The bacterium, HS-3, was isolated from a limestone cave wall that is intermittently submerged by an underground river. HS-3 has two distinct life phases; on a solid surface it self-organises into a layer-structured colony with the properties of a liquid crystal. After maturation, the HS-3 colony forms a semi-closed sphere housing clusters of ‘daughter’ coccobacillus (short rod-shaped) cells, which are released upon contact with water.
“The emergence of multicellularity is one of the greatest mysteries of life on Earth,” states corresponding author Kouhei Mizuno, a professor at the National Institute of Technology (KOSEN), Tokyo, Japan. “The point is that we already know the superior function and adaptability of multicellularity, but we know almost nothing about its origins. Established function and adaptability are not necessarily their own formative driving force. A curiosity of multicellularity is the conflict between the ‘benefits of individuals’ versus the ‘benefit of the group’ that must have existed in the early stage of the evolutionary transition. We don’t have a good existing model to study multicellularity except theoretical models.”
One such model is ‘ecological scaffolding’, which proposes that environmental factors provide a selection pressure to an evolving population, suggesting that Darwinian natural selection is applicable even to unicellular organisms.
Mizuno and his lab student Ohta first discovered HS-3 in 2008, when they isolated it from dripping water on a limestone cave wall in northern Kyushu Island, Japan. They were originally searching for lipid accumulating bacteria, but, when checking old agar plates of bacteria before disposal, Ohta noted a small colony with an unusually beautiful colour and texture. Most bacteria on agar show an opaque texture due to their disordered structure, but this colony appeared transparent, with an iridescent hue. Phenotypic comparisons with closely related species confirmed this colony as a novel species, HS-3, which the team gave the name Jeongeupia sacculi (meaning ‘cradle’).
The team used microscopies to analyse the colony growth. The cells started to reproduce simply as coccobacilli, but the occurrence of cell elongation caused the colony to form a single-layered structure, orientated like a liquid crystal. Bulges form particularly at the colony edge, relieving internal pressure and granting HS-3 the unique ability to maintain this two-dimensional liquid arrangement for a prolonged period, which may be a prerequisite for HS-3 to establish muliticellular behaviour.
Then, the colony then expanded to form additional layers. The internal filamentous cells buckled, generating vortex-structured domains. These domains and the liquid crystal-like arrangement explain the transparency observed in HS-3 colonies on agar. After two days, rapid cell reproduction occurred internally and the colony began to swell three-dimensionally, forming a semi-closed sphere housing the coccobacillus cells. After the fifth day, the internal cells were crowded-out of the colony, triggering a chain reaction of this event in adjacent colonies and thereby indicating some multicellular control.
As the cave wall sampling site of HS-3 was regularly subject to flowing water in the cave, the team submerged the mature semi-sphere colonies in water. The internal coccobacilli were released into the water, leaving behind the filamentous cell architecture. By plating these daughter cells on fresh agar, they discovered that the cells were able to reproduce the original filamentous structure, showing that the two distinct phases of HS-3’s life cycle are reversible, and may have arisen due to the changing conditions inside the cave.
“We needed 10 years to be sure that this was not a contamination of two different species and that it was not just a mutation.” says Mizuno. “First, we used a series of microscopic observations to film the entire process from a single cell to a colony, for which we developed our own methods. Then, we found that the morphological changes in cells and colonies were both controlled and reversible. Those data led us to believe that it is a ‘multicellularity’ of HS-3.”
“The first stage of HS-3’s life cycle suggests that the liquid crystal-like organisation is involved in the emergence of multicellularity, which has not been reported before. The existence of the second life stage implicates the involvement of dynamic water environment in the emergence of HS-3’s multicellularity” says co-corresponding author Kazuya Morikawa, a professor in the Division of Biomedical Science, University of Tsukuba, Japan.
“We have been surprised by the various curious properties that HS-3 encompasses, one of which is that the multicellular behavior of this new species fits well with the recently proposed ‘ecological scaffolding’ hypothesis. We now think that the leap towards multicellularity would be more elaborate and beautiful process than the one we have imagined so far.” commented Mizuno and Morikawa.
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