RpoN (σ 54 ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

Some bacteria are capable of forming flocs in which bacterial cells are self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here we use a floc-forming bacterium, Aquincola tertiaricarbonis RN12, as a model to explore the biosynthesis of EPS and the regulation of floc-formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in the floc-formation-deficient mutants generated by transposon mutagenesis and the gene functions have been further confirmed by genetic complementation analyses. Interestingly, the biosynthesis of exopolysaccharides remained in the rpoN1 -disrupted flocculation-defective mutants, but most of the exopolysaccharides were secreted and released rather than bound to the cells. Furthermore, the expression of exopolysaccharide biosynthesis genes seemed not to be regulated by RpoN1. Taken together, our results indicate that RpoN1 may play a role in regulating the expression of certain gene(s) involved in the self-flocculation of bacterial cells but not in the biosynthesis and secretion of exopolysaccharides required for floc-formation. IMPORTANCE Floc formation confers bacterial resistance to predation of protozoa and plays a central role in the widely used activated sludge process. In this study we have not only identified a large gene cluster for biosynthesis of extracellular polysaccharides, but also identified four rpoN paralogues, one of which ( rpoN1 ) is required for floc formation in A. tertiaricarbonis RN12. In addition, this RpoN sigma factor regulates the transcription of genes involved in biofilm formation and swarming motility as previously shown in other bacteria. However, this RpoN paralogue is not required for the biosynthesis of exopolysaccharides, which are released and dissolved into culture broth by the rpoN1 mutant rather than tightly bound to cells as observed during the flocculation of wild-type strain. These results indicate that floc formation is a regulated complex process and other yet-to-be identified RpoN1-dependent factors are involved in self-flocculation of bacterial cells by exopolysaccharides and/or other biopolymers.