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Previous Article | Next Article 
Applied and Environmental Microbiology, June 2000, p. 2548-2554, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Surface of Lactic Acid Bacteria: Relationships
between Chemical Composition and Physicochemical Properties
Christophe J. P.
Boonaert and
Paul G.
Rouxhet*
Unité de Chimie des Interfaces,
Université Catholique de Louvain, Louvain-la-Neuve, Belgium
Received 16 November 1999/Accepted 15 March 2000
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ABSTRACT |
The surface chemical composition and physicochemical properties
(hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv.
diacetilactis and Lactobacillus helveticus, have been
investigated using cells harvested in exponential or stationary growth
phase. The surface composition determined by X-ray photoelectron
spectroscopy (XPS) was converted into a molecular composition in terms
of proteins, polysaccharides, and hydrocarbonlike compounds. The
concentration of the last was always below 15% (wt/wt), which is
related to the hydrophilic character revealed by water contact angles
of less than 30°. The surfaces of L. lactis cells had a
polysaccharide concentration about twice that of proteins. The S-layer
of L. helveticus was either interrupted or crossed by
polysaccharide-rich compounds; the concentration of the latter was
higher in the stationary growth phase than in the exponential growth
phase. Further progress was made in the interpretation of XPS data in
terms of chemical functions by showing that the oxygen component at
531.2 eV contains a contribution of phosphate in addition to the main
contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about 
3.0 × 10
8 and 0.6 × 108 m2 s1 V1 for
L. lactis and L. helveticus, respectively. The
electrokinetic properties of the latter reveal the influence of
carboxyl groups, while the difference between the two strains is
related to a difference between N/P surface concentration ratios,
reflecting the relative exposure of proteins and phosphate groups at
the surface.
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INTRODUCTION |
In many instances, the behavior of
lactic acid bacteria is dependent on interfacial processes and thus on
cell surface physicochemical properties and chemical composition. A
better knowledge of these aspects would allow a deeper understanding of
the autolysis of lactic acid bacteria (29, 36) and the
production of texturing exopolysaccharides (4). It would
help in controlling the sedimentation of starters for commercial
production (7) and in understanding the roles of specific
and nonspecific interactions in phage attachment (7, 46,
56).
In dairy product manufacturing, adhesion of lactic bacteria to a
material may be the first step leading to biofilm formation, which can
be either deleterious (contamination, taste alteration, and biofouling
on heat exchangers) (17, 25) or beneficial (continuous
inoculation in yogurt or cheese making) (5). The adhesion
behavior of microbial cells has been shown to depend on the balance of
electrostatic and van der Waals interactions and on the hydrophobic
character of the surfaces involved (38, 42, 53, 54),
pointing to the possible influence of the respective zeta potentials
and surface hydrophobicities. Moreover, the production of extracellular
substances either at the cell surface or in the surrounding medium has
been shown to influence adhesion (14, 55).
The surface hydrophobicity and composition of lactic acid bacteria have
been studied primarily by microbial adhesion to hydrocarbon (7,
18, 40, 46, 56) and by biochemical analysis (7, 18, 22, 35,
45, 49, 50, 56), respectively. However, the relevance of these
methods is questionable for interfacial phenomena. Electrostatic
interactions may play a major role in the adhesion of microorganisms to
hydrocarbons (2, 51); in contrast, water contact angle
measures the surface hydrophobicity without the interference of
electrostatic interactions. Biochemical analysis refers to constituents
of the whole cell wall and not to the cell surface; in contrast
(16), X-ray photoelectron spectroscopy (XPS) provides
information on the element and chemical function compositions of the
outermost cell surface (2 to 10 nm). XPS is based on irradiation of the
freeze-dried sample by an X-ray beam and analysis of the kinetic energy
of the emitted photoelectrons (43, 44). The relevance of XPS
for probing microbial surfaces is supported by correlations between the
XPS results and cell surface properties and by relationships with the
cell behavior at interfaces (13, 14, 15, 16, 21, 44).
Lactococcus lactis and Lactobacillus helveticus
are two lactic acid bacteria important in cheese ripening. The former
is present in Cheddar and Saint-Paulin cheeses, while the latter is
found in Swiss-type and Italian cheeses (6, 8, 29, 48).
