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Updated: Mar 8, 2026
Microtiter Dish Biofilm Formation Assay
Published on: January 30, 2011
Contribution of the Pseudomonas fluorescens MFE01 Type VI Secretion System to Biofilm Formation
Mathias Gallique1, Victorien Decoin1, Corinne Barbey1,2
1LMSM, Laboratoire de Microbiologie Signaux et Microenvironnement, EA 4312, IUT d'Evreux, Université de Rouen, Normandy University, Evreux, France.
Abstract:
Type VI secretion systems (T6SSs) are widespread in Gram-negative bacteria, including Pseudomonas. These macromolecular machineries inject toxins directly into prokaryotic or eukaryotic prey cells. Hcp proteins are structural components of the extracellular part of this machinery. We recently reported that MFE01, an avirulent strain of Pseudomonas fluorescens, possesses at least two hcp genes, hcp1 and hcp2, encoding proteins playing important roles in interbacterial interactions. Indeed, P. fluorescens MFE01 can immobilise and kill diverse bacteria of various origins through the action of the Hcp1 or Hcp2 proteins of the T6SS. We show here that another Hcp protein, Hcp3, is involved in killing prey cells during co-culture on solid medium. Even after the mutation of hcp1, hcp2, or hcp3, MFE01 impaired biofilm formation by MFP05, a P. fluorescens strain isolated from human skin. These mutations did not reduce P. fluorescens MFE01 biofilm formation, but the three Hcp proteins were required for the completion of biofilm maturation. Moreover, a mutant with a disruption of one of the unique core component genes, MFE01ΔtssC, was unable to produce its own biofilm or inhibit MFP05 biofilm formation. Finally, MFE01 did not produce detectable N-acyl-homoserine lactones for quorum sensing, a phenomenon reported for many other P. fluorescens strains. Our results suggest a role for the T6SS in communication between bacterial cells, in this strain, under biofilm conditions.
Insights
Pseudomonas fluorescens uses Type VI secretion systems (T6SS) with Hcp proteins to kill prey and mature biofilms. T6SS also appears crucial for bacterial communication in this strain under biofilm conditions.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Cellular Communication
Background:
- Type VI secretion systems (T6SS) are essential virulence factors in Gram-negative bacteria, including Pseudomonas species.
- Hcp proteins are key structural components of the T6SS apparatus, mediating interbacterial interactions and toxin delivery.
- Pseudomonas fluorescens MFE01 utilizes T6SS and Hcp proteins for interbacterial killing and biofilm development.
Purpose of the Study:
- To investigate the roles of Hcp proteins (Hcp1, Hcp2, Hcp3) in Pseudomonas fluorescens MFE01.
- To determine the involvement of T6SS in biofilm formation and interbacterial interactions.
- To explore potential quorum sensing mechanisms in P. fluorescens MFE01.
Main Methods:
- Genetic manipulation of hcp genes (hcp1, hcp2, hcp3) and a T6SS core component gene (tssC).
- Co-culture experiments to assess interbacterial killing and biofilm inhibition.
- Biofilm formation assays and maturation analysis.
- Detection of N-acyl-homoserine lactones (AHLs) for quorum sensing analysis.
Main Results:
- Hcp3, in addition to Hcp1 and Hcp2, contributes to prey cell killing by P. fluorescens MFE01.
- Mutations in hcp genes impaired biofilm formation of a co-cultured P. fluorescens strain (MFP05) but not MFE01's own biofilm formation.
- All three Hcp proteins were necessary for the maturation of P. fluorescens MFE01 biofilms.
- Disruption of the T6SS core component gene (tssC) abolished both MFE01 biofilm production and its ability to inhibit MFP05 biofilm formation.
- P. fluorescens MFE01 did not produce detectable AHLs, suggesting a lack of typical quorum sensing.
Conclusions:
- The T6SS, through its Hcp proteins, plays a multifaceted role in Pseudomonas fluorescens MFE01, including interbacterial antagonism and biofilm development.
- The T6SS is essential for the maturation of P. fluorescens MFE01 biofilms and influences the biofilm formation of other strains.
- The findings suggest a potential role for T6SS in bacterial communication, particularly within biofilms, in strains lacking conventional quorum sensing mechanisms.
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