Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapG-dependent cleavage, biofilm formation, and cell surface localization.
The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Chelsea D
CD
Boyd
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
T Jarrod
TJ
Smith
Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Sofiane
S
El-Kirat-Chatel
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Peter D
PD
Newell
Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Yves F
YF
Dufrêne
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA georgeo@dartmouth.edu.
George A
GA
O'Toole
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018441:Biofilms / Q000254:growth & development*; D019295:Computational Biology; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D037102:Lectins / Q000235:genetics / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D037102:Lectins; C038692:adhesin, Pseudomonas