L. helveticus is also involved in other applications:
production of fermented milk with antihypertensive effect and
production of lactic acid from whey permeate (33). Moreover,
L. lactis and Lactobacillus spp. could be
considered probiotic agents (23, 41) and used as live
delivery vehicles for the administration of oral vaccines (19,
32).
In the present paper, the relationships between surface chemical
composition (XPS), hydrophobicity (water contact angle), and electrical
properties (microelectrophoresis) are examined for L. helveticus and L. lactis. These are taken as models of lactic acid bacteria, rodlike and cocci, respectively; moreover, the
L. helveticus strain possesses an S-layer (31).
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
L. helveticus
ATCC 12046 (Institut Pasteur, Paris, France) and L. lactis
subsp. lactis bv. diacetilactis LMG 9452 (LMG Culture Collection, Universiteit Gent, Ghent, Belgium) were stored at 20°C
in MRS (9) and M17 (47) media (Merck, Darmstadt,
Germany), respectively, containing 15% (vol/vol) glycerol (Vel,
Leuven, Belgium). L. helveticus and L. lactis
were incubated at 37 and 30°C, respectively, without agitation under
aerobic conditions. The volumes for the preculture and the culture were
10 (test tube) and 100 (Erlenmeyer flask) ml, respectively. Precultures
were carried out by inoculation with a frozen sample, and the cultures were inoculated at 1% (vol/vol) with an overnight preculture. The
cells were harvested (13,873 × g; 4°C; 10 min)
either in the exponential (after 4 and 8 to 9 h for L. lactis and L. helveticus, respectively) or the
stationary (after 24 h for both strains) growth phase.
Surface characterization.
The electrical properties of the
cell surfaces were assessed by microelectrophoresis (24)
with a Pen Kem Laser Zee Meter (model 500). For this purpose, cells
were resuspended in 1 mM KNO3 (UCB, Leuven, Belgium) at
concentrations of about 2 × 108 and 5 × 108 per ml for L. helveticus and L. lactis, respectively. The pH was adjusted with KOH (Janssen
Chimica, Beerse, Belgium) or HNO3 (Merck), with a new
suspension made for each determination.
Cell surface hydrophobicity was estimated by water contact angle
(
w) measurements on a cell lawn using the
sessile drop method (52). The cell lawns (30 to 100 layers)
were obtained by filtration on a filter (4.5-cm diameter) with a pore
diameter of 0.45 µm (HAWP filter; Millipore Co., Bedford, Mass.). To
standardize the moisture content, the filters were then transferred
onto agar for 30 min (1% [wt/vol] agar in 10% [vol/vol]
glycerol). Finally, the filters were mounted on a microscope slide, and
w (drop volume, 0.3 µl) was measured as a
function of drying time, using water of high-performance liquid
chromatography grade produced by a MilliQ plus system (Millipore). For
each filter, 10 measurements were made at different places (standard
deviations were less than 10% of the mean value).
The sample preparation for XPS analysis was as follows. The cell pellet
was resuspended in MilliQ water (Millipore) at a final concentration of
1011 cells per ml. Two milliliters of this suspension was
transferred into a glass flask (diameter, 3 cm; height, 2.5 cm)
precooled in liquid nitrogen. After 15 min, the flasks were either
freeze-dried immediately or stored at 80°C until they were
freeze-dried. The freeze-drying was carried out in an apparatus
specially designed by Leybold (Brussels, Belgium). The temperature of
the freeze-dryer shelf was maintained at 50°C for 3 h; then it
was increased to 5°C over 15 h and maintained at 5°C for 6 to 12 h; finally, it was increased to 23°C over 3 h. The
dehydrated cell powder was homogenized with a spatula and pressed with
a polyacetal cylinder cleaned with isopropanol. More details of the
procedure can be found in a previous paper (10).
