Identification of genetic determinants that promote biofilm growth under heterotrophic conditions in Cupriavidus necator using transposon enrichment

Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for auto- and heterotrophic bioprocesses, yet the genetic determinants governing its biofilm formation remain largely uncharacterized, particularly under process-relevant heterotrophic conditions. Here, we applied a forward-genetics transposon-enrichment approach to identify loci which promote surface-associated growth. A high-density mini-Tn5 mutant library (26,185 insertion clones, exceeding the >17,000 required for genome-wide coverage) was cultivated as a biofilm in a microfluidic flow-cell system on fructose for 168 h, and the surface-associated community was characterized by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several with documented links to biofilm formation in other bacteria, including the ferrous-iron uptake system (feoA/feoB), galU, and a GSDEF/EAL dual-domain protein. The gene B2043 (E6A55_RS29530), encoding this c-di-GMP-metabolizing protein, was selected for validation by markerless deletion. Under static conditions, the ΔB2043 mutant showed a 1.69 ± 0.06-fold increase in biofilm-associated biomass (p = 5.16 × 10<sup>−15</sup>). Under flow-through conditions, the mutant attached faster, entered exponential growth ∼10 h earlier, reached its biovolume plateau ∼16 h earlier than the wild-type, and formed distinct tower-like structures. These results identify B2043 as a negative regulator of biofilm formation acting predominantly during attachment, provide the first experimental evidence for c-di-GMP-dependent biofilm regulation in C. necator H16, and establish a functional-genomics framework — together with eleven further candidate loci — for engineering productive biofilms in this organism.

Content

Subjects

Biofilm formation

Cupriavidus necatorH16

Cyclic di-GMP

EAL-domain

Ralstonia eutrophaH16

Transposon insertion library

Transposon screen

DDC Class

579: Microorganisms, Fungi and Algae

660.2: Chemical Engineering

Funding References

Funding(s)

SFB 1615 - SMARTe Reaktoren für die Verfahrenstechnik der Zukunft

SFB 1615 - Teilprojekt A05: Maßgeschneiderte funktionale Elektrodenstrukturen für SMARTe Bioreaktoren

SFB 1615 - Teilprojekt C02: SMARTe kontinuierliche bioelektrochemische Prozesse

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https://creativecommons.org/licenses/by/4.0/

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publishedVersion

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