Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for autotrophic and heterotrophic bioprocesses. The genetic determinants that govern its biofilm formation, however, remain largely uncharacterised, particularly under heterotrophic conditions relevant to such processes.
In this work, the authors applied a forward genetics approach based on transposon enrichment to identify loci which promote growth on surfaces. A mini-Tn5 mutant library of 26,185 insertion clones, more than the 17,000 required to cover the whole genome, was cultivated as a biofilm on fructose in a microfluidic flow cell system for 168 hours, and the community growing on the surface was characterised by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several of them with documented links to biofilm formation in other bacteria, including the ferrous iron uptake system (feoA/feoB), galU and a protein with both GGDEF and EAL domains.
The gene B2043, which encodes this protein involved in the metabolism of c-di-GMP, was selected for validation by markerless deletion. Under static conditions, the deletion mutant formed 1.69 times more biofilm biomass than the wild type. Under continuous flow, the mutant attached faster, entered exponential growth about 10 hours earlier, reached its biovolume plateau about 16 hours earlier than the wild type and formed distinct structures resembling towers.
The results identify B2043 as a negative regulator of biofilm formation that acts predominantly during attachment and provide the first experimental evidence for the regulation of biofilm formation by c-di-GMP in C. necator H16. Together with eleven further candidate loci, they establish a functional genomics framework for engineering productive biofilms in this organism.
The study was carried out at the Institute of Technical Microbiology at Hamburg University of Technology within the Collaborative Research Centre CRC 1615 (SMART Reactors).
Janek René Weiler, Christian Jonas Lapp, Johannes Gescher, Miriam Edel (2026). Identification of genetic determinants that promote biofilm growth under heterotrophic conditions in Cupriavidus necator using transposon enrichment. Biofilm 12, 100395.