The XPS analysis was performed with an SSI X-Probe (SSX-100/206;
Surface Science Instruments, Mountain View, Calif.) equipped with an
aluminum anode and a quartz monochromator and interfaced with a
Hewlett-Packard 9000/310 computer, allowing analysis control, data
accumulation, and data treatment. Pressure during analysis varied
between 4 × 107 and 2.7 × 106
Pa. The analyzed area was an elliptical spot with a minor axis of 1,000 µm. Sample charging was stabilized with a flood gun (6 or 8 eV) and a
nickel grid placed 2 mm above the surface sample. The peaks were
recorded in the following sequence: survey spectrum, C1s,
O1s, N1s, P2p, K2p,
Na1s, and C1s again to check for the absence of
sample degradation and charging stability during analysis (12). The constant pass energy was 150 and 50 eV for the
survey spectrum and individual spectra, respectively. Under these
conditions, the resolutions determined by the full width at half
maximum (FWHM) of the Au4f7/2 peak of a standard gold
sample were about 1.6 and 1.1 eV, respectively. To assess the eventual
hydrocarbon contamination of the sample during freeze-drying, a
sorbitol specimen was included in each set of samples, starting with
the freezing step. Data treatment was performed as described by
Dufrêne and Rouxhet (13).
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RESULTS |
The surface properties, i.e., hydrophobicity (Table
1), and electrical properties (Fig.
1) of L. lactis and L. helveticus were not significantly affected by the growth phase of
the cells. The surfaces were hydrophilic whatever the strain, as
indicated by the water contact angle in the range of 21 to 29°. The
isoelectric points were around pH 2 and 3 for L. lactis and
L. helveticus, respectively. Between pH 2 and 5, the
electrophoretic mobility decreased continuously. Above pH 5, the
electrophoretic mobilities were about 3.0 × 108
and 0.6 × 108 m2 s1
V1 for L. lactis and L. helveticus,
respectively.
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TABLE 1.
Water contact angle (w)
determined on cell lawns of L. helveticus ATCC 12046 and L. lactis subsp. lactis bv.
diacetilactis LMG 9452
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FIG. 1.
Variation of electrophoretic mobility as a function of
pH for L. helveticus ATCC 12046 (circles) and L. lactis subsp. lactis bv. diacetilactis LMG 9452 (squares) cells harvested in the exponential (open symbols) and
stationary (closed symbols) growth phases. Measurements were made in 1 mM KNO3 solution on two independent sets of data; the bars
represent 95% confidence intervals.
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Table 2 presents the surface elemental
composition, determined by XPS, for L. helveticus and
L. lactis cells harvested in the exponential and stationary
growth phases. No significant difference was observed between the two
growth phases for L. lactis, while L. helveticus
cells were slightly richer in oxygen and poorer in nitrogen in the
stationary growth phase. Except for the N/C ratio, which was slightly
inferior, the outermost surface chemical composition of L. lactis was similar to that of L. helveticus in the
stationary growth phase. The O/C, N/C, and P/C ratios determined on
L. helveticus in the exponential growth phase were similar to those reported by Mozes and Lortal (36). The
concentration of phosphorus detected at the extreme cell surface was
not affected significantly by the strain or growth phase of the cells.
Potassium was also detected in a concentration similar to that of
phosphorus.
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TABLE 2.
XPS peak assignments and surface chemical compositions
for L. helveticus ATCC 12046 and L. lactis subsp.
lactis bv. diacetilactis LMG 9452 cells harvested in
exponential or stationary growth phase
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The sorbitol control did not reveal any abnormal surface contamination
due to freeze-drying. The carbon peaks were decomposed in three
components (FWHM = 1.45 eV), attributed to carbon singly bound to
carbon and hydrogen [C---(C,H) at 284.8 eV],
carbon singly bound to oxygen or nitrogen
[C---(O,N) at 286.2 eV, including ether,
alcohol, amine, or amide], and carbon making a double bond or two
single bonds with oxygen (at 287.9 eV, denoted C==O, including amide, carbonyl, carboxylate,
acetal, or hemiacetal) (20, 44). The oxygen peaks were
decomposed in two components (FWHM = 1.63 eV), attributed mainly
to oxygen making a double bond with carbon (at 531.2 eV, denoted
O531.2, including carboxylic acid,
carboxylate, ester, carbonyl, or amide), and to oxygen involved in
hydroxide or ether functions (O---C at 532.6 eV). The nitrogen peaks were decomposed in two components (FWHM = 1.53 eV), attributed to nonprotonated nitrogen
(Nnonproton at 399.8 eV, involved in
amine or amide) and to protonated nitrogen (Nproton at 401.3 eV, involved in
ammonium or protonated amine). The phosphorus peak appearing at 133.4 eV was attributed to phosphate groups.
