Aug 21, 1991 - For the SAT, bacteria were "salted out" (aggregat- ed) withammonium sulfate (21). More hydrophobic bacteria aggregate at relatively low ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1991, p. 3193-3199
0099-2240/91/113193-07$02.0O/0 Copyright C 1991, American Society for Microbiology
Vol. 57, No. 11
Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria MARIA L. SORONGON,1 ROBERT A. BLOODGOOD,2 AND ROBERT P. BURCHARDl* Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21228,1 and Department of Anatomy and Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia 229082
Received 27 December 1990/Accepted 21 August 1991
The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure. an assay
The gliding bacteria, a taxonomically heterogeneous assemblage, demonstrate translocational and other movements only when they are associated with substrata (4, 15, 29). It has generally been assumed that extracellular polymers constituting an amorphous "slime" or an organized sheath mediate the adhesion of these bacteria (for examples, see references 4, 16, and 26). Alternatively, integral cell envelope components may function as adhesins (5, 20). Earlier work suggests that the nature of the substratum is a determinant in the adhesion and motility of these bacteria (1, 6). A variety of gliding bacteria adhere tenaciously to substrata with low critical surface energies (i.e., hydrophobic) and tenuously on surfaces with high critical surface energies (hydrophilic). We proposed that adhesion of these bacteria involves, at least in part, hydrophobic interactions (a water exclusion mechanism) (6). Additionally, van der Waals forces, hydrogen bonding, and electrostatic interactions may mediate adhesive interactions. The significance of hydrophobic interactions in nonspecific adhesion of bacteria and the observations summarized above led to the prediction that the surfaces of gliding bacteria are hydrophobic. On the basis of studies of bacterial adherence to hydrocarbons (BATH) (33), Wolkin and Pate (41) reported that gliding bacteria from three genera varied in their hydrophobicities. A number of other methods to determine hydrophobicity are also available (for examples, see references 7, 10, and 25). We report here a study of the hydrophobicity of the surfaces of several aquatic and terrestrial gliding bacteria and several adhesion-defective mutants and revertants of one of these, by using hydrophobic interaction chromatography (HIC) (37) and a salt aggregation test (SAT) (21) in addition to the BATH assay. Attempts were made to correlate the results with adhesion to moderately
*
Corresponding author.
hydrophobic glass and with changes in arrays of cell surfaceexposed proteins. MATERIALS AND METHODS Bacterial strains. The bacteria examined in this study and their origins and descriptions are presented in Table 1. Culture conditions. Bacteria were cultured in the media listed in Table 1. Most of the cultures were incubated overnight in shallow layers of medium without agitation at 25°C. Myxococcus xanthus and Flexibacter sp. strain FS-1 were incubated at 30°C. Logarithmic-phase cultures of FS-1 provided filaments; fragments were generated during the stationary phase (8). Flexibacter maritimus was grown in CAS medium (6) under three culture conditions: in static or shake (100 rpm) liquid cultures and on agar. Because of the cohesiveness of agar-grown F. maritimus lyl-1H, the bacteria were dispersed by use of a tissue homogenizer. Starvation conditions. Logarithmic-phase bacteria were centrifuged (10,000 x g, 10 min), resuspended in 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.0) containing 1.0 mM MgCl2, and incubated overnight at 25°C. BATH. For the BATH assay, the method of Rosenberg (33) was used. Bacteria in growth medium were diluted to an A540 of 0.2 (final volume, 3 ml) and mixed with different volumes (0.12, 0.24, 0.36, and 0.48 ml) of hexadecane by vortexing. After phase separation, the optical density (A400) of the aqueous phase was determined. Results (see Fig. 1) were recorded as the percentage of cells that partitioned into the aqueous phase, in keeping with previous studies. In the bar graphs, the one value recorded for the BATH assay is the percentage of bacteria from 3 ml of suspension that partitioned into 0.48 ml of hexadecane. SAT. For the SAT, bacteria were "salted out" (aggregated) with ammonium sulfate (21). More hydrophobic bacteria aggregate at relatively low concentrations of salt. Volumes (25 ,ul) containing 2 x 109 bacteria suspended in culture 3193
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TABLE 1. Bacteria examined in this study Strain
Cytophaga sp. strain RB1057 Cytophaga sp. strain RB1058 Cytophaga johnsonae FDC 405 Cytophaga johnsonae FDC 444 Cytophaga sp. strain U67 and adhesion mutantsa Flexibacter maritimus lyl-1R, lyl-1S, and ly1-1Hb Flexibacter sp. strain FS-1 Myxococcus xanthus DK1622 Microscilla sp. strain RB1
Growth medium (reference)
Sorer
CAS (6) CAS
This laboratory This laboratory
C62 (15) C62 C62
P. Christensen P. Christensen J. Henrichsen
CAS
D. Baxa
YE/2 (9) CT (12) HSM (6)
D. White (35) D. Kaiser (17) This laboratory
reference
a Isolation described by Burchard and Bloodgood (5). b Substrains described in Results.
