What is to be multicellular? A question from a novel cave bacterium

We know two main types of multicellularity which until recently were enough to classify all bacterial multicellular-like behaviours. But a novel cave bacterium — Jeongeupia sacculi — is a discovery which goes beyond this framework and urges to redefine multicellularity
What is to be multicellular? A question from a novel cave bacterium

For a guy who loves nature,
you'd think he'd be more into animals.
When they have more than one cell,
Jack sort of loses interest.
("Nim's Island")

I am an evolutionary bioinformatician, and my core area of expertise is the evolution of multicellularity. It is a fascinating topic which would need an entire book to explain (and maybe, I will even write such a book). But if you needed a quick guide, the "general theory" of multicellularity appeared relatively simple.  

Clones and aggregates

First case of multicellularity is when an entire organism emerges from a single cell due to the cell division process. All cells of the resulting organism are clones — so this type of multicellularity is referred to as clonal multicellularity

We all are clear examples of clonal multicellularity — as well as plants, fungi, and all groups of algae. Bacterial domain also includes some groups of clonal multicellular organisms, the most prominent of which are filamentous cyanobacteria. Their filaments include tens of cells which are tightly interconnected and exchange their metabolites by microplasmodesms — analogues of plant plasmodesms. Moreover, some filamentous cyanobacteria (specifically the representatives of Nostocales) possess cell differentiation capability. Their filaments are normally differentiated into heterocytes and vegetative cells which carry different biochemical functions: the former fix atmospheric nitrogen and the latter carry out photosynthesis. It is very simple multicellular organism, but its key traits are strikingly similar to ours: both Nostoc punctiforme and Homo sapiens are clonal multicellular organisms in which cells are differentiated to several types and connected with intercellular contacts. From the perspective of multicellularity evolution, both organisms are built in the same way, so are also some giant sulfur bacteria (Thioploca spp. and Beggiatoa spp.), all algae and plants, and fungi. 

These cyanobacteria from Mind The Graph collection are an example of clonal multicellularity — and all of us are the second example. In this case, each multicellular organism develops from a single cell. 

But there is the second, less prominent and known, case of multicellularity, called aggregative multicellularity. The best example of it is social amoeba Dictyostelium dicoideum. In a sated state it is... just an amoeba, but upon starvation, hundreds of amoebas come in flocks to one point and aggregate to form a multicellular organism. It further transforms itself into a fruiting body which in turn sporulates and gives rise to a new generation of amoebas. The essential trait of aggregative multicellularity is developing a whole organism by aggregation of genetically heterogenous individual cells. 

Dictyostelium life cycle
Dictyostelium discoideum is an example of aggregative multicellularity: its developmental cycle starts by gathering lots of free-living amoebas together to form a complex organism which can even move as a whole. This cycle is concluded by the formation of a fruiting body and the release of spores from it. Each spore will give a rise to a new amoeba. The disctyostelium's ability to run such cycles makes possible its use as a model organism for testing embriofoetal toxicity. Created with BioRender.com.

The "aggregative" world is not less diverse than the "clonal" world. Aggregative multicellularity emerged on Earth independently not less than 6 times, and dictyostelid slime moulds are just one of these evolutionary branches. Eukaryotic aggregative multicellular organisms include also, for example, a slime mould Physarum polycephalum known by its ability to solve mathematical problems from theory of graphs. This type of multicellularity is also represented in the bacterial domain: myxobacteria form brightly coloured fruiting bodies by aggregation when they starve — just like dictyostelids. So, bacterial multicellularity does exist, and it was clearly divided by two types

It was before the discovery of a surprising novel bacterium. 

Accidental discovery

In a talk for American science community "The Science Society" (available on Clubhouse), Prof. Kouhei Mizuno confessed that he had been obsessed by the origin of life problem since his childhood. As fate would have it, his career was associated with screening bacteria for biotechnology and industrial purposes. But his favourite topic found him years on.

This time, his team looked for a bacteria which can synthesize polyhydroxyalkanoates, naturally occurring polyesters with a potential use as a biodegradable plastics. The researches screened bacterial cultures from a limestone cave in the Hirao carst plateau in northern Kyushu Island, Japan. But one culture had a very unusual appearance. The most bacterial colonies are opaque or turbid, but this transparent culture with an iridescent hue seemed to have unusual optical properties. 

Further analysis revealed the cause: the new bacterium, called Jeongeupia sacculi, forms highly ordered colonies that resemble nematic liquid crystals which are built from the living bacterial cells rather than molecules. But this was not the last surprise. These ordered colonies were subsequently found to reproduce as a whole — in contrast to most other bacteria where each cell reproduces for itself. 

A normal colony of Jeongeupia sacculi consists of ordered rod-shaped cells. As it matures, central cells begin to differentiate into another morphological type — coccobacilli. Now, the strangest thing is that these cells are released en masse in a jiff — once the full colony is submerged by water. The coccobacilli spread in the water, sedimenting and starting new colonies in new places. This amazing life cycle is driven by water: the massive release of coccobacilli does not occur until water submerges colonies, and the moment of submergence triggers this spectacular conclusion.