Table 2 presents the results of these decompositions. L. helveticus stationary cells and L. lactis cells in both
growth phases presented high C---(O,N)/C and
O---C/C ratios (around 0.5 and 0.4, respectively). For L. helveticus cells,
C---(O,N)/C and O---C/C
ratios increased 25% during culture, while the C---(C,H)/C ratio decreased. The
C==O/C and
O531.2/C ratios were not affected
significantly by the strain or growth phase of the cells. Finally, most
of the nitrogen was involved in nonprotonated amine or amide functions.
Figure 2 presents the plot of the molar
concentration ratios C==O/C and
O531.2/C as a function of
Nnonproton/C. If these three ratios
were exclusively due to amide functions, O==C---N, the graphs should
give a straight line with unit slope. The higher
C==O/C ratio may be due to the presence of
acetal functions, present in polysaccharides. The part of
C==O/C due to acetal (subscript Ac) may be
evaluated as previously described (13, 44). Figure 2A shows
that [C==O/C (C==O)AcC] is very close to
Nnonproton/C for L. helveticus and L. lactis; the same is found for other
gram-positive and gram-negative bacteria analyzed with a spectral
resolution similar to that used here. This indicates that most of the
nonprotonated nitrogen may be attributed to the peptidic link of
proteins. The higher value of
O531.2/C with respect to
Nnonproton/C (Fig. 2B) will be
discussed below.
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FIG. 2.
Molar concentration ratio with respect to total carbon,
as a function of the molar ratio of nonprotonated nitrogen to total
carbon (Nnonproton/C), of carbon
making one double or two single bonds with oxygen
(C==O/C) (open symbols) and after deduction of
the acetal contribution (closed symbols) (A) and of oxygen responsible
for the peak at 531.2 eV (O531.2/C,
mainly due to O==C/C) (open symbols) and after
deduction of 2 P/C (closed symbols) (B). Circles, L. helveticus ATCC 12046, including data from Mozes and Lortal
(36); squares, L. lactis subsp. lactis
bv. diacetilactis LMG 9452; triangles and diamonds, other gram-positive
(triangles) (16) and gram-negative (diamonds) (13, 27,
39) bacteria analyzed earlier.
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The XPS data were converted into concentrations of model
compounds (i.e., proteins, polysaccharides, and hydrocarbonlike
compounds) as previously described (13, 16). Table
3 presents the elemental compositions and
carbon concentrations of the three model constituents considered. In
this method of modeling the molecular composition, peptidoglycans are
considered a mixture of proteins and polysaccharides, and
(lipo)teichoic acids are considered a combination of hydrocarbonlike compounds and polysaccharides. Figure 3
presents the results obtained for different samples of L. helveticus and L. lactis harvested in the exponential
and stationary growth phases. The cell surface was very poor in
hydrocarbonlike compounds (less than 15% [wt/wt]); it was
essentially made of proteins and polysaccharides. For L. helveticus, the polysaccharide/protein ratio increased between the
exponential and stationary growth phases and showed an appreciable variation from one culture to another. This ratio was higher for L. lactis; it did not change during culture and showed
a weak variability from one culture to another.
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TABLE 3.
Chemical compositions of model constituents (molar ratio
with respect to total carbon) considered for deduction of the molecular
composition of cell surfaces
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FIG. 3.
Diagram showing the surface composition of L. helveticus ATCC 12046 (circles) and L. lactis subsp.
lactis bv. diacetilactis LMG 9452 (squares) in terms of
model compounds, as deduced from XPS. Pr, protein; PS, polysaccharide;
HC, hydrocarbonlike compounds. Cells harvested in the exponential
(closed symbols) and stationary (open symbols) growth phases are
represented.