0
80 00 z
%
~~~~~~~~1.2MJ
20-
R
S
H
FIG. 5. Hydrophobicity of F. maritimus lyl-I substrains R, S, and H grown under three different culture conditions. Symbols and scales are as described in the legend to Fig. 2.
VOL. 57, 1991
HYDROPHOBICITY OF GLIDING BACTERIA
FIG. 6. SDS-polyacrylamide slab gel (4 to 16% gradient) of whole-cell polypeptides of F. maritimus lyl-1 substrain R, grown in shake (lanes 1) and static (lanes 2) liquid cultures and on agar (lanes 3) and lyl-1 substrain S grown in shake (lanes 4) and static (lanes 5) cultures, that were surface iodinated as described in the legend to Fig. 4. The gel was loaded to equalize Coomassie blue staining. (A) Coomassie blue-stained gel. The open circle marks a 24-kDa polypeptide. Cracks occurred during drying of the gel. (B) Autoradiogram. Arrows point to 33-, 58-, and 115-kDa polypeptides from shake-culture-grown bacteria; closed circles mark 19.5-, 24-, and 41-kDa species from agar-grown bacteria.
adhered weakly. M. xanthus (columns F), a terrestrial glider, demonstrated little adhesion, was hydrophilic by the BATH assay, and was relatively hydrophobic by HIC and SAT.
DISCUSSION
These studies demonstrate that even closely related gliding bacteria may vary significantly in surface hydrophobicity
as measured by any one of the three assays that we employed. As with other bacteria (10, 25, 38), consistency of results among all three hydrophobicity assays was only observed in strains with very hydrophilic surfaces (e.g., C. johnsonae 405 and some mutants derived from Cytophaga sp. strain U67). Additionally, individual assays identified significant differences in cell surface properties between two strains of a single species (e.g., C. johnsonae FDC 405 and 444). Differences in the distribution of hydrophobic components on the bacterial surface may account for these disparities. While the SAT may provide a measure of overall surface hydrophobicity, the BATH and HIC assays may indicate the presence of hydrophobic domains on an otherwise hydrophilic cell surface (38). This domain concept may explain the observation that Flexibacter aurantiacus cells orient perpendicularly to the surface of oil droplets (23). Polar hydrophobic domains may also account for the formation of rosettes of M. xanthus cells (3, 30). Accordingly, we predicted a high hydrophobicity value by the BATH assay for M. xanthus. However, we found a very low hydrophobicity value by such an assay, in agreement with one previous report (41) and in contrast with a second (19). Other evidence suggests that hydrophobic sites are also distributed on lateral surfaces of several groups of gliding bacteria, including benthic cyanobacteria (13) and other members of the genus Flexibacter (41). It is possible that cell surfaces of the gliding bacteria were modified by the hydrophobicity assay procedures. For example, hexadecane used in the BATH assay may extract constituents from the cell envelope (10, 34). The adhesion-defective mutants of Cytophaga sp. strain U67 were isolated by enriching for cells that partitioned into the aqueous phase in the BATH procedure (5). With the exception of the behavior of strain Adhl in the SAT, generally hydrophilic surface properties were demonstrated by these mutants. Among these strains, results from the BATH assay provided particularly good correlation with adhesion to glass. Comparable mutants of C. johnsonae demonstrated similar properties (41).
