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A D H E R E N C E O F B A C T E R I A TO H Y D R O C A R B O N S : A SIMPLE M E T H O D F O R
MEASURING CELL-SURFACE HYDROPHOBICITY
bon (n-hexadecane, n-octane or p-xylene). Following The cell-stabilized emulsions did not break even after
10 min preincubation at 30C, the mixtures were agi- several days; however, addition of 5% (v/v) iso-
tated uniformly on a Thermolyne Maxi Mix (Sybron) propranol brought about an immediate coalescence of
for 120 s. After allowing 15 min for the hydrocarbon droplets and release of cells into the aqueous phase
phase to rise completely, the aqueous phase was care- (Fig. 1, right tube).
fully removed with a Pasteur pipette and transferred Results illustrating the adherence of various bac-
to a 1 ml cuvette. Light absorbance was determined teria to the test hydrocarbons are presented in Figs.
at 400 nm, using a Gilford Model 240 spectropho- 3 - 6 . Fig. 3 compares the affinity ofE. coli with
tometer. those of two Acinetobacter strains. E. coli B showed
no significant affinity towards hexadecane or octane
and little or no affinity towards xylene. Similar
3. Results results (not presented) were obtained with M. lyso-
deikticus, E. aerogenes, B. subtilis and P. aeruginosa.
The basic experiment used to measure bacterial While Acinetobacter RAG-1 showed high affinity
hydrophobicity is shown qualitatively in Fig. 1. Hexa- towards all three hydrocarbons, Acinetobacter BD
decane was layered onto a turbid aqueous suspension exhibited higher affinity for octane and xylene than
of Acinetobacter RAG-1 (Fig. 1, left tube) and then for hexadecane. More than 97% of the RAG-1 and
mixed for 120 s; on standing a clear bottom layer and over 99% of the BD strain cells were removed from
a "creamy" upper layer formed (Fig. 1, middle tube). the aqueous phase by 0.2 ml octane.
Microscopic examination of the upper layer revealed The affinities of two species of Staphylococci
an oil-in-water emulsion consisting of hexadecane towards the test hydrocarbons are presented in Fig. 4.
droplets covered with patches of bacteria (Fig. 2). Although S. albus did not adhere to hexadecane or
Decrease in absorbance of the lower aqueous phase xylene, S. aureus exhibited a high affinity towards all
was used as a measure of cell surface hydrophobicity. the test hydrocarbons.
Although S. marcescens grown to early exponen-
tial phase showed relatively low affinity towards
hexadecane and xylene and none towards octane,
pink-pigmented early stationary phase cells adhered
strongly to all three hydrocarbons (Fig. 5).
Loss of oligosaccharide components on the outer
surface ofE. coli rough mutant J-5 [19] was accom-
panied by a significant increase in its affinity towards
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Fig. 6. o, E. coli K-12 (1.558); o, E. coli J-5 (1.408).
Figs. 3-6. Aff'mityof bacteria towards hydrocarbon as a function of hydrocarbon volume. Aqueous bacterial suspensions were
mixed with varying volumes of hexadeeane, octane and xylene as described in Materials and Methods. Results are expressed as
percentage of the initial absorbanee (A 400) of the aqueous suspension as a function of hydrocarbon volume. Initial absorbance is
shown in parentheses.
each of the three test hydrocarbons, as compared between Acinetobacter cells and bulk hydrocarbon is
with E. coli K-12 (Fig. 6). due to general hydrophobic interactions rather than
specific recognition of the substrate. P. aeruginosa
PAS 279 did not adhere significantly to the test hy-
4. Discussion drocarbons under the conditions studied, despite its
ability to grow on hexadecane. This result suggests
A simple quantitative method has been described that affmity for hydrocarbon may vary among hydro-
for studying the outer cell surface of bacteria, based carbon-degrading bacteria. Further studies on the
on the affinity of these cells for liquid hydrocarbons. relationship between adherence to hydrocarbon and
Large differences in the aftrmities of various bacteria growth of hydrocarbon-degrading bacteria are under-
for hydrocarbons have been demonstrated using this way.
