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Title:
Development of an Adhesion Assay and Characterization of anAdhesion-Deficient Mutant of Pseudomonas fluorescens
DISCUSSION: The reproducibility of the adhesion data obtained inthe sand column assay indicates that the assay can distinguishbetween strains which adhere differentially to an inert matrix.Soil pseudomonads and two E. coli strains demonstrated a wide rangeof adhesive abilities. All soil isolates adhered in higherpercentages than the enteric strains. Although use of soil as thecolumn matrix would have been more similar to the environment fromwhich these strains were isolated, we found that soil columnsfiltered rather than measured adhesion -of the bacteria. Thisfeature of soil columns may partially explain results of others whostudied soil adsorption of Azospirillum brasilense and recovered nocells from washed soil columns (2). In this work, however, A.brasilense applied to a sand column did not adhere and was almostcompletely removed by the wash procedure. In contrast, P.fluorescens in our assay remained attached to the sand columndespite repeated washes. This result indicates a relatively strongmechanism of adhesion by these pseudomonads. In optimizing theassay, we found that logarithmically growing cells adhered inhigher percentages than stationaryphase cultures.-However, minimalmedium was a better growth medium for attachment than was L broth.These findings suggest that growth phase and nutrient state affectthe attachment potential of these cells. Moreovei, adhesion wasbest if cells were washed free of growth medium before inoculation.These results agree with previous reports that bacterial attachmentto 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 topolystyrene better than those in the stationary phase (15), andvibrios inoculated in salt solution rather than rich medium adheredmore readily (11). Increasing the ionic strength of a suspendingbuffer also decreases the electrostatic repulsion between twosurfaces of like charge. If both the bacterial cell and theattachment surface are negatively charged, purely on the basis ofphysicochemical properties, increasing ionic strength shouldincrease adhesion (30). In our assay, however, increasing the saltconcentration in either the buffer in which the cells were added orin the wash buffer did not significantly affect adhesion. Theseresults suggest that electrostatic repulsion is not important inadhesion of PfO-1 to sand. VOL. 56, 1990 118 DEFLAUN ET AL. Thedesign of the assay allowed us to screen thousands of mutants andto identify Tn5-induced chromosomal mutations in P. fluorescenswhich caused reduced ability to adhere to sand. Preliminaryevidence suggests that this screening process is also efficient forP. putida soil isolates, although an adhesion mutant has not beenverified from this group (unpublished data). The adhesiondeficiency phenotype suggested an alteration in the cell surface.This was confirmed by membrane protein profiles. A 34-kDa majorprotein in the outer membrane of the wild-type Pf0-1 strain and inthe TnS mutants which retain wild-type adhesion ability was missingin PfO-5. Preliminary studies of the second adhesion-deficientPfO-1::TnS mutant, PfO-10, indicate that although Tn5 is located ata different site on the chromosome, it lacks the same 34-kDa outermembrane protein as PfO-5. This is additional evidence that thisprotein is important for adhesion in this strain. Transmissionelectron microscopy and protein purification identified thewild-type protein as flagellin. Although pili or fimbrialstructures are more commonly associated with bacterial adhesion,flagella have been implicated in the adhesive abilities of certainVibrio strains (1, 4). Adhesion of P. fluorescens to a soil amoebahas also been attributed to polar flagella as revealed by lightmicroscopy. Electron microscopy suggested that this attachment wasnot confined to the flagellar tip but involved other surfaces ofthese appendages (26). Flagella of P. fluorescens were found to beessential for colonization of potato roots; this was attributed tolack of motility in flagellumless mutants (12). Our findings arethe first to link a defined mutation site with the absence of thismotility structure in P. fluorescens and reduced adhesion to aninert surface such as sand. The molecular weight of the flagellumprotein in P. fluorescens seems to vary with the strain. Theflagella from a P. fluorescens isolate from potato roots which werepurified by the same method that we used had a molecular mass of 58kDa on a polyacrylamide gel (12), which is much larger than the34-kDa molecular mass that we found for our purified flagellin. Anapproximate molecular weight obtained by sedimentation coefficientsfor P. fluorescens flagella was 38 kDa, although this was thoughtto be an underestimate (31). Antisera produced against thispurified flagellin did not react with the flagella or flagellin ofall of the P. fluorescens strains tested, indicating that there areindeed subspecies differences in flagella (31). The residualadhesive ability of PfO-5 (40 to 50%), which adhered at a higherpercentage than the E. coli strains tested (8 to 22%), indicatesthat other proteins may be responsible for attachment. AlthoughPfO-5 does not have flagella, such adhesins may still be present onthe cell surface and account for the residual attachment observedin this strain. Linkage between the Tn5 insertion and theadhesiondeficient phenotype of PfO-5 was demonstrated by the markerexchange technique. Placement of TnS into the same chromosomal sitein unmutated strain PfO-1 caused the adhesion deficiency phenotypeof Pf0-5. The TnS marker thus affords a means for identifying andcloning the gene(s) responsible for flagellar synthesis andunderstanding its role in adhesion.
ACKNOWLEDGMENTS This work was supported by grant BSR 8606657from the National Science Foundation. We appreciate the technicalassistance of Judith Reichler, who performed the electronmicroscopy, and we thank Blaine Metting for careful reading of themanuscript.