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DISCUSSION |
Relationships among surface functional groups.
Figure 2A
confirms that the higher values of the C==O/C
ratio compared to the Nnonproton/C
ratio may be attributed to the acetal of polysaccharides (Fig. 2A). The
difference between the O531.2/C and
Nnonproton/C (Fig. 2B) ratios did not receive attention before. When this difference is plotted as a
function of the phosphate concentration (Fig.
4), a correlation is found (correlation
coefficient, 0.61); the trend is preserved if other bacteria are
included in the comparison. This difference may therefore be
interpreted in the light of previous data obtained on
phosphate-containing compounds. The O1s peaks of
AlPO4, CaHPO4, and
Ca2P2O7 were reported to appear at
531.6, 531.6, and 531.4 eV, respectively (26, 28). On the
other hand, spectra of KH2PO4, CaHPO4, and FePO4 (G. Vereecke, personal
communication) showed that the O1s peak of phosphate has
two components appearing in the ranges of 530.9 to 531.5 and 532.4 to
532.9 eV, respectively, with the relative intensities given in Table
4. Table 4 also gives the relative
intensities expected if the low binding energy peak is attributed to
P==O or
P---O and the high binding energy
peak is attributed to P---OH. A comparison of
experimental and expected data supports this assignment. In addition,
Table 4 gives the data obtained for glucose-6-phosphate (20)
and shows that they are in agreement with the assignment of the low
binding energy component to P==O and
P---O and the assignment of the
high binding energy component to P---O---C and
P---OH.
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FIG. 4.
Difference between the molar concentration ratios of
oxygen responsible for the peak at 531.2 eV and nonprotonated nitrogen
with respect to total carbon,
[O531.2/C Nnonproton/C], as a function of the
molar ratio of phosphate to total carbon (P/C). L. helveticus ATCC 12046, including data from Mozes and Lortal
(36) (circles), L. lactis subsp.
lactis bv. diacetilactis LMG 9452 (squares), and other
gram-positive (triangles) (16) and gram-negative (diamonds)
(13, 27, 39) bacteria analyzed previously are represented.
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TABLE 4.
Proportions of the components in the O1s XPS
peak for phosphate salts (G. Vereecke, personal communication) and for
glucose-6-phosphate (20) and proportions of the expected
types of oxygen
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In the case of bacteria, the excess of
O531.2/C with respect to
Nnonproton/C may thus be tentatively
attributed to the oxygen of phosphate groups, involved in
(lipo)teichoic acids or in phospholipids. In these moieties, two oxygen
atoms of phosphate are singly bound to carbon and are expected to
contribute to the component at 532.6 eV. One oxygen is doubly bound to
phosphorus, and one is in the form
P---O; these are expected to
contribute to the component at 531.2 eV, together with
O==C. Consequently, the
O==C/C ratio should be corrected as follows:
O==C/C = [O531.2/C] 2 P/C. Figure 2B
shows that this correction brings
O531.2/C closer to
Nnonproton/C;
O==C/C may thus be mainly assigned to proteins.
The above discussion of relationships between functional groups, as
detected by XPS, supports the accuracy of the sensitivity factors and
peak decomposition. Furthermore, it demonstrates the relevance of XPS
for quantifying the main classes of molecular constituents at the
surfaces of microorganisms.
Surface molecular composition.
The results of the modeling of
XPS data in terms of proteins, polysaccharides, and hydrocarbonlike
compounds (Fig. 3) may be discussed in the light of previous
biochemical studies. The cell surface composition of L. lactis shows a large excess of polysaccharide with respect to
protein. This is in line with the results (7) obtained with
loosely associated cell surface material of another strain of L. lactis subsp. lactis bv. diacetilactis. Moreover, it is
also in agreement with the fact that the phage receptors present at the
surfaces of other strains of L. lactis were found to be
located mainly on carbohydrate moieties (22, 35, 45, 50).