technique. The large increase in hydrophobicity observed in
Previous observations have suggested that the abil- S. marcescens with increasing age of the ceils agrees
ity to adhere to bulk hydrocarbon is a characteristic with previous reports [8,13]. The small, but signifi-
of hydrocarbon-degrading microorganisms [ 11 ]. We cant increase in the affmity of the rough mutant
have shown that S. aureus and early stationary phase E. coli J-5 as compared with that ofE. coli K-12 indi-
S. marcescens cells adhere to a variety of fiquid hy- cates the increased hydrophobicity of the rough mu-
drocarbons despite their inability to degrade them. tant, presumably due to the increased exposure of
Thus, the ability of bacterial cells to adhere to hydro- inner core regions of the LPS layer. Similar results
carbon is not restricted to hydrocarbon-degrading have been obtained with rough mutants of Salmonella
bacteria. Moreover, both Acinetobacter strains typhimurium [1,14].
adhered strongly to octane and xylene, hydrocarbons The method described here may prove useful in
which they are incapable of metabolizing. This indi- enabling the separation of certain cell mixtures, and
cates that the adherence of these hydrocarbon- in separating hydrophobic cell components. In addi-
degrading microorganisms is not limited to metabo- tion, this technique may provide a means of enriching
lizable hydrocarbons, and suggests that direct contact for and selecting mutants which are altered in their
33
surface hydrophobicity. A search for such mutants is [6] Marshall, K.C. and Cruickshank, R.H. (1973) Arch. Mi-
currently in progress, with a view towards investigat- krobiol. 91, 29-40.
ing the relationship between the metabolism o f hy- [7] Stanley, S.O. and Rose, A.H. (1967) J. Gen. Microbiol.
48,9-23.
drophobic substrates and modifications in cell surface [8] Blanchard, D.C. and Syzdek, L.D. (1978) Limnol.
hydrophobicity. Oceanogr. 23,389-400.
[9] Miura, Y., Okazaki, M., Hamada, S.-I., Murakawa, S.-I
and Yugen, R. (1977) Biotech. Bioeng. 19, 701-714.
Acknowledgements [10] McLee, A.G. and Davies, S.L. (1972) Can. J. Microbiol.
18,315-319.
[ 11 ] Kennedy, R.S., Finnerty, W.R., Sudarsanan, K. and
We thank Z.L. Zosim, L. Goldstein and E.A. Bayer Young, R.A. (1975) Arch. Microbiol. 102, 75-83.
for stimulating discussions and constructive criticism. [12] K~ippeli,O. and Fiechter, A. (1976) Biotech. Bioeng.
18,967-974.
[13] KjeUeberg, S., Lagercrantz, C. and Larsson, Th. (1980)
FEMS Microbiol. Lett. 7, 41-44.
References [14] Magnusson, K.-E. and Johansson, G. (1977) FEMS Mi-
crobiol. Lett. 2, 225-228.
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Van Oss, C.J. (1975) Immunol. Commun. 4,429-442. Edebo, L. (1978) Acta Pathol. Microbiol. Scand. Sect. B
[2] Peters, L., Andiiker, L., Edebo, L., Stendahl, O. and 86,113-120.
Tagesson, C. (1977) Acta Pathol. Mierobiol. Scand. [17 ] Reisfeld, A., Rosenberg, E. and Gutnick, D. (1972)
Sect. B 85,308-316. Appl. Microbiol. 24,363-368.
[3] Smyth, C.J., Jonsson, P., Olsson, E., S~derlind, O., [18] Horowitz, A., Gutnick, D. and Rosenberg, E. (1975)
Rosengren, J., Hjert6n, S. and Wadstr6m, T. (1978) Appl. Microbiol. 30, 10-19.
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