Same same but different - Remarkable diversity behind identical 16S rRNA gene sequences

Co-existing strains of intracellular symbionts differ extensively in their gene content. Can this be an advantage for their host?

Go to the profile of Rebecca Ansorge
Oct 14, 2019
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Diversity matters – this likely applies to almost every aspect of our lives. It matters in our society, at work, in politics, in animals, plants and of course in bacteria. When it comes to measuring diversity in environmental communities, we are usually interested in three questions. Who are the members? What can they do? and What are they doing? For decades, microbial ecologists have been answering the first question using the gold standard 16S rRNA gene, a single marker sequence that is encoded by every bacterial cell. While I regard this approach to be extremely important as an assessment of community composition, I am always a little disappointed when this is where it stops. It’s like meeting a room full of people and knowing only their family name. I am mostly interested in their different jobs, talents and opinions. This is similar for bacteria. Knowing the 16S sequence does not always tell us their capabilities. For example, two highly related strains of the same bacterial species can mean the difference between making us sick or not1. But environmental microbial communities are often very complex and therefore it can be challenging to take a glimpse behind the 16S rRNA curtain.

The Dubilier lab studies animal-microbe symbioses using the deep-sea mussel Bathymodiolus as model system. These mussels are among the dominating fauna at deep-sea hydrothermal vents and cold seeps and harbor a very simple symbiont community, making them an ideal system to investigate symbiont diversity beyond 16S rRNA resolution. A Bathymodiolus species commonly associates with one or two 16S rRNA gene types, suggesting that it lives together with one or two symbiont types. These symbionts reside intracellularly in the mussels’ gills and use chemical energy that is plentiful in these habitats, such as reduced sulfur compounds, to produce their host’s nutrition in a process termed chemosynthesis. Although this symbiosis represents a great system for high-resolution analyses of the symbiont diversity, retrieving these mussels from their natural habitats far below the ocean surface is anything but simple. Surely, I did not mind this, as it did give me the opportunity to join two research expeditions to these habitats, get entirely fascinated by these stunningly beautiful and special environments and to learn about the importance of scientist diversity on a research vessel (oh yes, also here diversity matters!). The samples for this project originated from a huge collaborative effort involving a lot of people on different research cruises.

So, we investigated the micro-diversity of the sulfur-oxidizing symbiont type. Why? Well, there were a few former studies using marker sequences other than the 16S rRNA that indicated that there is some degree of heterogeneity beyond an identical 16S rRNA sequence2. But only after we sequenced the metagenomes of 18 mussels from 4 hydrothermal vent sites in the Atlantic, we realized that this diversity was much higher than we expected. Standard genome binning approaches did not resolve the different symbiont strains and thus most of our information relied on single nucleotide polymorphism data and sequencing coverage. I stared hours (or was it days?) at the polymorphism counts wondering what this diversity really meant. Does it matter? How many different symbiont types are there really? Do they have different roles? We finally found a method to determine the number of symbiont types based on polymorphism linkage and frequencies and from now on we referred to these types as ‘strains’. In fact, we found as many as 16 strains in single mussels. But every step was new territory for us, and we used simulations and different sequencing approaches to make sure we can trust this number. Imagine the room full of people – before we just knew that all of them have the same family name (identical 16S sequence) but now we found 16 people with a different first name. Nice to meet y’all! It was not long though, until that voice in the back of my mind (might have been the voice of my advisers) whispered ‘16 strains – so what?’. We kept digging and were really baffled when we found that co-existing strains differed extensively in the genes they encode (and express). This meant that not all these strains can do the same things but instead differ in many of their capabilities. For example, strain A can use hydrogen as an energy source whereas strain B in the same mussel cannot. So not only has every person in our imaginary room a different first name but they all have different talents.

This raised a whole array of new questions – how do all of these talents co-exist? Is there cooperation, or competition, or both – and above all, does this impact the host? According to most evolutionary theories we should not even be seeing this kind of symbiont strain diversity inside single hosts. Based on few well-studied model organisms competition for the same host-derived resource is expected to destabilize mutualistic symbioses. And this was where it all came together – the symbionts of Bathymodiolus mussels don’t get their energy resources from the host but from the environment instead. This means, keeping the symbionts satisfied seems to be a very cheap task for the host as long it keeps them in a ‘good spot’ full of reduced chemicals. Even better, having a consortium of symbionts with different skills may turn out as an advantage for the mussel that becomes more adaptable to the fluctuating conditions that are typical for these environments. So, in the end it seems as if it is all a matter of costs – as long as these are low for the host, a larger strain diversity can be tolerated and may prove advantageous. We are convinced that this is a principle that should also apply to other systems that involve low costs for the hosts, such as those where the symbionts’ energy comes from the environment. Indeed, strain diversity has been reported for the symbionts of corals that thrive on sun light3.

So yes, of course diversity matters. But how this diversity manifests itself and what impact it has in a community is a very complex topic. It is no longer enough to know who is there but to understand the degree, importance and functional implications of microbial diversity we need to step beyond 16S rRNA and combine information from theory and cultivated model organisms with the observations of populations in their natural environment. Even, or especially so, if this brings us to places like the deep sea…!

If you want to know more about the strain differences that we found in Bathymodiolus mussels or want to see how they are partitioned in the mussels’ gill tissue, check out our publication here.

Video material was recorded and is owned by MARUM - Center for Marine Environmental Sciences, University of Bremen

Like the Bathymodiolus mussel, also a study like this one is not the result of a single person but rather the symbiotic group effort of excellent scientists (with different names and talents), and everyone of them played an essential role to bring this all together. These were a lot easier to identify than the symbiont strains – thank you Stefano Romano, Lizbeth Sayavedra, Miguel Ángel González Porras, Anne Kupczok, Halina Tegetmeyer, Nicole Dubilier, and Jillian Petersen for bringing this project to life!

References

1. Ahmed, N., Dobrindt, U., Hacker, J. & Hasnain, S. E. Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention. Nat. Rev. Microbiol. 6, 387–394 (2008).

2. Won, Y.-J. et al. Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus. Appl. Environ. Microbiol. 69, 6785–6792 (2003).

3. Rowan, R. & Knowlton, N. Intraspecific diversity and ecological zonation in coral-algal symbiosis. Proc. Natl. Acad. Sci. 92, 2850–2853 (1995).


Poster image (c) Miguel Ángel González Porras

Go to the profile of Rebecca Ansorge

Rebecca Ansorge

PostDoc, Max Planck Institute for marine Microbiology

2 Comments

Go to the profile of Francisco Rodriguez-Valera
Go to the profile of Rebecca Ansorge
Rebecca Ansorge about 1 month ago

Thanks for the read! We do briefly discuss phages as one of the possible factors shaping the symbiont strain diversity in our system. Check out the supplement of the paper :)