Mutations in Flavobacterium johnsoniae sprE Result in Defects in Gliding Motility and Protein Secretion

Abstract

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Transposon mutagenesis was used to identify sprE, which is involved in gliding. Mutations in sprE resulted in the formation of nonspreading colonies on agar. sprE mutant cells in wet mounts were almost completely deficient in attachment to and movement on glass, but a small percentage of cells exhibited slight movements, indicating that the motility machinery was not completely disrupted. SprE is a predicted lipoprotein with a tetratricopeptide repeat domain. SprE is similar in sequence to Porphyromonas gingivalis PorW, which is required for secretion of gingipain protease virulence factors. Disruption of F. johnsoniae sprE resulted in decreased extracellular chitinase activity and decreased secretion of the cell surface motility protein SprB. Reduced secretion of cell surface components of the gliding machinery, such as SprB, may account for the defects in gliding. Orthologs of sprE are found in many gliding and nongliding members of the phylum Bacteroidetes, suggesting that similar protein secretion systems are common among members of this large and diverse group of bacteria.

Flavobacterium johnsoniae RemA Is a Mobile Cell Surface Lectin Involved in Gliding

“…The bound spheres moved rapidly along the cell surface (see Movie S4 in the supplemental material), suggesting that RemA is propelled along the cell by the gliding "motor," as previously suggested for SprB (26). As previously reported (26,(31)(32)(33)(34), and as shown in Table 2 and in Movie S4 in the supplemental material, Protein G-coated spheres did not bind to cells unless specific antiserum was added. They also failed to bind to wild type cells that did not express Myc-tagged RemA.…”

“…Antibodies against myc tag peptide (1 l of a 1:10 dilution of a 1-mg/ml stock), 0.5-m-diameter protein G-coated polystyrene spheres (1 l of a 0.1% stock preparation; Spherotech, Inc., Libertyville, IL), and bovine serum albumin (1 l of a 1% solution) were added to 7 l of cells (approximately 5 ϫ 10 8 cells per ml) in MM. The presence of protein G on the spheres and the addition of bovine serum albumin eliminated nonspecific binding of spheres to cells, as previously reported (26,(31)(32)(33)(34). The cells were introduced into a tunnel slide and examined by phase-contrast microscopy at 25°C.…”

“…A novel protein secretion system, the Por secretion system (PorSS), is required for delivery of SprB to the cell surface, and also for secretion of an extracellular chitinase (33)(34)(35). A strain carrying myctagged remA and carrying a deletion of the region spanning the porSS genes gldN and gldO was constructed to determine whether the PorSS is involved in secretion of RemA.…”

“…Instead, they rely on a novel motility apparatus composed of proteins that are unique to the phylum Bacteroidetes. Nineteen proteins involved in motility have been identified (2,3,20,26,27,31,34). SprB is a cell surface protein that appears to be propelled rapidly by the motility machinery (26).…”

“…SprB is a cell surface protein that appears to be propelled rapidly by the motility machinery (26). Some of the motility proteins (GldK, GldL, GldM, GldN, SprA, SprE, and SprT) are thought to comprise a protein secretion system, the PorSS, that is needed for delivery of SprB to the cell surface and for secretion of an extracellular chitinase (33)(34)(35). The PorSS is not related to the type I to type VI protein secretion systems.…”