0
10 ()
80
*
c 0 J_
.2
0.4
60
0.8
0
40-
1.2
q
0
0l.
Lo
X:
3197
z 20-
IT -2.0
A
B
C
D
TE
F
FIG. 7. Hydrophobicity and adhesion of several strains of gliding bacteria. (A) C. johnsonae FDC 405; (B) C. johnsonae FDC 444; (C) Cytophaga sp. strain RB1057; (D) Cytophaga sp. strain RB1058; (E) Microscilla sp. strain RB1; (F) M. xanthus DK1622. For panels C and D, some SAT values were >2.0 (t). Symbols and scales are as described in the legend to Fig. 2.
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The differences in hydrophobicity and adhesion among Cytophaga sp. strain U67 and its mutants and revertants may be explained by variations in arrays of surface-exposed proteins among these strains (5). Each of the adhesiondefective mutants demonstrated arrays of surface-exposed polypeptides that differed from those of the wild type. Some of the revertants, selected by the ability to glide on agar, demonstrated wild-type patterns of surface-exposed proteins. On the basis of similarities to the wild type of these proteins from two revertants, Adh2Rev2 and Adh3Rev, we predicted that they would resemble the wild type in adhesion and hydrophobicity. Adh3Rev did behave as predicted. Adh2Rev2 was also similar to the wild type in adhesion and SAT and HIC results, yet was more hydrophilic than the wild type by the BATH assay. We expected that two other revertants of Adh2 with surface protein patterns resembling that of their adhesion-defective parent (5) would have similar
hydrophobicity patterns. Adh2Rev4 behaved as predicted in both its hydrophobicity and its adhesion to glass. Surprisingly, in light of its poor adhesion, this strain demonstrates
gliding motility
on agar.
Adhesion and hydrophobicity of
Adh2Revl were intermediate between those of Adh2 and U67. The imperfect correlation of cell surface hydrophobicity, adhesion, and arrays of surface-exposed proteins among these strains might be explained in part by the presence of other surface polymers not detectable by the iodination protocol (e.g., lipopolysaccharide and extracellular polysac-
charides). Cell surface hydrophobicity changes during fragmentation of Flexibacter sp. strain FS-1 filaments. In this bacterium, adhesion to glass correlates very well with HIC and SAT results (Fig. 3). The decrease in adhesion that occurs during
fragmentation may provide a mechanism of dispersal for this bacterium in its natural habitat. These changes in hydrophobicity and adhesion may reflect an alteration in arrays of
surface-exposed proteins that
occurs
during fragmentation
of FS-1 filaments (Fig. 4B). Some of the surface-exposed
proteins that distinguish filaments and fragments may be major cell components in that bands of similar molecular weight were resolved by one-dimensional gel electrophoresis (Fig. 4A). Alternatively, some of these labelled proteins may actually be minor constituents that electrophoretically comigrated with one or more major proteins that were not accessible to surface-immobilized iodination. The similarity in filament and fragment arrays of total cell proteins contrasts with the differences in their surface-exposed proteins, suggesting that topological reorganization of the cell envelope occurs during filament fragmentation. Starvation has been reported to effect both increases and decreases in bacterial surface hydrophobicity. Both strategies are ecologically rational (18, 39). Starvation of Cytophaga sp. strain U67 and one of its adhesion-defective mutants resulted in significant increases in hydrophobicity as determined by the BATH assay. This change in surface properties may facilitate adhesion of these aquatic bacteria to substrata which are known to concentrate nutrients from the bulk water phase (for a review, see reference 40). Other culture conditions also may effect changes in cell surface characteristics as demonstrated by results of our studies of F. maritimus grown on agar and in shake and static liquid culture (Fig. 5 and 6). Specific hydrophobicity assays may be useful predictors of adhesion for closely related strains (e.g., BATH assay for Cytophaga sp. strain U67 and its adhesion-defective mutants). However, since the gliding bacteria are so taxonomically diverse, it may not be surprising that we could identify
