Department of Molecular Ecology

Inspiration and focus of our department

Long be­fore plants and an­im­als had evolved, single-celled mi­croor­gan­isms have shaped Earth and ever since they re­mained es­sen­tial for the hab­it­ab­il­ity of our blue planet. Whereas only thou­sands of spe­cies of Bacteria and Archaea have been de­scribed by cul­tiv­a­tion-based meth­ods, we are identi­fy­ing mil­lions with mo­lecu­lar techniques. Today high through­put se­quen­cing is provid­ing ample data on spe­cies rich­ness and even­ness, yet quan­ti­fic­a­tion of the in­di­vidual spe­cies re­mains dif­fi­cult. Here we ap­ply fluor­es­cence in situ hy­brid­iz­a­tion for the mi­cro­scopic iden­ti­fic­a­tion of single mi­cro­bial cells and their loc­al­iz­a­tion in the en­vir­on­ment. Functional assays, i.e. tracing the uptake of substrates by single cells, together with com­par­at­ive gen­ome ana­lyses al­low us to de­duce the ecological role and bio­chem­ical po­ten­tial of not yet cul­tiv­ated Bacteria and Archaea. Beyond quantitative descriptions of microbial communities of individual samples, we use long term datasets to predict the functional niches and annual recurrence of marine microbes. Besides a full taxonomic description, the predicted niches are confirmed by isolation and physiological characterisation of strains.

Uncultivated microbes in need of their own taxonomy

Taxonomy encompasses the identification, classification and nomenclature of organisms. As such, taxonomy is a prerequisite for ecology. Only based on accurate taxonomic concepts and methods, can the diversity and composition of complex microbial communities be accurately described and monitored. Our department has a long tradition of developing and applying new taxonomic methods. We have pioneered the in situ identification, quantification and localization of not-yet cultivated bacteria and archaea by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes (Amann et al. 1995; Amann & Fuchs 2008). We have a long-standing collaboration with Wolfgang Ludwig and Ralf Westram, the main developers of the ARB program that is widely used for the reconstruction of 16S rRNA-based phylogenetic trees and for the design of oligonucleotide probes. Emerging from that program, we also have long been the home of the much-used, curated 16S rRNA database SILVA (Quast et al. 2013; Yilmaz et al. 2014). This has now been transferred to the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. 

Since the majority of microorganisms have not yet been cultivated, there was a need for thresholds to assess whether their 16S rRNA fingerprints indicate the existence of new species, genera, families, orders, classes or even new phyla. We contributed to widely used standards of 16S rRNA-based classification (Yarza et al. 2014). Today, the genomic revolution is enabling very detailed descriptions of new taxa without cultivation, and it is evident that order must be brought to the rather uncontrolled alphanumerical naming of "novel" (Konstantinidis et al. 2017). Single cell genomics and/or metagenome-assembled genomes, often combined with single cell identification by FISH, discloses the true diversity of microorganisms, which is breathtaking, yet certainly not endless (Amann & Rossello-Mora 2016). Currently, others and we have identified a pressing need to do proper taxonomy of abundant and functionally important clades of environmental bacteria and archaea. As such, we have recently described several new marine taxa including the species Candidatus Prosiliicoccus vernus (Francis et al. 2019) and the genus Candidatus Abditibacter (Grieb et al. 2020). However, since the rank of Candidatus is preliminary and the nomenclature lacks priority, others and we are convinced that genomic information needs to be accepted as type material for the permanent description of novel microorganisms (Konstantinidis et al. 2020). Ecologists and taxonomists require a roadmap for naming uncultivaed Archaea and Bacteria (Murray et al. 2020) that is compatible with the current Code of Nomenclature requiring live pure cultures as type material. We believe that this will not hinder the enrichment and cultivation work of others and us that is more needed than ever. We now not only know that something is out there but also can predict what it is doing, characterising its ecology while we wait for cultivation efforts to provide a more detailed inspection. These "ghosts" have names and we know their genomes.

