Both widespread PEP‐CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP‐CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide‐spread family of high copy number‐per‐genome PEP‐CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK‐PrsR two‐component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc‐forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post‐translational modifications, could not rescue the long‐rod morphology of the rpoN mutant. Consistently, PEP‐CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc‐forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.