no one measure of surface hydrophobicity that would predict their capacity as a group to adhere to coverslip glass, a substratum of intermediate critical surface energy. It is likely that the adhesion of these bacteria cannot be explained by hydrophobic interactions alone. Rather, adhesion is likely to be mediated by an interplay of hydrophobic and hydrophilic surface components as it is in other prokaryotes (7, 22, 28, 34, 38, 41). Achievement of a complete understanding of the mechanism(s) of adhesion of the gliding bacteria is likely to be complicated by their moving cell surface components, evidence for which has been reported in the genera Cytophaga and Flexibacter (20, 27, 31). ACKNOWLEDGMENTS This research was supported by Office of Naval Research contract N00014-88-K-0158 to R.P.B. and by a grant from the National Science Foundation (DCB-8905530) to R.A.B. We thank those who supplied bacterial strains and gratefully acknowledge the expert technical assistance of Nancy L. Salomonsky. REFERENCES 1. Arlauskas, J., and R. P. Burchard. 1982. Substratum requirements for bacterial gliding motility. Arch. Microbiol. 133:137141. 2. Baxa, D. V., K. Kawai, and R. Kusuda. 1986. Characteristics of gliding bacteria isolated from diseased cultured flounder, Paralichthys olivaceous. Fish Pathol. 21:251-258. 3. Burchard, R. P. 1974. Studies on gliding motility in Myxococcus xanthus. Arch. Microbiol. 99:271-280. 4. Burchard, R. P. 1981. Gliding motility of prokaryotes: ultrastructure, physiology and genetics. Annu. Rev. Microbiol. 35:497-529. 5. Burchard, R. P., and R. A. Bloodgood. 1990. Surface proteins of the gliding bacterium Cytophaga sp. strain U67 and its mutants defective in adhesion and motility. J. Bacteriol. 172:3379-3387. 6. Burchard, R. P., D. Rittschof, and J. A. Bonaventura. 1990. Adhesion and motility of gliding bacteria on substrata with different surface free energies. Appl. Environ. Microbiol. 56: 2529-2534. 7. Busscher, H. J., A. H. Weerkamp, H. C. van der Mei, A. W. J. van Pelt, J. P. de Jong, and J. Arends. 1984. Measurement of the surface free energy of bacterial cell surfaces and its relevance to adhesion. Appl. Environ. Microbiol. 48:980-983. 8. Costenbader, C. J., and R. P. Burchard. 1978. Effect of cell length on gliding motility of Flexibacter FS-1. J. Bacteriol. 133:1517-1519. 9. Dayrell-Hart, B., and R. P. Burchard. 1979. Association of flexing and gliding in Flexibacter. J. Bacteriol. 137:1417-1420. 10. Dillon, J. K., J. A. Fuerst, A. C. Hayward, and G. H. G. Davis. 1986. A comparison of five methods for assaying bacterial hydrophobicity. J. Microbiol. Methods 6:13-19. 11. Dull, C.-L., and R. P. Burchard. Unpublished data. 12. Dworkin, M. 1962. Nutritional requirements for vegetative growth of Myxococcus xanthus. J. Bacteriol. 84:250-257. 13. Fattom, A., and M. Shilo. 1984. Hydrophobicity as an adhesion mechanism of benthic cyanobacteria. Appl. Environ. Microbiol. 47:135-143. 14. Gerhart, D. J., D. Rittschof, I. R. Hooper, K. Eisenman, A. E. Meyer, R. E. Baier, and C. Young. Submitted for publication. 15. Henrichsen, J. 1972. Bacterial surface translocation: a survey and a classification. Bacteriol Rev. 36:478-503. 16. Humphrey, B. A., M. R. Dickson, and K. C. Marshall. 1979. Physicochemical and in situ observations on the adhesion of gliding bacteria to surfaces. Arch. Microbiol. 120:231-238. 17. Kaiser, D. 1979. Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 76: 5952-5956. 18. Kjelleberg, S., and M. Hermansson. 1984. Starvation-induced effects on bacterial surface characteristics. Appl. Environ. Microbiol. 48:497-503.
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