Skip to main content

Host-microbe interactions involving microbial dark matter: Biology and evolution of a ubiquitous group of intracellular bacteria.

Periodic Reporting for period 1 - Hidden life (Host-microbe interactions involving microbial dark matter: Biology and evolution of a ubiquitous group of intracellular bacteria.)

Reporting period: 2017-01-01 to 2018-12-31

The kingdom Bacteria represents the most widespread and diverse life form on Earth, and is playing major roles, from regulation of biogeochemical cycles, to pathogenesis. The vast majority of bacterial representatives are yet unculturable, being thus considered “microbial dark matter” (MDM). While metagenomics investigations have provided insights into MDM diversity and possible importance for ecosystems functioning, its ecology and biology remains largely unexplored.
Indubitably, there is a raising interest for questions asked by MDM presence in the environment. The understanding and characterization of MDM is of primary concern in the field of bacteriology and microbial ecology. MDM representatives are thought to represent a large –if not the major– fraction of bacterial diversity, yet their ecology, biology and role in different ecosystems has been barely investigated. Therefore, MDM representative could have major implication in crucial processes, such as geo biochemical cycles, and pathogenicity towards humans or (micro)organisms of economic interest.
TM6 is a candidate phylum standing as a prominent part of the MDM. Members of TM6, like many other MDM phyla, are described as small sized bacteria harboring reduced genomes and being unable to cultivate axenically in laboratory conditions. TM6 were first discovered in peatbogs (from the German Torf, Mittle Schicht, hence the designation TM6), but numerous subsequent studies reported TM6 occurrence worldwide in diverse environments such as soils, sediments, fresh and marine water and biofilms. In drinking water biofilms, TM6 was shown to represent up to 11% of the bacterial community, and members of this phylum were repeatedly found from drinking water distribution systems.
The overall goal of the project was to uncover the ecology and biology of TM6 bacteria, as a phylum model of MDM. A targeted analysis of a range of environments enabled the assessment of TM6 ecological niches, while genomic characterisation provided useful information on the evolutionary history, as well as the biology of these poorly understood bacteria. Finally, the comprehensive analysis of the lifecycle of selected TM6 isolates uncovered original mechanisms involved in infection, replication, host interaction, and host resources subversion. This project provided the first detailed analysis of the yet-unknown life style of a major MDM phylum.
Since the beginning of the project, several tasks from the work packages described within the proposal were successfully completed.

We collected 450,000 rRNA gene sequences related to the TM6 phylum, originating from publicly available metagenomes. Using this dataset along with associated environmental metadata, we showed that TM6 is distributed in a wide range of environments, although it occurred at highest frequencies in soil, fresh and marine water, as well as sediments. Interestingly, these environments represent hotspots for protists such as amoebae, which could serve as hosts for supporting TM6 growth. By extracting all TM6 sequences covering the variable region V4 of the 16S rRNA gene, we then showed that TM6 is a moderately diversified phylum, comprised of 1,390 operational taxonomic units at 97% similarity.

In parallel, TM6 whole genomes were recovered from publicly available metagenomes, and the TM6 isolate available was sequenced in house. In total, 22 (near)complete TM6 genomes were collected for further investigations. By means of comparative genomics, we determined that all TM6 genomes analyzed were extremely reduced in their metabolic capabilities. In line with these observations, a wide array of transporters involved in aminoacid and energy import were identified, allowing the bacteria to efficiently subvert host resources for replication. These features clearly indicate that TM6 are extremely dependent on other (micro)organisms for thriving, and are thus adopting a parasitic lifestyle.
In parallel, using the TM6 model Vermiphilus pyriformis that we were able to grow and maintain in laboratory conditions, we described in details the bacterium lifecycle within its natural host, the amoeba Vermamoeba vermiformis. Using fluorescence in situ hybridization along with confocal microscopy, a detailed analysis of the infection process was performed, allowing to characterize and better understand the replication cycle of TM6 bacteria. Using this approach, we showed that V. pyriformis completes its replication cycle 48 hours, and efficiently colonizes the whole amoebal population. V. pyriformis also showed dramatic morphological changes during its replication, transiting from a small infection stage (400 nm), to a filamentous replication phase (up to 5µm), before switching back to an infectious phase, preceding the bacterial excretion from the amoeba host. These features highlight an unusual cell division process.
The progress achieved during the project were presented in form of a poster at the 16th International Symposium of Microbial Ecology (ISME), held at Montréal in 2016. An oral presentation of this work was also done during the “Let’s talk about Symbiosis” workshop organized by the University of Vienna and the Medical University of Vienna, in 2016. Actions for outreach were also undertaken, notably a participation to the 2017 edition of KinderUni (

Ultimately, this MSCA funded project allowed me to integrate a new and stimulating laboratory. This experience greatly expanded my professional network, as well as the array of techniques used in my field of expertise. Thanks to this, I successfully passed the selection, and was appointed to a permanent position of Assistant Professor at the University of Poitiers, hence the anticipated termination of the present project.
The MSCA support greatly helped me to become an independent researcher, by obtaining a position in full adequation with my career goals.
The results generated by the project yielded several unexpected results that are furthering the knowledge in the ecology and biology of TM6 phylum.
Our approach allowed to massively collect TM6 related sequences and their associated environmental data. It resulted in the most precise and comprehensive description of TM6 environmental tropism, which was previously fragmented and poorly understood. This environmental cartography will allow to target specific environments for greater chance of TM6 isolation, thus contributing to a wider understanding of the TM6 phylum.
Through a comparative genomics approach, we also showed that TM6 most certainly use an unprecedented strategy to initiate and maintain its interaction within their hosts. These results are of high importance, as they describe an original bacterial adaptation to intracellular life. These mechanisms are of prime importance for bacteria to colonize and thrive within eukaryotic hosts. These strategies are found among various bacteria, which include environmental bacteria, as well as several pathogens of humans and other macro-organisms of economic importance.
Using one of the only TM6 isolate available for cultivation in laboratory conditions, we also found evidence for an unusual bacterial cell division process, which suggest that TM6 may contribute to a better understanding of alternative pathways and mechanisms involved in bacterial replication.
Fluorescent in situ hybridization of the amoeba Vermamoeba vermiformis infected by TM6 bacteria