SAR11 are the most abundant microbes on Earth, dominating marine ecosystems and standing as icons for genome-streamlined oligotrophs. However, less is known about the freshwater lineage SAR11-IIIb (also known as LD12 lineage or ‘Ca. Fonsibacter’1, later renamed to ‘Ca. Allofontibacter’ and now once more to its hopefully final name Fontibacterium), mainly because of challenges in cultivation. While marine SAR11 were cultivated over 20 years ago2, the first freshwater SAR11-IIIb strain wasn’t isolated until 20181. These ultra-small, strictly pelagic oligotrophs don’t grow on agar plates or in rich media and their reduced (streamlined) genome includes multiple auxotrophies that add to other unknown growth requirements.
In this blog post, I won’t focus much on the results of our study but will instead share the background stories behind our global sampling campaigns. Those interested in the ecology and biogeography of Fontibacterium can refer to our recent study in Nature Microbiology.
Just a quick wrap-up: We isolated seven new strains, tested hypotheses derived from genomic analyses in growth assays, and conducted global and seasonal metagenomic analyses including unpublished time series datasets (LIMNOS) kindly provided by our collaborator Hans-Peter Grossart. Our global sampling focused mainly on the underrepresented Southern Hemisphere and yielded many high-quality MAGs (metagenome-assembled genomes), allowing detailed phylogenomic analyses, the description of several new species, and the discovery of endemic species in the African Great Lakes and species confined to the Northern or Southern Hemisphere.
The plan
We were fortunate to isolate several Fontibacterium strains by extensive dilution-to-extinction cultivation a few years back3, and since these microbes are highly abundant in freshwater lakes (accounting for up to 1/3 of all microbes)4, I decided to write a grant proposal for the Czech Science Foundation in spring 2021. Gathering all publicly available genomes of freshwater SAR11-IIIb revealed two things: High-quality genomes are rare (as these abundant, microdiverse microbes are hard to assemble from short-read metagenomes), and there are huge white spots on the global map, mainly on the Southern Hemisphere. At this time, only one published culture from the USA existed1, along with a handful of SAGs and MAGs from a few lakes in North America, Europe, Asia, and one MAG from Lake Tanganyika in Africa5. The Tanganyika MAG was deeply branching in an initial phylogenomic tree, but the data remained inconclusive as it was represented by only a single genome. Therefore, I planned to sample another African Great Lake (Lake Malawi) and diverse freshwater and brackish sites on the under-sampled Southern Hemisphere (along with a transect on the Baltic Sea, not used in this study). The main goal was to increase the number of high-quality genomes on a global scale through combined long- and short-read metagenome sequencing. As we already had several axenic strains, my PhD student Clafy Fernandes could also start right away with detailed lab experiments and genomic analyses. Luckily, the proposal was granted, and we started our sampling campaigns in 2022.
The global hunt: Behind the scenes
Our journey began close to home, and thanks to experience from many lake sampling projects in Europe (the paper also includes data from Central European lakes sampled earlier), our trip to Northern Poland was a piece of cake. We (Rohit Ghai, Fran Kostanjšek and I) caught a night train to Gdynia, where our friend Kasia Piwosz from the National Marine Fisheries Research Institute awaited us. She arranged a research vessel for Rohit, Fran, and her colleague Adam Woźniczka to sample the Vistula Lagoon, while Kasia, Mariusz Zalewski and I took water samples from the Vistula River and the coastal Baltic Sea. Everything went smoothly, and as we finished lab work in one day, we had some spare time to explore Gdańsk and enjoy a sailing trip with Kasia’s husband Michał.
In autumn 2022, we traveled to Japan to sample four deep lakes with our collaborators Yusuke Okazaki and Shin-ichi Nakano from Kyoto University. Everything was meticulously organized by Yusuke, all permits were taken care of, and even a typhoon passing Kyoto caused only a few days delay. A research vessel was organized for Lake Biwa, and afterwards our sampling equipment was shipped ahead while we (Markus Haber, Cecilia Chiriac – both accompanied by their partners – Yusuke, his colleague Shin Sho and I) took the bullet train from Kyoto to the Fukushima region to Lakes Inawashiro and Chuzenji and then further north to Lake Toya on Hokkaido Island. For deep lake sampling, Yusuke used his custom deep-water sampler (essentially a tuna fishing rod with a 5-l Niskin bottle) to “fish for microbes”. Besides sampling and admiring the beautiful landscapes, we visited many cultural sites in Kyoto, Osaka and Tokyo and Yusuke and Shin introduced us to the fantastic Japanese cuisine with Sushi, Sashimi, Ramen, Okonomiyaki, Takoyaki, Tempura, Tonkatsu, Wagyu steaks and many more delicacies.
