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

Abstract

Some bacteria are capable of forming flocs, in which bacterial cells become 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 extracellular polysaccharides 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 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 a 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 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 remaining tightly bound to cells, as observed during the flocculation of the 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 via exopolysaccharides and/or other biopolymers.

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

“…On the other hand, little EPS was extracted from the culture supernatants of the MMBΔ rpoN mutant, which is inconsistent with our previous finding that the transposon insertional rpoN1 (the rpoN ortholog) mutants of A. tertiarcarbonis RN12 released large amounts of exopolysaccharides into the culture broth (Yu et al ., ). This inconsistency may be due to the severe growth defect and pleiotropic effects caused by the deletion of the only rpoN gene in Z. resiniphila MMB while other three rpoN paralogs could somehow substitute for the cellular functions of rpoN1 in A. tertiarcarbonis RN12.…”

“…Previously, we have demonstrated that the bound exopolysaccharides of wild‐type A. tertiaricarbonis RN12 strain and the released polysaccharides extracted from culture supernatants of the floc‐forming deficient rpoN1 mutant are highly similar in the monosaccharide composition and chemical structure (Yu et al ., ). Similarly, GC‐MS results showed that the bound EPS, extracted from the cell pellets of the wild‐type MMB strain, and released soluble EPS, extracted from culture supernatants of MMBΔ prsK and MMBΔ prsR mutants, were similarly composed of glucose, galactose and mannose (Supporting Information Fig.…”

“…Wild‐type and mutant strain were grown at 28°C with shaking (220 rpm). EPS of wild type and mutant was extracted from both cell pellets and culture supernatants as previously described (An et al ., ; Yu et al ., ). The EPS samples were precipitated by adding three volumes of ethanol per sample volume, and dried by freeze‐drying for Fourier transformed‐infrared (FT‐IR) spectroscopy analysis (Hong et al ., ) as well as TMS derivatizaion and GC‐MS analysis (Ng et al ., ; York et al ., ).…”

“…S6). Recently we had demonstrated that one of the four RpoN paralogues, RpoN1, regulated the floc formation but not exopolysaccharide biosynthesis in a flocforming A. tertiaricarbonis RN12 strain (Yu et al, 2017). The synthesized exopolysaccharide chains were not tightly bound to the bulk of cell aggregates but were released into the cultivation broth.…”

“…Deletion of either prsK or prsR gene resulted in floc formation deficiency, which prompted us to generate the rpoN mutant. Deletion of rpoN led to pleiotropic phenotypes as we previously demonstrated in a floc‐forming Aquincola tertiaricarbonis strain (Yu et al ., ). A regulatory circuit for PEP‐CTERM gene transcription emerged in our previous and current analyses.…”