Cave bacterium developmental cycle
Jeongeupia sacculi also has an intricate developmental cycle. Its colony consists of filamentous cells ordered in liquid crystal-like structure. In the middle of this colony, a second kind of cells arises — coccobacilli. They are released and spread when all colony is submerged by water — and give rise to new colonies. Created with BioRender.com
Colony life cycle by K. Mizuno
Colonies of Jeongeupia sacculi undergo visible changes during the life cycle. Starting with a homogeneous spot, a colony soon evolve  largea cluster of coccobacilli in its center, and it begins to differ visibly from the edges. Coccobacilli, which act like spores in this bacterium, are "blown up" and spread when a colony is submerged by water. Image kindly provided by Prof. Kouhei Mizuno 

Obviously, such reproduction "as a whole" if a keystone trait of multicellular organisms — in all of them, only a fraction of cells continue to produce offspring while other cells are destined to die. Complex developmental cycles are also common in multicellular species, but not in unicellular ones. So, it seems fair to say that Jeongeupia sacculi is a kind of multicellular. But what kind exactly?

Discussion on a portal 

I am a freelance scientific journalist working with several Russian pop-sci outlets. When I came across the article on Jeongeupia sacculi on Twitter, I immediately pitched it to the editor of online science magazine Elementy.ru and received an approval to write a news article featuring this discovery. In this new article, I wrote:

"It is not aggregative multicellularity - there is no phase in the life cycle of the bacterium where cells aggregate into a single multicellular organism. It is also difficult to call it clonal: there is no evidence that each colony comes from a single coccobacillus. We are facing just a highly ordered colony of cells. The only thing that indicates its multicellular nature is that it behaves as a single organism during reproduction".

This article triggered an interesting and significant discussion how to regard and classify this new multicellular-like behaviour. 

"Why can't it be considered clonal multicellularity after all?
"there is no evidence that each colony comes from a single coccobacillus"
How else could a colony in a Petri dish originate? There, each colony usually grows from a single bacterium, doesn't it? And then a colony must be made up of clones,"

a user with a nickname "Mopp" asked.

There were also the second opinion in the same thread. A user with a nickname "mol_biol" (whose name is Alexey) argued

"I cannot rule out that it may easily behave according to an aggregative multicellular strategy as well, merging into a single colony. Again, there is no evidence to contradict this. Why shouldn't this thing be able to do both?"

Prof. Kouhei Mizuno kindly joined this discussion. Responding to the questions on aggregative or clonal type of Jeongeupia's multicellularity, he mentioned that his team really observed forming of an entire colony from a single coccobacillus, and provided a preprint link containing a respective video. It could be highly suggestive for the clonal nature of the novel way to be multicellular. However, Prof. Mizuno mentioned some extent of uncertainty in his comment for my blog: 

"Which category (clonal or aggregative) the HS3 <the strain name of Jeongeupia sacculi — G.K.>  can belong to is a problem. In the published data, a single cell developed an ordered structure, which is regarded as a clonal multicellularity. However, we assume that some unknown intercellular attracting force might enable the collective behavior that forms a nematic liquid crystal-like pattern in a colony, suggesting an aggregative characteristic. We need further studies".

What about biofilms?

A bacterium with very primitive multicellularity (even compared to cyanobacteria and myxobacteria – acknowledged geniuses of bacteria multicellularity) and even an uncertain type of social behaviour vaguely resembles another type of multicellular-like systems in bacteria — biofilms. They are well known as complex systems and more than a large piles of bacterial cells. Their intricate signalling and resilient extracellular matrix are extensively studied, but they are usually left aside when discussing  evolutionary origins of multicellularity or its classification. However, they were reckoned among the examples of aggregative multicellularity in an article by N.A. Lyons and R. Kolter in Current Opinions in Microbiology. This indicates the intention to fit biofilms in a current paradigm of multicellularity studies. In this regard, biofilms are somewhat similar to Jeongeupia sacculi with their uncertain place in the general "theory of multicellularity". 

Viktor Kotiuk, MD, PhD, Ukrainian orthopedic surgeon from Carolina Medical Center (Warsaw, Poland), assumes that this fact could attract the attention of medical specialists in surgical disciplines. In his comment on my Elementy.ru article on social media, he expressed an opinion that the fundamental studies of the nature of multicellularity are significant even for clinicians working in applied field only: 

"Actually, doctors should know more about such things to better understand how bacteria adapt inside the body as well. At least how they can adapt. In the past, we treated infectious complications simply by isolating the pathogen and determining its sensitivity in vitro, but now we are taking into account all kinds of biofilms, which, although not multicellular organisms, are much more complex and sophisticated systems than single bacteria, and in which antibiotics no longer "want" to act as in vitro, and some bacteria from biofilms cannot be identified by standard sampling and seeding methods at all. I think the study of such cellular systems will make it possible to find something similar elsewhere, and possibly in the human body as well. But at the very least, it will improve our understanding of the adaptability of bacteria".

This comparison to biofilms raises one more problem. Till recently, we thought about multicellularity like about a binary parameter that could be present or absent. An organism could be unicellular or multicellular, and tertium non datur, even taking into account primitive forms of multicellularity like dictyostelids and cyanobacteria. But, if we regard biofilms from the "multicellular" point of view, it will turn out that a huge amount of bacteria are multicellular rather that unicellular, and multicellularity is much more common in this "simple" world that we have failed to notice.

And, if we could ignore this problem till recently, the discovery Jeongeupia sacculi urges to revisit it. This bacterium, which belongs to a "grey zone" between colonial and multicellular beings, challenges our view on these forms of existence. 

In terms of linguistics, "multicellular" is a relative adjective, and thus should not have any degrees of comparison. But a novel bacterium reminds us that there might be a complex continuum between unicellularity and multicellularity. It is a single species of this kind so far, but similar discoveries could be expected. And, maybe, one day we will have to say "more multicellular" or "less multicellular". Who knows? 





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