Next came Australia, where we selected eight distinct freshwater and brackish lakes in collaboration with Dave Hamilton from the Rivers Institute in Brisbane. Sampling was a bit more challenging; we (Clafy Fernandes, Paul Layoun and I) rented a canoe to sample two reservoirs near Brisbane, bought a small inflatable raft, and went on a road trip along the Sunshine Coast from Brisbane to Bundaberg. Three of the sites were on K’gari (Fraser Island), that is famous for its dune lakes, and luckily, we received sampling permits and the blessings from the Butchalla people just in time. The dune lakes were spectacular, but I never thought that we would have to hike with our inflatable boat through rain forests and endless sand dunes to reach them - and to carry liters and liters of water for metagenomic filtration back to our lodge. Also, our raft turned out to be rather flimsy, first a paddle broke resulting in a troublesome time to reach the shore on Lake Boomanjin, and finally it sank during our last sampling on Lake Cootharabra. Fortunately, this lagoon is very shallow (~1 m), so I could simply walk back to the shore while dragging the remains of the raft with our precious water samples. We balanced these sampling adventures by chilling on beautiful beaches, observing kangaroos, opossums, dingoes, mantas, sharks and other native fauna in their natural habitat and enjoying delicious sea food.
Lake Malawi was originally scheduled for 2022 but postponed because I severely underestimated the time-consuming paperwork for all the permits (Nagoya Protocol) and public health concerns. Although COVID19 was no longer a threat in Europe, it was still an ongoing pandemic in Africa as vaccines were not supplied equally on the globe and, moreover, Malawi faced a Cholera outbreak after being hit by a tropical Cyclone in 2022. By autumn 2023, we had all permits and the conditions were safe. We (Markus Haber, Tanja Shabarova and I) headed to Malawi, where Maxon Ngochera and his PhD student Paul from the Department of Fisheries in Lilongwe joined us to four sites along a north-south transect. Maxon organized boats from local fishermen and a winch with 200 m rope. Lake Malawi was challenging to sample because of its immense size (580 km length, 700 m depth) and stormy weather with high waves forced us to repeatedly modify our schedule. Besides rough conditions causing sea sickness for some of us, we also faced other troubles, sometimes our YSI probe failed, sometimes the winch, but Malawian fishermen were very helpful and well trained, they easily hauled the 10-l Niskin bottle by hand from 200 m depth. The lake and surrounding landscape were breathtaking, and we also spent a considerable amount of time snorkeling and diving with the endemic cichlids and adoring the local fauna and flora (it’s a bird watching paradise), we enjoyed fresh local fruits and freshly caught fish with Nsima, and met the most friendly people. Unfortunately, we brought back more than great samples and souvenirs: Two of us were later diagnosed with schistosomiasis (parasitic flatworms), and I was also tested positive for strongyloidiasis; it seems that we spent just too much time in the warm and clear waters of Lake Malawi. Thankfully, we recovered swiftly after medical treatment.
Our final destination was South America. Also here, we had to considerably modify our initial plans, first we had to postpone the trip because of VISA issues, then we had to change the sampling sites. Originally, I planned to sample deep Patagonian lakes with collaborators from Bariloche in Argentina. However, after the elections in 2023, the new president significantly reduced scientific funding, and our colleagues were forced to cancel our planned visit because of severe budget cuts. Desperately, I reached out to Cecilia Alonso (a former colleague from my PhD times in Zurich), now at the Centro Universitario de la Región Este in Uruguay, and she immediately offered help by proposing to sample some very interesting coastal lagoons. As our flight tickets were already bought and Uruguay is easy to reach by ferry from Buenos Aires, we (Markus Haber, Vojta Kasalický and I) were on our way to South America three weeks later. Cecilia didn’t promise too much; the coastal lagoons were amazing, but also shallow and easy to sample by boat with local fishermen or by kayak; at Laguna Negra we could simply walk to our sampling spot, dragging the kayak with equipment behind us. Only one minor incident happened when the kayak flipped in Laguna de Briozzo, fortunatley, Juan Zanetti is not only microbial ecologist, but also a lifeguard and he swiftly rescued our sampling bottle from sinking. And as on all other sampling trips, we enjoyed great food (the best steaks ever!), stunning landscapes and even had a few extra days for sightseeing in Montevideo, Buenos Aires and the Iguazu Falls.
Back in Czechia, we sequenced 42 samples using both Oxford Nanopore and Illumina technologies. Our global endeavor resulted in a large collection of high-quality MAGs (incl. circular and near-complete genomes), nearly doubling the number of publicly available Fontibacterium genomes. Additional analyses of diverse other MAGs are ongoing, and we hope that our metagenomes from so far under-sampled regions will help resolve the global biogeography of many more taxa and are of great value to the scientific community. None of this would have been possible without our collaborators, thank you all for your invaluable support!
References:
1. Henson, M. W., Lanclos, V. C., Faircloth, B. C. & Thrash, J. C. Cultivation and genomics of the first freshwater SAR11 (LD12) isolate. ISME J. 12, 1846-1860 (2018).
2. Rappé, M. S., Connon, S. A., Vergin, K. L. & Giovannoni, S. J. Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418, 630-633 (2002).
3. Salcher, M. M. et al. Bringing the uncultivated microbial majority of freshwater ecosystems into culture. ResearchSquare (2025). https://doi.org/10.21203/rs.3.rs-6237413/v1
4. Salcher, M. M., Pernthaler, J. & Posch, T. Seasonal bloom dynamics and ecophysiology of the freshwater sister clade of SAR11 bacteria 'that rule the waves' (LD12). ISME J. 5, 1242-1252 (2011).
5. Tran, P. Q. et al. Depth-discrete metagenomics reveals the roles of microbes in biogeochemical cycling in the tropical freshwater Lake Tanganyika. ISME J. 15, 1971–1986 (2021).