Strict aerobic bacteria specifically use oxygen as Keywords Lactococcus lactis; proton motive force; redox; reducing activity; thiol groups Correspondence R.. lactis culture was mediated
Trang 1Contribution of exofacial thiol groups in the reducing
activity of Lactococcus lactis
D Michelon1, S Abraham1, B Ebel1, J De Coninck1, F Husson1, G Feron2, P Gervais1
and R Cachon1
1 Laboratoire de Ge´nie des Proce´de´s Microbiologiques et Alimentaires, AgroSup Dijon, Universite´ de Bourgogne, Dijon, France
2 Unite´ Mixte de Recherche 1129 FLAVIC ENESAD-INRA-UB, Dijon, France
Introduction
Electrochemical measurement of the oxidoreduction
potential (Eh) in liquid media has been used for eight
decades [1] Recent studies have emphasized that the
characterization of redox conditions is of both
scien-tific and practical significance in water [2], food
prod-ucts [3–6], soils [7] and sediments [8] In such complex
natural ecosystems, both the mechanisms involved in
the redox equilibrium and the possible influence of
microbial reductive activities on the measured redox
potential remain poorly investigated
The most important reaction catalyzed by microbial
cells is energy generation from the oxidation of organic
substrates and the corresponding dehydrogenation
steps in the glycolysis and citric acid cycles that gener-ate reduced electron carriers (NADH, FADH2) The latter are re-oxidized by electron transfer to oxidants (i.e respiratory metabolism) or to metabolic intermedi-ates (i.e fermentative metabolism) The use of external oxidants such as electron acceptors and, in some cases, the production of reduced compounds, might explain the reducing capacity (i.e decrease in Eh) measured in cultures of microorganisms
The implication of microorganisms in the redox equilibrium varies according to whether they are aerobic, facultative anaerobic or obligate anaerobic [9] Strict aerobic bacteria specifically use oxygen as
Keywords
Lactococcus lactis; proton motive force;
redox; reducing activity; thiol groups
Correspondence
R Cachon, Laboratoire de Ge´nie des
Proce´de´s Microbiologiques et Alimentaires,
AgroSup Dijon, Universite´ de Bourgogne,
site INRA, 17 Rue Sully, 21065 Dijon,
France
Fax: +33 3 80 69 32 29
Tel: +33 3 80 69 33 73
E-mail: remy.cachon@u-bourgogne.fr
(Received 17 July 2009, revised 22 February
2010, accepted 8 March 2010)
doi:10.1111/j.1742-4658.2010.07644.x
Lactococcus lactiscan decrease the redox potential at pH 7 (Eh7) from 200
to )200 mV in oxygen free Man–Rogosa–Sharpe media Neither the con-sumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in Eh7by the bacteria Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria After the final Eh7 ()200 mV) was reached, only thiol-reactive reagents could restore the initial Eh7value Inhibition of the proton motive force showed no effect on maintaining the final Eh7 value These results suggest that maintaining the exofacial thiol (–SH) groups in a reduced state does not depend on an active mechanism Thiol groups appear to be displayed by membrane proteins or cell wall-bound proteins and may participate in protecting cells against oxidative stress
Abbreviations
AMdIS, 4-acetamido-4¢-maleimidylstilbene-2,2¢-disulfonic acid, disodium salt; BIAM, N-(biotinoyl)-N ¢-(iodoacetyl)ethylenediamine;
CCCP, carbonyl cyanide m-chlorophenyl; DCCD, N,N ¢-dicyclohexylcardiimide; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); Dsb, disulfide bond formation protein in Escherichia coli; E0¢, midpoint oxidation reduction potential; E h , redox potential; Eh7, redox potential at pH 7; FNR, transcription factor fumarate nitrate reductase; GSH, glutathione; MRS, Man–Rogosa–Sharpe; NEM, N-ethylmaleimide; PMF, proton motive force; PMSF, phenylmethanesulfonyl fluoride.
