Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

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Abstract
Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha - and Betaproteobacteria and Actinobacteria . A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea , and Aquaspirillum -related organisms) and Actinobacteria -related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes ), some Betaproteobacteria (e.g ., Aquaspirillum -related filaments), Gammaproteobacteria ( Thiothrix ), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae ). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.