Question

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Title:
Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

DISCUSSION: The reproducibility of the adhesion data obtained in the sand column assay indicates that the assay can distinguish between strains which adhere differentially to an inert matrix. Soil pseudomonads and two E. coli strains demonstrated a wide range of adhesive abilities. All soil isolates adhered in higher percentages than the enteric strains. Although use of soil as the column matrix would have been more similar to the environment from which these strains were isolated, we found that soil columns filtered rather than measured adhesion -of the bacteria. This feature of soil columns may partially explain results of others who studied soil adsorption of Azospirillum brasilense and recovered no cells from washed soil columns (2). In this work, however, A. brasilense applied to a sand column did not adhere and was almost completely removed by the wash procedure. In contrast, P. fluorescens in our assay remained attached to the sand column despite repeated washes. This result indicates a relatively strong mechanism of adhesion by these pseudomonads. In optimizing the assay, we found that logarithmically growing cells adhered in higher percentages than stationaryphase cultures.-However, minimal medium was a better growth medium for attachment than was L broth. These findings suggest that growth phase and nutrient state affect the attachment potential of these cells. Moreovei, adhesion was best if cells were washed free of growth medium before inoculation. These results agree with previous reports that bacterial attachment to surfaces is one response to conditions of nutrient limitation (18, 21). Similar findings have been reported for marine bacteria (11, 15). Logarithmically growing marine pseudomonads adhered to polystyrene better than those in the stationary phase (15), and vibrios inoculated in salt solution rather than rich medium adhered more readily (11). Increasing the ionic strength of a suspending buffer also decreases the electrostatic repulsion between two surfaces of like charge. If both the bacterial cell and the attachment surface are negatively charged, purely on the basis of physicochemical properties, increasing ionic strength should increase adhesion (30). In our assay, however, increasing the salt concentration in either the buffer in which the cells were added or in the wash buffer did not significantly affect adhesion. These results suggest that electrostatic repulsion is not important in adhesion of PfO-1 to sand. VOL. 56, 1990 118 DEFLAUN ET AL. The design of the assay allowed us to screen thousands of mutants and to identify Tn5-induced chromosomal mutations in P. fluorescens which caused reduced ability to adhere to sand. Preliminary evidence suggests that this screening process is also efficient for P. putida soil isolates, although an adhesion mutant has not been verified from this group (unpublished data). The adhesion deficiency phenotype suggested an alteration in the cell surface. This was confirmed by membrane protein profiles. A 34-kDa major protein in the outer membrane of the wild-type Pf0-1 strain and in the TnS mutants which retain wild-type adhesion ability was missing in PfO-5. Preliminary studies of the second adhesion-deficient PfO-1::TnS mutant, PfO-10, indicate that although Tn5 is located at a different site on the chromosome, it lacks the same 34-kDa outer membrane protein as PfO-5. This is additional evidence that this protein is important for adhesion in this strain. Transmission electron microscopy and protein purification identified the wild-type protein as flagellin. Although pili or fimbrial structures are more commonly associated with bacterial adhesion, flagella have been implicated in the adhesive abilities of certain Vibrio strains (1, 4). Adhesion of P. fluorescens to a soil amoeba has also been attributed to polar flagella as revealed by light microscopy. Electron microscopy suggested that this attachment was not confined to the flagellar tip but involved other surfaces of these appendages (26). Flagella of P. fluorescens were found to be essential for colonization of potato roots; this was attributed to lack of motility in flagellumless mutants (12). Our findings are the first to link a defined mutation site with the absence of this motility structure in P. fluorescens and reduced adhesion to an inert surface such as sand. The molecular weight of the flagellum protein in P. fluorescens seems to vary with the strain. The flagella from a P. fluorescens isolate from potato roots which were purified by the same method that we used had a molecular mass of 58 kDa on a polyacrylamide gel (12), which is much larger than the 34-kDa molecular mass that we found for our purified flagellin. An approximate molecular weight obtained by sedimentation coefficients for P. fluorescens flagella was 38 kDa, although this was thought to be an underestimate (31). Antisera produced against this purified flagellin did not react with the flagella or flagellin of all of the P. fluorescens strains tested, indicating that there are indeed subspecies differences in flagella (31). The residual adhesive ability of PfO-5 (40 to 50%), which adhered at a higher percentage than the E. coli strains tested (8 to 22%), indicates that other proteins may be responsible for attachment. Although PfO-5 does not have flagella, such adhesins may still be present on the cell surface and account for the residual attachment observed in this strain. Linkage between the Tn5 insertion and the adhesiondeficient phenotype of PfO-5 was demonstrated by the marker exchange technique. Placement of TnS into the same chromosomal site in unmutated strain PfO-1 caused the adhesion deficiency phenotype of Pf0-5. The TnS marker thus affords a means for identifying and cloning the gene(s) responsible for flagellar synthesis and understanding its role in adhesion.

ACKNOWLEDGMENTS This work was supported by grant BSR 8606657 from the National Science Foundation. We appreciate the technical assistance of Judith Reichler, who performed the electron microscopy, and we thank Blaine Metting for careful reading of the manuscript.

Answer 1

Sand column assay can distinguish between strains depending on differences in adhesion properties on to an inert matrix. Soil pseudomonads and two strains of E. coli exhibited different adhesive abilities. Soil isolates have high ability of adhesion than enteric species. The soil is used as column of adhesion and this made easy for adhesion of soil isolates to the matrix and result in huge number of isolates adhering to matrix. Azospirillum brasilense applied to a sand column did not adhere and is removed by washing. P. fluorescens remained attached to the sand column despite of repeated washes. The adhesion properties of P. fluorescens is attributed a specific protein and mutants lacking the proteins did not exhibit the adhesion properties. Therefore, TnS marker has an important role in identifying and cloning the gene(s) responsible for flagellar synthesis and understanding its role in adhesion.