Trang 2terminal electron acceptors in respiration, which restricts
the range of redox potentials to values close to the
oxi-dant values [10] Anaerobes have higher reducing
capacities; they can decrease the Eh from )200 to
)600 mV [9] They can reduce external terminal
elec-tron acceptors such as NO3), SO4 ), Mn(III⁄ IV) and
Fe(III) [11] but, in many cases, their reducing
capaci-ties can be explained by the production of strongly
reducing end-products, such as H2(midpoint oxidation
reduction potential, E0¢ =)420 mV) [12]
Enterobac-teria such as Escherichia coli can produce H2 during
mixed fermentation, regardless of the pH [13,14] For
this bacterium, it has also been suggested that, under
aerobic conditions, other reducing mechanisms might
also be involved [15]
Lactic acid bacteria have no respiratory chain and
no strong peroxidase activity (catalase)), but can
partly tolerate oxygen They obtain most of their
energy from lactic acid fermentation; reducing
equiva-lents (NADH) produced during glycolysis are used to
reduce pyruvate to lactic acid Among the lactic acid
bacteria, some species have low reducing capacities
(e.g Lactobacillus bulgaricus, Streptococcus thermophilus)
[3,16], whereas higher reducing species such as
Lactococcus lactis are able to decrease the Eh7 to
)200 mV (Eh7: Eh calculated at theoretical pH 7) [3]
L lactis can eliminate oxygen by water-forming
NADH oxidase [17] using NADH produced during
glycolysis, thus leading to a decrease in Eh
Neverthe-less, oxygen disruption by L lactis cannot explain the
decrease in Eh from oxidant to reducing values
Removing oxygen from a liquid media using nitrogen
gas does not decrease the Eh to reducing values [4,18]
According to the Nernst equation, Eh is decreased by
59 mV for one log of oxygen concentration, and it is
generally observed that degassing the medium only
decreases the Eh by 100–150 mV These results
strongly suggest that other mechanisms may be
involved in the reducing activity of L lactis, and that
reducing molecules leading to an Eh7 of)200 mV may
be implicated This bacterium does not produce
hydro-gen or H2S, which are the main reducing molecules
produced by microorganisms; thus, it is an attractive
model for investigating new mechanisms involved in
the reducing activity of microorganisms This was the
aim of the present study
Results
Redox activity of L lactis
Figure 1 presents the evolution over time of Eh7 and
pH in Man–Rogosa–Sharpe (MRS) media under
anaerobic conditions The initial pH and Eh7 values were respectively 6.5 ± 0.1 and 204 ± 34 mV Eh7 was the first parameter that changed, with a maximal reducing rate equal to )367 mVÆh)1 After 5.5 h, the reduction stage was finished and the Eh7remained sta-ble at approximately)200 mV until the end of fermen-tation Acidification began 1 h after the start of the reduction step, with a final pH of 4.7
These results were obtained in oxygen free med-ium The reducing activity of L lactis has only been previously reported for cultures in static aerobic batch conditions [3] We can thus conclude that the aptitude of L lactis to decrease Eh is not dependent
on the presence of oxygen Moreover, the final Eh7 values were the same ()200 mV) despite the fact that the culture media (MRS⁄ milk) had different initial
Eh7 values (100–240 mV in MRS; 230 mV in milk); consequently, L lactis reduction is not so much characterized by the amplitude of the decrease in Eh7 but rather by the final Eh7 Lastly, a major part of the acidification occurred once the final Eh7 had already been reached During both the acidification stage and after the final pH was reached, the final
Eh7 was stable
Implication of cell components in the decrease in
Eh The bacterial cells were removed from the culture media by filtration after the minimal Eh7 ()200 mV) was reached (Table 1) For the three stains of L lactis, the Eh7 measured in the filtrate was not significantly different from the initial Eh7of the sterile MRS media Moreover, maximal care was taken in the experiment
to avoid introducing oxygen into the filtrate Conse-quently, the restoration of the initial Eh7 in the filtrate
4.0 4.5 5.0 5.5 6.0 6.5 7.0
–250 –150 –50 50 150 250
Eh7
Time (h)
(A) (B)
Fig 1 Time course evolution of (A) Eh7and (B) pH during culture
of Lactococcus lactis (TIL46) under anaerobic conditions (N2) Experiments were performed in triplicate; average curves are shown (SD for Eh: ± 22 mV; SD for pH: ± 0.1 pH units).
