Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu 525-8577, Japan1
Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162-0826, Japan2
Ocean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164-8639, Japan3
Author for correspondence: H. Morisaki. Tel: +81 77 561 2767. Fax: +81 77 561 2659. e-mail: morisaki{at}se.ritsumei.ac.jp
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ABSTRACT |
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Keywords: cell attachment, motility, surface polymers, electrostatic repulsion, soft particle
Abbreviations: EPM, electrophoretic mobility; SSW, synthetic seawater
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INTRODUCTION |
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These findings clearly answer a long-standing question: does motility facilitate attachment? However, another unsolved problem remains: how the bacteria can overcome the energy barrier, estimated to be about 102 times greater than the thermal kinetic energy of the bacterial cell or about 106 times greater than the cell swimming energy according to the DLVO theory (Marshall et al., 1971 ), due to electrostatic repulsion between the cells and glass (commonly both of them are negatively charged).
Ohshima and co-workers (Ohshima et al., 1993 ; Ohshima, 1994
, 1995
) pointed out that the electrokinetic behaviour of soft, polyelectrolyte-covered colloidal particles is different from that of rigid particles having no polymer, and proposed a new theory which can analyse the surface polymers on soft particles. This theory was applied to bacterial cells (typical examples of soft particles) and the polymer layers on bacterial cells were characterized (Takashima & Morisaki, 1997
; Bos et al., 1998
).
The aim of this study was to analyse the surface polymers on the cells of V. alginolyticus by the soft-particle theory and to clarify the effect of polymers on the interaction between the cells and glass surfaces. We report here that the energy barrier due to the electrostatic repulsion between bacterial cells and a glass surface becomes much smaller than that usually expected, even less than the thermal kinetic energy, when the bacteria are rich in polymers at the cell surfaces. This finding reasonably explains the dependence of the cell attachment rate on the swimming speed. Moreover, it provides us with a new way to understand the interaction between surfaces when either or both of them have a polymer layer. Our findings demand reconsideration of the mechanism of bacterial attachment and subsequent biofilm formation causing various problems such as food contamination, ship biofouling and pipeline corrosion.
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METHODS |
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Electrophoretic mobility (EPM) measurement.
The EPM of the cells was measured at 25 °C with system 3000 (Pen Kem). The cell suspension in 400 mM NaCl solution was mixed (1:39, v/v) with a phosphate buffer solution (pH 7·4). The ionic strength of the buffer was varied, while its osmotic pressure was maintained constant, by changing the ratio of 0·154 M phosphate buffer, a sucrose solution (0·5 M) and distilled water.
Attachment measurement.
Precleaned cover glasses (24x32 mm, NEO no. 1; Matsunami, Osaka, Japan) were used as the attachment substratum. They were washed by ultrasonication and dried before being packed and shipped by the manufacturer. We compared several methods of precleaning (two kinds of detergent and HCl/ethanol treatment), but there was virtually no difference in the degree of attachment. Therefore we used the cover glasses without any pretreatment. One piece of cover glass was placed in a cell suspension (10 ml) in a test tube (30x60 mm) with shaking at 100 r.p.m. on a rotary shaker at 25 °C. After 1 h, the glass was taken out and washed twice by dipping and shaking it gently in SSW; only the cells whose attachment energy is greater than the energy of the applied shear force during the washing procedure would remain. One drop of DAPI (4',6-diamidino-2-phenylindole) solution (approx. 1 µg ml-1) was placed on the glass surface, which was observed with a fluorescence microscope (Olympus BH-2) within 30 min. Cells in at least 10 randomly chosen fields were counted at a magnification of x1000 and the counts averaged. Assays for each experimental condition were repeated twice to confirm the reproducibility of the results.
Swimming speed measurement.
