Structure and community composition of sprout-like bacterial aggregates in a Dinaric Karst subterranean stream

The Vjetrenica cave in the Dinaric Karst hosts a worldwide extraordinarily high cave biodiversity. Beside a diverse and specialized cave fauna, sprout-like formations attached to the bed of the cave stream were observed and described, but not further characterized, almost a century ago. Here we investigated these sprout-like microbial aggregates by the rRNA approach and detailed microscopy. Based on fluorescence in situ hybridization and ultrastructural analysis, the sprout-like formations are morphologically highly organized, and their core consists of a member of a novel deep-branching lineage in the bacterial phylum Nitrospirae. This organism displays an interesting cellular ultrastructure with different kinds of cytoplasmic inclusions and is embedded in a thick extracellular matrix, which contributes to the stability and shape of the aggregates. This novel bacterium has been provisionally classified as "Candidatus Troglogloea absoloni." The surface of the sprout-like aggregates is more diverse than the core. It is colonized by a bacterial biofilm consisting primarily of filamentous Betaproteobacteria, whereas other microbial populations present in the crust include members of the Bacteriodetes, Gammaproteobacteria, Actinombacteria, Alphaproteobacteria, and Planctomycetes, which are intermingled with mineral inclusions. This study represents the first thorough molecular and ultrastructural characterization of the elusive sprout-like bacterial aggregates, which are also found in other cave systems of the Dinaric Karst. The discovery of Ca. Troglogloea absoloni contributes to the known biodiversity of subterranean ecosystems and especially of macroscopic structures formed in caves by microorganisms, whose composition and ecological function often remain enigmatic.