It is well established that the S-layer present at the surface of
L. helveticus ATCC 12046 is made of unglycosylated proteins (30, 31, 36). According to the amino acid composition of the
S-layer protein of L. helveticus determined by Lortal et al. (31), the ratio Nproton/C
should be about 0.030 compared with the ratio of 0.013 ± 0.002 measured by XPS. The observation of polysaccharide material at the
L. helveticus extreme surface may not be due to contribution
of material present below the S-layer, as the latter is expected to
have a thickness of about 9 nm (31). It may thus be
attributed either to a fragmentation of the S-layer, allowing the
underlying material to be at the surface, or to the protrusion of
polysaccharides and/or (lipo)teichoic acids through the S-layer. The
enrichment of the extreme surface in polysaccharide material for
stationary-phase cells could be due either to their synthesis during
growth or to a further fragmentation of the S-layer.
Relation between surface composition and physicochemical
properties.
Water contact angle measurements (Table 1) indicated
that the surfaces of L. lactis and L. helveticus
were hydrophilic whatever the growth phase. This is in agreement with
previous studies, based on microbial adhesion to solvents, which showed
a hydrophilic character for other strains of L. lactis
subsp. lactis bv. diacetilactis (7) and of
Lactobacillus (40). In previous works, cell
surface hydrophobicity, assessed by the water contact angle, was
directly correlated with the concentration of nitrogen or carbon
involved in hydrocarbon form and inversely correlated with the
concentration of oxygen (3, 13, 16, 34, 37). In this study,
the hydrophilic character of the two strains is in agreement with the
low concentration of hydrocarbon and the high concentration of oxygen
measured by XPS. However, the differences observed in the
polysaccharide/protein ratios when the two strains or the two growth
phases are compared are not accompanied by significant differences in
surface hydrophilicity.
L. lactis has a lower isoelectric point and an
electrophoretic mobility above pH 5 four times larger than L. helveticus; similar results were obtained on cells in exponential
and stationary growth phases (Fig. 1). In previous works, positive
correlations were found between the electrophoretic mobility at pH 4 and the phosphorus content of the extreme cell surface (37).
For a set of Lactobacillus species, Millsap et al.
(34) showed that the isoelectric point was directly
correlated with the nitrogen surface concentration and inversely
correlated with the oxygen surface concentration. The electrokinetic
properties of bottom-fermenting brewing yeasts were shown to be
controlled by phosphate, resulting in a low isoelectric point, while
those of top-fermenting yeasts were determined by the balance between
the protonated amino and carboxylate groups in proteins, giving a
higher isoelectric point (11).
In order to tentatively relate the electrokinetic properties to the
surface chemical composition, the surface charge concentration of the
exponential L. helveticus cell was computed, as described previously for yeasts (11). This was done by using the
surface chemical composition given by XPS and the amino acid
composition of its S-layer protein, determined from biochemical
analysis (31). Since the latter did not allow a distinction
between aspartic acid and asparagine or between glutamic acid and
glutamine, two extreme situations were considered: only aspartic and
glutamic acid or only asparagine and glutamine. In this charge
computation, three ionized groups were considered to determine the
surface electrical properties: phosphate groups (pKa = 2.15), involved in (lipo)teichoic acids, and carboxylate
(pKa = 3.95) and protonated amino
(pKa = 9.67) groups of proteins.
Figure 5A1 and A2 shows the variation of
the concentration of ionized functions and of the resulting charge as a
function of pH for the surface of L. helveticus in the
exponential growth phase, considering the two extreme situations and
the intrinsic acidity constants given above. Actually, the acidity
constant should be replaced by an apparent constant,
Kapp, obtained by multiplying the intrinsic
acidity constant by exp (F
/RT), where F is the
Faraday constant, is the local electrical potential, R
is the gas constant, and T is the temperature. The
electrical potential at the slip plane, the zeta potential, may be
deduced from the electrophoretic mobility through multiplication of the latter by 12.85. Using Kapp and assuming that
the local electrical potential was equal to the zeta potential provided
curves (not shown) which were not significantly different from those in
Fig. 5A1 and A2, respectively. In reality, the surface potential
differs from the zeta potential due to specific ion adsorption and the separation between the surface and the slip plane at which the zeta
potential is measured. In order to examine the sensitivity of the
results to the local potential, the concentrations of the ionized
groups and of the resulting charge were computed by assuming a local
electrical potential equal to 10 times the zeta potential. The results
obtained are given in Fig. 5B1 and B2. Comparison with Fig. 5A shows
that, in the pH range of interest, the local potential has a strong
influence on deprotonation of RCOOH; it has no effect on deprotonation
of RNH3+ and hardly any on that of
R2HPO4.