Twenty microlitres of cell suspension (preincubated for 30 min in SSW of the particular composition to be used) was placed onto a glass slide, and the swimming speed of planktonic cells was measured as follows. The movements of planktonic cells were immediately recorded with an inverted microscope (Diaphot 300; Nikon) at a magnification of x400 and a high-speed video camera (HSV500 HR; Nac) for 2 min. The focus was set at the top of the surface layer. The images obtained were analysed by using Movias 2D software (Nac). From one frame, 20 cells were randomly chosen, regardless of their motility. The distance travelled in 40 ms was consecutively measured for up to 1 s. When a cell moved out of the layer being focused on, measurement was stopped and the data that had been collected until this time point were used. Assays were repeated twice.
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RESULTS AND DISCUSSION |
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We measured the EPM of V. alginolyticus cells in medium at different ionic strengths. The EPM of the three strains of V. alginolyticus changed along with the ionic strength of the medium as shown in Fig. 1. At a low ionic strength the EPM became slightly less negative with the increment in the ionic strength, but the EPM levelled off at a high ionic strength. This pattern of EPM change is totally different from that usually expected, as follows. For colloidal particles, including bacterial cells, we have calculated their zeta potential (
) from their electrophoretic mobility (µ) by the Smoluchowski equation: µ=
r
0
/
, where
r is the relative permittivity of the medium in which the cells suspended,
0 is the permittivity of a vacuum and
is the viscosity of the medium. We have also used the equation
0=
/
r
0
to relate the surface potential (
0) of the particles to the surface charge density (
), where
is the DebyeHückel parameter, depending mainly on the square root of electrolyte concentration. Combination of these equations by approximating the zeta potential with the surface potential gives:
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Ohshima (1994 , 1995
) has pointed out that the Smoluchowski equation cannot be applied to soft particles having polymers at their surfaces: the equation seems to be limited to rigid particles having no polymer. He solved the problem concerning soft particles whose EPM approaches a non-zero value with increasing ionic strength by presenting a theory formulated as follows.
![]() | (2) |
where DON is the Donnan potential of the polymer layer,
m can be interpreted as the DebyeHückel parameter of the polymer layer,
is a parameter whose reciprocal has the dimension of length corresponding to a softness of the polymer layer, z is the valence of charged group in the polymers, e is the electric unit charge and N is the density of the charged groups in the polymer layer.
Ohshimas theory can explain the EPM change along with the ionic strength by considering the presence of charged polymer layer at the cell surface. We can characterize the polymer layer by determining its charge density and softness to fit the theoretical curve from equation (2) to the actual EPM change as shown in Fig. 1. In equation (2),
0,
DON and
m are functions of ionic strength, and the value defined by the first term in this equation decreases along with the increment in the ionic strength. However, the second term, zeN/
2, gives a constant value regardless of the change in ionic strength. Hence, the EPM levels off to a non-zero value defined by the second term at a high ionic strength (Ohshima, 1994
, 1995
). The theory also gives us the electric potential at the outermost fringe of the polymer layer, i.e. the surface potential. The surface potential of V. alginolyticus obtained from this theory (Fig. 2
) was surprisingly small compared to that calculated by putting the EPM value of the cells shown in Fig. 1
into the Smoluchowski equation: the potential from the Smoluchowski equation ranged from -20·2 to -24·4 mV in the ionic strength range 0·01 to 0·15 M. Fig. 2
also shows that, above a critical ionic strength, the bacterial cells can surmount the energy barrier between the cells and glass, because the cell-surface potential is so small as to make the energy barrier less than the cell thermal kinetic energy.
