With a Little Help From My Microbes

Mussels in the deep sea can only survive there thanks to symbiotic bacteria living inside of them. Researchers at the Max Planck Institute for Marine Microbiology in Bremen have now succeeded for the first time in simultaneously identifying individual bacteria in the symbiosis and measuring which metabolites they convert. This enables a new understanding of many biological processes. The researchers now present their results in Nature Mi­cro­bi­o­logy.

Bac­teria in our en­vir­on­ment can be dif­fi­cult to study: They are tiny and of­ten live un­der con­di­tions hard to re­cre­ate in the lab, for ex­ample in the deep sea or as sym­bionts in an an­imal host (or both, as the sym­bi­otic bac­teria in the present study). In­vest­ig­a­tions of the bac­terial gen­ome tell us what the mi­crobes are the­or­et­ic­ally cap­able of. What they ac­tu­ally do, however, is not re­vealed. Thus, sci­ent­ists study the so-called meta­bolome of the bac­teria: It com­prises every meta­bol­ite the cells pro­duce or con­sume, for ex­ample pro­teins, sug­ars or fats.

A team of re­search­ers around Be­ne­dikt Geier and Manuel Liebeke from the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy in Bre­men has now de­veloped a method to identify in­di­vidual bac­teria and at the same time de­term­ine which meta­bol­ites are present in the cells. With the new method they in­vest­ig­ate how bac­teria live and sur­vive as sym­bi­otic ten­ants in deep-sea mus­sels. Liebeke and his group ana­lysed hun­dreds of meta­bolic products on an area smal­ler than one square mil­li­meter. This en­ables them to un­der­stand how the sym­bi­otic mi­crobes live and com­mu­nic­ate in their host. “We vir­tu­ally take a snap­shot of bac­teria at work – just as it func­tions in its nat­ural en­vir­on­ment, here within a single an­imal cell,” says Liebeke. “And we can do this with an im­press­ive res­ol­u­tion of a few mi­cro­met­ers, about ten times thin­ner than a hu­man hair.”

Snap-frozen for the best snap­shot: Not just what is hap­pen­ing, but also who is in­volved!

"For our ana­lyses, we use mus­sel tis­sue that has been snap-frozen and can thus be cut into wafer-thin slices,“ Be­ne­dikt Geier ex­plains. "From these slices, we take a snap­shot of the chem­ical com­pounds of the cells us­ing a spe­cial mass spec­tro­metry tech­nique called MALDI-MS ima­ging. When ana­lys­ing this snap­shot in de­tail, we are able to dis­tin­guish many dif­fer­ent meta­bol­ites on a very small area.“ They provide in­form­a­tion about which meta­bol­ites the bac­teria use for what pur­pose and how they co­habit with their mus­sel host. In ad­di­tion to the MALDI-MSI at the Max Planck In­sti­tute in Bre­men, Liebeke and his team used a new MS ima­ging pro­to­type at the Jus­tus Liebig Uni­versity in Gießen in close co­oper­a­tion with Pro­fessor Spen­gler, which en­abled par­tic­u­larly high-res­ol­u­tion in­sights.

Cor­rect con­clu­sions from the im­ages of the meta­bol­ites are only pos­sible if we also know who pro­duces or uses them. “To date, we have only been able to meas­ure the meta­bol­ites," ex­plains Geier, "but we did not know whether any bac­teria were in­volved and if so, which ones.” To solve this prob­lem, the re­search­ers ad­ded a second tech­nique, the so-called fluor­es­cence in situ hy­brid­iz­a­tion, or FISH, to identify in­di­vidual bac­terial cells in the same sample. “The com­bin­a­tion with FISH was the key for us to in­ter­pret the high-res­ol­u­tion MALDI-MS im­ages in a mean­ing­ful way and cor­rel­ate them with the bac­teria in the mus­sel tis­sue.”

From the deep sea, now on cam­era

For the present study, Geier and his col­leagues used samples from black smokers in the deep sea – tower­ing chim­neys where hot, min­eral-rich wa­ter gushes out of the sea­floor. An­im­als and bac­teria can only sur­vive there in sym­bi­otic com­munity. Geier in­vest­ig­ates the co­ex­ist­ence of bac­teria and mus­sels, in par­tic­u­lar the close link­age of their meta­bol­ism, as part of his PhD-thesis. With the new method, he was able to show that the com­pos­i­tion of lip­ids in the mus­sel dif­fers sig­ni­fic­antly in body re­gions with and without bac­terial ten­ants. “Up to now, we were not able to gain such in­sights as the samples were ho­mo­gen­ised, that is vir­tu­ally blen­ded, be­fore ana­lysis,“ Geier ex­plains. “Moreover the fact that our method works on samples com­ing dir­ectly from the en­vir­on­ment and not from the lab un­der­lines its great po­ten­tial,“ he con­tin­ues.

All plants and an­im­als as well as us hu­mans live in as­so­ci­ation with mi­croor­gan­isms, shar­ing meta­bol­ites through close in­ter­ac­tions. “Ap­ply­ing this method in other host-mi­crobe in­ter­ac­tions will al­low for many ex­cit­ing new in­sights into the secret life of mi­crobes. I am curi­ous to see whether we can use this ima­ging ap­proach for look­ing into the shared chem­istry between mi­crobes and or­gans in whole an­im­als. There is still a lot to dis­cover!”

Reference
Benedikt Geier, Emilia M. Sogin, Dolma Michellod, Moritz Janda, Mario Kompauer, Bernhard Spengler, Nicole Dubilier, Manuel Liebeke. Spatial metabolomics of in situ host–microbe interactions at the micrometre scale. Nature Microbiology, 2020; DOI: 10.1038/s41564-019-0664-6.

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