Tuesday, September 06, 2016

Symplasmata: a curious case of multicellularity in bacteria


Cells in a symplasmatum and surrounded by a capsule,
seen with transmission electron microscopy.

'Curiouser and curiouser’, famously said Lewis Carrol’s Alice, as she was experiencing some very peculiar events in Wonderland. I have sometimes felt like Alice when I was studying the curious behavior of the bacterium Pantoea agglomerans [1], during my time in the Lab Leveau at UC Davis.

At first sight, Pantoea agglomerans looks quite ordinary. It grows as rods a few micrometers long, it can swim with flagella and it feeds on all sorts of sugars. It belongs to the family Enterobacteriaceae, and thus it is a distant cousin of E. coli. You can find P. agglomerans in all sorts of environments, but it is particularly good at colonizing the surface of plants, and in certain cases it competes with pathogens and thus keeps its plant host healthy (that is, it can serve as a biocontrol agent). Because it is a very good leaf colonizer, we have used it in many studies of bacterial life in the ‘phyllosphere’ (the aerial surfaces of plants), such as the one described in this previous post

Now here’s what special, and actually seemingly unique, about Pantoea bacteria. Under certain conditions, instead of dividing and spreading as individual cells, the bacteria stay close together and form an aggregate containing up to hundreds of tightly packed cells. Aggregation is not uncommon in bacteria but, in the case of Pantoea, cells are constrained by a fibrillar layer, and surrounded by a thick capsule made of polysaccharides, which indicates some level of cooperation and resources sharing (see image on top of the post). The resulting sausage-shaped structures are called symplasmata [2]. Interestingly, the species name 'agglomerans' (forming into a ball), which was coined by the great Dutch microbiologist and botanist Martinus Beijerinck in a paper dating from 1888, probably refers to the species' ability to form symplasmata [3]. Although symplasmata have been known for a very long time, their importance and function in the environment is still a mystery. We have observed symplasmata on bean leaf surfaces, and others have described them attached to the roots of rice plants (Achouak et al., 1994). What is their ecological role? Does it benefit the plant as well? We do not know yet. 



Symplasmata among single cells, seen with light microscopy.
Growing populations always consist of a mix of individual
cells and multicellular symplasmata (we don't know yet why).
However, we now better understand how they manage such a feat as making a symplasmatum in the first place, and we just published our findings in the journal Scientific Reports, in a paper entitled 'Symplasmata are a clonal, conditional and reversible type of multicellularity'. It took us two breakthrough moments to achieve this. The first one was to find laboratory growth conditions that would allow for symplasmata formation, in order to study them in controlled conditions, without the need of a plant. This was relatively easy: the development of symplasmata is induced in minimal medium with glucose as sole carbon source (if you try to grow them in a rich medium, nothing happens...). Thanks to this convenient way of producing symplasmata in the lab, we found out important facts. First, symplasmata stem from the growth of a single bacterium, hence they are clonal multicellular clusters. Secondly, symplasmata formation is conditional to environmental factors. If you add amino acids, increase the pH or the temperature, no symplasmata are formed. Finally, symplasmata formation is reversible. Under certain conditions cells burst out of their capsule and resume their solitary lifestyle!

The second breakthrough involved a much bigger effort. We did a classic genetic study, that is, we looked for mutants of Pantoea agglomerans that were unable to form symplasmata, in order to identify the genes that underlie the clustering phenotype. It would take us forever to find a natural mutant, so we sped up the process using a transposon mutagenesis (we inserted a piece of DNA in the cell that would randomly inactivate a gene on the chromosome). We screened about 5,000 of these mutants, individually, looking at them under the microscope --- fortunately I was not alone to do this!... (Huge thanks to Mila and Irina from Lab Leveau!) This screening allowed us to identify half a dozen protein-coding genes that were directly involved in the formation of symplasmata, including one that helps build the capsule surrounding symplasmata, and one which is a global regulator of gene expression. In addition, we also studied how the whole gene expression profiles changed in symplasmata compared to single cells, and in the wildtype compared to the symplasmata-defficient mutant. Hopefully, this new knowledge will open the way for further studies of symplasmata formation and their ecological role in plant or soil habitats.

Symplasmata and single cells, seen with scanning electron microscopy.
The capsule was removed by the preparation, but the layer surrounding
the cells appear as an opaque membrane.
Notes
[1]. The strain that I have used in the lab, Pantoea agglomerans 299R, has been reclassified recently as Pantoea eucalypti 299R, but I have kept the previous name in this post for historical reasons.
[2]. Actually, I found out that they were called that way a long time after I had started studying them... I had even entertained the idea that I was the first one to describe it!... But I couldn't have come out with such a cool name as symplasmata, so all is for the best.
[3]. Since Beijerinck only mentioned the species in passing in that paper (as 'Bacillus agglomerans'), and without reference to its aggregative behavior, we are left with speculations regarding the original meaning of ‘agglomerans’. But it seems reasonable to assume the link with symplasmata.

Acknowledgements
This research was supported by a VIDI grant to Johan Leveau from the Netherlands Organisation for Scientific Research and by a fellowship to Robin Tecon by the Swiss National Science Foundation.

References

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