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Suppose bacterial cells (concentration c) to be suspended and a glass plate immersed in a medium. When the cells travel distance L, on average, in a unit time without the aid of flagellum plus distance V with flagellum rotation, the frequency of cell collision to the glass surface (area S) in a unit time is cS(L+V)F1, where F1 is the fraction of the cells moving toward the glass. If a part of the collisions, F2, results in cell attachment to the surface, the attachment rate A(V) is cS(L+V)F1F2. Division of the attachment rate of the cells swimming with speed V by that with V0 gives A(V)/A(V0)=(L+V)/(L+V0). When L<<V0 and V, this equation reduces to:
![]() | (3) |
Equation (3) means that the attachment rate increases proportionally to the swimming speed. We obtained Fig. 4 by plotting A(V)/A(V0) vs V/V0 from the results of Kogure et al. (1998)
for V. alginolyticus. The relative attachment rate of strain YM4, possessing a polar flagellum rotated by Na+ motive force, increased along with the relative swimming speed; we observed a similar relationship for strain NMB86, which possesses only a polar flagellum and lacks chemotactic ability (data not shown). Thus, Fig. 4
clearly indicates that the relation in equation (3) holds. This is due to the fact that the swimming cells can freely access the glass surface because of the absence of the energy barrier between them. When the rotation of the flagellum was specifically inhibited by adding amiloride, the attachment rate decreased dramatically (Kogure et al., 1998
). The reduced attachment rate, although to a small extent, increased proportionally to the cell concentration of strain YM4 (data not shown). This also indicates the absence of the barrier.
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Attachment mechanism of V. alginolyticus cells
An irreversible attachment with the premise of a high energy barrier between bacterial cells and surface has been explained as follows. A secondary energy minimum 510 nm away from a substratum surface may act to halt bacterial cells long enough to result in an irreversible attachment by means of polymer fibrils, for which the electrostatic repulsion is much smaller compared with a cell, bridging the gap between the cells and surface (Rogers, 1979 ). However, we cannot explain the irreversible attachment of V. alginolyticus by this mechanism, for the following two reasons. First, the depth of the secondary minimum for the cells was not sufficient to halt the cells subject to turbulence by thermal kinetic energy. The depth was about -1 kT even at 400 mM, the highest electrolyte concentration examined, the calculation being based on the DLVO theory by using the same parameters as Fig. 3
and a bacterial surface potential of -20 mV. Second, the three V. alginolyticus strains are mutants lacking lateral flagella. The polar flagellum also had no role as the bridging polymer, because the attachment rate was comparable between a strain (NMB94) having a non-rotating flagellum and a strain (YM18) without a polar flagellum (Kogure et al., 1998
). The high energy barrier preventing the cells from approaching closely to a surface does seem to be present under low ionic strength conditions even for a soft particle (see Fig. 2
). However, under conditions of high ionic strength, the barrier becomes less than the thermal kinetic energy of the bacterial cells when the cells are rich in polymers at their surfaces, as noted by Kogure et al. (1998)
. Thus, we must reconsider the attachment mechanism of bacterial cells under high ionic strength conditions such as in seawater.
Cell attachment of other kinds of bacteria
We examined the relationship between the cell attachment rate to the glass surface and the swimming speed for other bacterial strains, Alcaligenes spp. 172 and 201, Alteromonas sp. WB30 and Pseudomonas aeruginosa. As observed for V. alginolyticus, the EPM of these strains did not fall to zero but approached a certain negative value at high ionic strength (data not shown), indicating the existence of a polymer layer at their cell surfaces. For all these strains, the electrostatic energy barrier becomes less than the thermal kinetic energy at ionic strengths above 7080 mM. At an ionic strength of 100 mM (maintained by varying the addition of KCl while Na+ concentration was varied), as shown in Fig. 5, the attachment rate increased along with the swimming speed for Alcaligenes sp. 201 and Alteromonas sp. WB30 as well as V. alginolyticus YM4. It is noteworthy that the increment in the attachment rate of these two strains appears to be greater than that of V. alginolyticus YM4. The factor F2, the fraction of cells resulting in attachment after collision to the glass surface, may be greater for these strains compared with V. alginolyticus YM4. In contrast, P. aeruginosa and Alcaligenes sp. 172 hardly attached to the glass surface and showed no such linear relation between the attachment rate and the swimming speed. The cell surfaces of these strains may have a low affinity, and thus a small F2 value, for the glass surface. This may make it difficult to observe the relationship between the attachment rate and the swimming speed. Except for these cases, the attachment of the bacterial cells seems to be facilitated by the cell motility.
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ACKNOWLEDGEMENTS |
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Received 19 January 1999;
revised 7 June 1999;
accepted 29 June 1999.