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FIG. 5.
Plot of the concentrations of
RNH3+ (a),
R2PO4 (b), RCOO
(c), and resulting charges (d) as a function of pH for
exponential-phase L. helveticus ATCC 12046 cells. The
acid-base equilibria are assumed to be unaffected by electrical
potential (A1 and A2) or affected by a local potential equal to 10 times the zeta potential (B1 and B2). The S-layer protein is assumed to
contain no acidic amino acids (A1 and B1) or no asparagine or glutamine
(A2 and B2).
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A comparison of Fig. 1 and 5 shows that the S-layer protein of L. helveticus contains a certain proportion of aspartic and glutamic
acids or that the polysaccharides protruding through the S-layer carry
carboxylate groups. Otherwise (Fig. 5A1), the total charge
concentration of the cell surface would remain positive over the whole
pH range, in contradiction to negative electrophoretic mobilities above
pH 3. Moreover, it appears in Fig. 5A2 that the carboxyl groups
determine the shape of the charge density-versus-pH curve and,
consequently, the shape of the electrophoretic mobility curve of
L. helveticus.
The Nnonproton/P ratio differs
markedly between L. helveticus (14 and 16) and L. lactis (8 and 7). The two strains follow the correlation between
N/P and the electrophoretic mobility at pH 4 reported for other
microorganisms (1, 37). The more negative character of
L. lactis compared to L. helveticus is thus related to the absence of an S-layer and a better exposition of the
underlying materials at the surface. A more detailed interpretation in
terms of the amount and nature (isoelectric point) of proteins (or
polypeptides), of (lipo)teichoic acids, and of the amount of
carboxylate borne by polysaccharides would require the examination of a
larger collection of strains and biochemical data.
Conclusion.
The interpretation of the XPS data has been
improved by showing that the oxygen peak component appearing around
531.2 eV contains a contribution of oxygen from phosphate (P==O and
P---O), together with oxygen making a double bond with carbon.
The surfaces of L. lactis and L. helveticus are
essentially made of proteins (or polypeptides) and polysaccharides. The
hydrophilic character of the two strains, as evidenced by the water
contact angle, is in agreement with a high concentration of
polysaccharide and with a low concentration of hydrocarbonlike
compounds. For L. helveticus, the polysaccharide/protein
ratio increases between the exponential and stationary growth phases,
which is attributed to either a fragmentation of the S-layer or a
synthesis of polysaccharides and/or (lipo)teichoic acids protruding
through the S-layer. The polysaccharide/protein ratio is higher for
L. lactis and does not change during culture.
The electrokinetic properties of L. helveticus reveal the
influence of carboxyl groups involved in the protein of the S-layer or
borne by polysaccharides. On the other hand, the more negative character of L. lactis is related to a lower N/P ratio at
the surface. The approach followed here may be used to relate the behavior of lactic acid bacteria to their surface chemical compositions and to understand the influence of culture conditions, strains, and mutation.
| |
ACKNOWLEDGMENTS |
We thank C. Dupont-Gillain, M. Genet, Y. Dufrêne, and J. Delcour for valuable discussions and G. Vereecke for providing XPS spectra of phosphate-containing compounds.
This work was supported by the Foundation for Training in Industrial
and Agricultural Research (FRIA), the National Foundation for
Scientific Research (FNRS), the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme), and the Research Department of Communauté
Française de Belgique (Concerted Research Action).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Chimie des Interfaces, Université Catholique de Louvain, Croix du
Sud 2/18, B-1348 Louvain-la-Neuve, Belgium. Phone: 32 10 47 35 89. Fax: 32 10 47 20 05. E-mail: rouxhet{at}cifa.ucl.ac.be.
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Abstract
Introduction