Diversity, visualisation and cultivation

One of the primary ques­tions of ecology is 'who or what is there?'. In mar­ine mi­cro­bial eco­logy, we typ­ic­ally em­ploy high-through­put se­quen­cing of the 16S rRNA gene to provide a win­dow into the di­versity of the microbial com­mu­nity. From this, we can de­term­ine the key mi­cro­bial taxa present and be­gin to for­mu­late hypotheses about eco­lo­gical pro­cesses tak­ing place. More re­cently, a sig­ni­fic­ant ad­vance­ment in long-read se­quen­cing tech­no­logy, with the advent of the PacBio Sequel II platform, now allows us to se­quence the ge­netic ma­ter­ial from en­vir­on­mental pop­u­la­tions (meta­ge­n­om­ics) and sim­ul­tan­eously re­trieve full-length 16S rRNA genes. In com­par­ison to short 16S rRNA gene amp­l­ic­ons, these full-length se­quences can be used in more ro­bust and ac­cur­ate phylo­gen­etic ana­lyses. 

The next fun­da­mental com­pon­ent of ecology is assessing the abund­ance of a spe­cific pop­u­la­tion in the en­vir­on­ment. Trivi­ally speak­ing, the more in­di­vidu­als there are, the more im­port­ant they are for the eco­sys­tem and the higher the im­pact on the nu­tri­ent cycle, re­source ex­plor­a­tion and meta­bolic in­ter­ac­tions. This is where our method FISH is an in­dis­pens­able tool for the enu­mer­a­tion of taxo­nom­ic­ally well-defined mi­croor­gan­isms. As most mi­crobes lack a con­spicu­ous mor­pho­logy, the only re­li­able marker to dis­tin­guish between them is the ri­bosomal RNA mo­lecule, which is highly con­served and abund­ant in every or­gan­ism. Us­ing small fluor­es­cently la­belled oli­go­nuc­leotides com­ple­ment­ary to the rRNA, we can tailor probes spe­cific for the taxa of in­terest to visu­al­ize and count them by epi­fluor­es­cence mi­cro­scopy. Be­sides a nu­mer­ical quan­ti­fic­a­tion, FISH provides a clue about cel­lu­lar struc­tures and phys­ical in­ter­ac­tions with other mi­crobes. We can use high-res­ol­u­tion mi­cro­scopy like Con­focal Laser Scan­ning Mi­cro­scopy (CLSM) and STim­u­lated Emis­sion De­ple­tion (STED) microscopy to study intracellular properties or the uptake of, e.g., substrates. Using FISH and high-resolution microscopy, we can determine accurate cell-sizes and biovolumes of uncultivated microbes.

Though we can ob­tain ample amounts of in­form­a­tion about the eco­logy of mi­croor­gan­isms from en­vir­on­mental samples, we ultimately still require pure cultures or enrichments in the lab. Cul­tiv­a­tion is still, in most cases, the only way to ex­per­i­ment­ally verify pre­dic­tions and hy­po­theses for­mu­lated from ge­n­omic se­quence in­form­a­tion. However, the isol­a­tion of mar­ine mi­croor­gan­isms can be a chal­len­ging task, as it is dif­fi­cult to re­cre­ate the nat­ural en­vir­on­ment and spe­cific con­di­tions re­quired for a pop­u­la­tion to grow. There­fore, we use li­quid me­di­ums and en­rich­ments along with phys­ical sep­ar­a­tion of in­di­vidual cells through di­lu­tion and mo­lecu­lar iden­ti­fic­a­tion meth­ods (in house de­veloped spe­cific PCR and in situ hy­brid­isa­tion probes) to isol­ate abund­ant mi­crobes from nat­ural com­munit­ies. Once a strain is in pure cul­ture, the source is un­lim­ited and physiolo­gical traits and in­di­vidual en­zymes can be stud­ied.

Functional characterisation