at them with antibacterial weapons. Through billions of years of evolution, they developed various strategies for resource competition, self-defense, and killing. It happens that interbacterial combat can result in more than death or victory. In 2015, researchers found that right after the killing act, bacteria can take up genetic material from their dead enemies and incorporate it into their genome. While we have long been aware that some bacteria can take up foreign DNA − an ability called natural competence − the answer to the question of why they do that depends on what the fate of that DNA might be. Some bacteria might use the DNA as a food source, others could use it to repair their own genetic material, and still others could exploit the acquisition of new genes to increase their adaptive potential (by analogy to the benefits of sexual reproduction). So, which DNA would be most desirable for a given bacterium? If DNA is to be used solely as food, it should not matter where it comes from. If exogeneous DNA is used to fix damage in the recipient's genome, the donor should be closely related to the recipient. And if exogeneous DNA is to serve as a source of new genes, it should come from someone more distantly related. Researchers from Biotechnical Faculty at the University of Ljubljana have recently found which DNA is preferred by the soil bacterium Bacillus subtilis, providing an important new insight into bacterial evolution. The answer was hidden in 1 cubic cm of soil from the riverbank of the Slovenian river Sava. From this tiny soil sample, Polonca Štefanič isolated and characterized dozens of B. subtilis strains that became foundational for pioneering microbial ecology studies in Ines Mandić Mulec's lab (Fig. 1A).
Discrimination between right and wrong mating partner
I was lucky to join the Mandić Mulec group as a PhD student, around the time when they observed a fascinating behavior of their isolates. When swarming on semi-solid surfaces, some B. subtilis strains formed a peculiar boundary when they met (Fig. 1B). I recall our consternation when we found that similar work was in progress in Roberto Kolter's lab. This was soon followed by the exciting and stimulating research atmosphere that ensued when the two groups joined forces. This collaboration led to the discovery of the first combinatorial kin-discrimination system which operates to discriminate among very closely related strains. Identical, or nearly identical, strains (kin) can mix when they encounter each other. But thanks to the combinatorial system, when less related strains (non-kin) meet, they compete using several molecular tools. Competition is manifested as a boundary visible in the swarming assay.
What does kin discrimination have to do with natural competence and DNA uptake? In their recent work, Štefanič et al., show that genetic exchange and natural transformation are greatly increased at the boundary formed when two non-kin strains meet. This increase in recombination does not occur when two kin strains meet (Fig. 2). The authors performed swarming assays with six B. subtilis strains of different genetic relatedness and measured the frequency of genetic exchange. They found that the efficiency of genetic exchange increased with increasing genetic distance between the isolates. During the exchange, one strain always seemed to act as DNA donor and the other as DNA recipient, with the latter having higher competitive fitness at the boundary. The fierce competition at the boundary was also evident from the morphology of cells, which looked like empty bags, as compared to the healthy rods found at the boundary of kin strains (Fig. 3). Therefore, within the relatedness scale in which the B. subtilis kin-discrimination system operates, the efficiency of genetic exchange increases with the genetic distance between the isolates. For some strains that could take up DNA when paired with non-kin, transformation rates were below the limit of detection when paired with kin, indicating that some sort of inbreeding barrier exists in bacteria (for more on the inbreeding angle, check this blog).
Harass the competitor and open up for its DNA
What I find most striking is the mechanism which leads to the increased genetic exchange between the two non-kin strains. As a first guess, one could think that killing the donor strain at the boundary leads to more DNA being released to the environment and thus more DNA being available for uptake by the recipient. But it is much more complicated! The authors found that there is plenty of exogenous DNA at the meeting points of both kin and non-kin strains. So, why is the DNA uptake from the non-kin strain more efficient? It turns out that when two non-kin strains encounter each other, one of them suffers "cell-envelope stress" (Fig. 4A). This is somehow sensed by the other strain and results in it inducing its natural competence. Therefore, the recipient first attacks the donor via the combinatorial kin-discrimination system, and then specifically reacts to its stress-response by increasing natural competence (Fig. 4BC). Since the meeting of two kin strains is stress-free, natural competence is not upregulated when the closest siblings encounter each other. These findings strongly support the idea that B. subtilis takes up exogenous DNA to expand its genetic repertoire, likely speeding up adaptation to new environments.
The last, "cherry on the cake" experiment in this study confirms the increase adaptation idea. The authors show that a pair of non-kin strains, each equipped with a gene conferring resistance to a different antibiotic, readily form recombinants harboring both resistance genes and are thus able to invade an area where both antibiotic resistance genes are required for growth. This phenomenon does not occur when two kin strains meet (Fig. 5).
It is time to revisit the long-standing idea that naturally competent bacteria prefer their closest relatives as DNA donors, at least for B. subtilis. The study of Štefanič et al. clearly shows that B. subtilis prefers DNA from the non-kin, less related strains. Let's remember that the B. subtilis combinatorial kin-discrimination system operates on fine scales of genetic relatedness and that the non-kin strains are still relatives that belong to the same species – perhaps an ideal compromise between genetic novelty and risks of deleterious effects of foreign DNA uptake. The link between competition and genetic exchange sheds new light on the evolution of naturally competent bacteria. When fighting and mating go together, the winner of an interbacterial combat may never be the same again…
Anna Dragoš is an Assistant Professor at the Biotechnical Faculty, University of Ljubljana, Slovenia. She graduated in 2014, from Ines Mandić-Mulec lab, spent six years as a postdoctoral fellow in Ákos T. Kovács' group working on B. subtilis evolution in biofilms. Currently she is heading her independent research group on host control by phage using B. subtilis as a model system.