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How is nitrogen-fixing symbiosis transmitted in bacteria?

A new experimental evolution study has shown that rhizobia transmit their symbiotic nitrogen-fixation ability to taxonomically distant bacteria through a horizontal transfer mechanism that is particularly effective. Genes involved in symbiosis are transferred at the same time as genes for evolutionary acceleration, allowing the recipient bacterium to rapidly develop symbiotic properties. Interview with Catherine Masson-Boivin.

Soybean plant. Root nodules. Rhizobium © BRUNEAU Roland
By Pascale Mollier, translated by Daniel McKinnon
Updated on 12/02/2014
Published on 10/15/2014

Flowering mimosa plant at Volubilis archaeological site, Morocco. © INRA, BOSSENNEC Jean-Marie
Flowering mimosa plant at Volubilis archaeological site, Morocco © INRA, BOSSENNEC Jean-Marie

What are you working on?

Catherine Masson-Boivin: We are studying nitrogen-fixing symbiotic bacteria known as rhizobia. The bacteria are associated with legumes, and allow legumes to convert airborne nitrogen into a useable form. The relationship gives legumes an environmental and agricultural advantage as they do not require nitrogen fertilisers. The possibility of transferring this nitrogen-fixing ability to other non-legume crops, particularly grains, is a current research focus, and has serious significance to agriculture. For this reason, a considerable body of research deals with the complex mechanisms of nitrogen fixation symbiosis and with understanding how the mechanisms work. The symbiosis involves an adaptation of the bacteria to the host plant, the formation of nodules – a space shared by both bacteria and plant where the bacteria multiply – on the plant’s roots, and the development within the nodules of a metabolic factory to fix nitrogen. How can we approach such a level of complexity? It so happens that there are rhizobia in many genera of taxonomically distant bacteria. Studying how the capacity for symbiosis is transmitted between these bacteria is a good way to understand the mechanisms involved.

What have you found?

C. M-B: We conducted an original experimental evolution study to look at how a generalist bacterial pathogen from the Ralstonia genus, which multiplies in plant vessels, is gradually transformed into a symbiotic bacterium specific to mimosa plants. To do so, we transferred the symbiotic plasmid of a rhizobium from the Cupriavidus genus to Ralstonia and then selected natural variants of the chimera able to form rudimentary nodules on the mimosa. We then performed repeated cycles of bacteria–mimosa coculture. At the end of 17 cycles – extraordinarily quickly in evolutionary terms – the bacteria had become symbiotic and able to infect nodules in a very successful manner. At this stage, however, the bacteria have not yet acquired the ability to fix nitrogen from the air.

What is happening at the molecular level?

C. M-B: We know that the capacity for symbiosis can be transferred between distant bacteria via the horizontal transfer of symbiotic plasmids, which are easily transferred circular strands of DNA that carry part of the genes essential for symbiotic functions. In general, however, this transfer is not sufficient. We believe that, to acquire symbiotic functions, the recipient bacterium’s genome must first be restructured to allow the bacterium to adapt to it its legume host. When sequencing the evolved chimera bacteria, we discovered that they had particularly high rates of mutation in the plant culture medium, at least five times higher than normal. The elevation of the mutation rate creates a burst of phenotypic diversity in the bacterial population. This favours the emergence of beneficial variants, the most beneficial of which are selected by the plant.

How can you explain this hypermutability?

C. M-B: We demonstrated that this high rate of mutation is caused by the presence of the plasmid. When we looked at the plasmid, we were able to identify a mutagenesis cassette with specific DNA polymerases that are able to replicate damaged DNA. They produced errors when doing so, which creates the mutations. This hypermutability is seen only with bacteria in the plant culture media; it is not seen in bacteria inside nodules. Interestingly, hypermutability is not seen in enriched culture media either. This means it is a type of environmentally dependent mutagenesis known as “stress-induced mutagenesis”. Here, stress may be caused by, for example, the lack of nutrients in the plant culture medium. We also showed that this hypermutability allows bacteria to adapt more effectively. We compared populations that evolved from bacteria carrying the cassette to those that did not and found that bacteria carrying the cassette outcompeted non-carriers to establish symbiosis with the plant.

To summarise, symbiotic genes are transferred at the same time as genes that accelerate mutations favourable to adaptive remodelling of the recipient bacterium’s genome and to its adaptation to plant symbiosis.

What is next for your work?

C. M-B: In just a few growing cycles (approximately 400 bacterial generations), we went from a pathogenic bacteria to a symbiotic legume bacteria (symbiotic in the sense of “living together”). The mechanism responsible for the extremely rapid evolution appears to be important in the horizontal transfer of symbiotic nitrogen fixation and thus in the appearance of new rhizobium strains, judging by the overrepresentation of plasmid mutagenesis cassettes in these bacteria. It is important to note that this cassette is not seen in the source rhizobium (Cupriavidus), which must have an extinction mechanism to prevent deleterious mutations once a certain evolutionary stage has been reached.

Our work is ongoing as we have not yet been able to obtain chimera bacteria capable of fixing nitrogen. We will select the most promising evolved bacteria and will continue to pursue our experimental evolution study.

Scientific contact(s):

Associated Division(s):
Plant Health and Environment, Plant Biology and Breeding
Associated Centre(s):


Remigi P, Capela D, Clerissi C, Tasse L, Torchet R, et al. 2014. Transient Hypermutagenesis Accelerates the Evolution of Legume Endosymbionts Following Horizontal Gene Transfer. DOI:10.1371/journal.pbio.1001942