Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein

Abstract

SummaryWe report the identification of an ATP-binding cassette (ABC) transporter and an associated large cellsurface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for l arge a dhesion p rotein. The LapA protein, with a predicted molecular weight of ~ 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membranelocalized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBCencoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae . The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…These products resemble an RTX-like transport system, which transports RTX (repeats in toxin) cytotoxins as well as Slayer proteins directly from the cytoplasm to the extracellular milieu (Lally et al, 1999). Interestingly, another cell surface protein secreted by an ABC transporter and promoting irreversible attachment during biofilm formation was recently identified in Pseudomonas fluorescens (Hinsa et al, 2003). We suggest that adhesive interactions are perturbed for the V. parahaemolyticus mfp mutants, as the structural integrity of the mutant biofilms was compromised.…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…A similar system has recently been identified in Pseudomonas putida (50). Time-lapse microscopy studies determined that ⌬lapA mutants (as well as other lap mutants) are unable to progress from reversible to irreversible attachment on abiotic surfaces that these organisms likely encounter in the environment (62). Localization of the adhesin LapA is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG.…”

“…Release of LapA from attached cells promotes their detachment from the substratum and bacteria returning to the free-swimming mode of growth. The model is based on structural and functional analyses (50,62,109,116,118).…”

The motor domains of ABC-transporters

“…For example, in the type I secretion system, the 107 kDa toxin HlyA is transported via a tunnel-like system which consists of the ABC transporter, haemolysin B (HlyB), HlyD and TolC (Holland et al 2005). The largest allocrite known today is an S-layer protein, secreted by the ABC-dependent, Type I pathway, with a molecular weight of 900 kDa (Hinsa et al 2003).…”