Trang 3was provoked by removing the bacterial cells from the
culture media rather than by the dissolved oxygen
These results led us to assume that the drop in Eh7 in
the L lactis culture was mediated by the whole cells,
thus demonstrating that the decrease in the redox
potential to a low value was not caused by the
produc-tion of end-products with reducing metabolisms
L lactis is able to maintain a low Eh7 until the end
of fermentation and for 24 h [16] The role of the
active mechanisms in maintaining a low reducing Eh7
was thus investigated Only the effects of compounds
that could modify the cell activity were investigated
The activity of L lactis is dependent on the
mainte-nance of the proton motive force (PMF), which is
involved in the membrane transport systems This
PMF is composed of a pH gradient and an
electro-chemical gradient Four energetic inhibitors were used
to target PMF activity: nigericin (a K+⁄ H+
exchan-ger) and valinomycin (an ionophore) were used
together to destroy the PMF; carbonyl cyanide
m-chlorophenyl (CCCP) (a protonophore) was used to
cancel the pH gradient; and
N,N¢-dicyclohexylcardii-mide (DCCD) acts as a specific proton pump
F0F1-ATPase inhibitor DCCD, CCCP and a
nigeri-cin⁄ valinomycin mixture were added just after the end
of reduction (Eh7)200 mV) (Fig 2) Using such
inhibitors, the PMF, ATP synthesis and primary and
secondary transport systems collapsed, and the
glyco-lytic flux and acidification of the medium by lactic acid
synthesis stopped Despite inhibition of the metabolism
and, consequently, the decrease in ATP and NADH
levels, the low reducing potential remained stable
(Fig 2)
Thiol groups and the decrease in Eh
The role of thiol groups in the decrease in Eh was
investigated using thiol-reactive reagents
[N-ethylmale-imide (NEM),
4-acetamido-4¢-maleimidylstilbene-2,2¢-disulfonic acid, disodium salt (AMdiS)] They
contain maleimide, which can bind with thiol groups
in an irreversible reaction that may suppress the con-tribution of thiol groups to the redox equilibrium NEM can diffuse across a cytoplasmic membrane Consequently, thiol groups on both sides of the mem-brane and in the cytosol are neutralized by this thiol reagent By contrast, AMdiS can only neutralize acces-sible thiol groups exposed on the external bacterial surface The addition of NEM or AMdiS to a reducing culture of L lactis rapidly increased the Eh7 and restored the initial Eh7value (Fig 3) These data show the implication of thiol groups in the decrease in Eh mediated by L lactis and their exofacial localization
As shown in Fig 4, thiol groups were labeled with a membrane impermeable fluorescent thiol-reactive reagent and observed using an upright fluorescent microscope The latter procedure allowed us more par-ticularly to visualize the fluorescent rim on the surface
of L lactis, confirming the presence of exofacial thiol groups
Table 1 Effect of filtration on the E h7 value of MRS media reduced by three different strains of Lactococcus lactis Filtration was carried out at pH 6 E h7 i, redox potential in degassed sterile MRS with (n = 81); E h7 r, redox potential in culture media when the minimal E h7 was reached (for each strain, n = 3); Eh7f, redox potential in filtrate (for each strain, n = 3).
Strains
Eh7(mV)
a
ANOVA (P < 0.05, n = 3), values in a column with the same superscript letter are not significantly different.bANOVA test (P < 0.05, n = 3), values in a row with the same superscript letter are not significantly different.
3 3.5 4 4.5 5 5.5 6 6.5 7
–250 –200 –150 –100 –50 0 50 100 150 200 250
Eh7
Time (h)
(A) (C)
(D) (B)
Fig 2 Effect of inhibitors on Eh7and pH during lactic acid fermen-tation by Lactococcus lactis TIL 46 (typical curves) Nigericin and valinomycin mixture or DCCD or CCCP were added when the mini-mal E h7 was reached Curves (A) and (B) are the pH and E h7 in the experiments with the addition of inhibitor (the arrow indicates the time of addition); curves (C) and (D) are the pH and E h7 in the control experiment.
Trang 4Evolution of exofacial thiol groups during
medium reduction by L lactis
The concentration of accessible exofacial thiol groups
was monitored during the growth of L lactis
(Fig 5A) Before the reduction phenomenon began,
the concentration of thiol groups was below 1 lm, and
increased to 12 lm at the end of growth The latter
thiol concentration was correlated to the reducing
activity Indeed, a decrease in Eh7 was linked to an
increase in exofacial thiol groups (Fig 5A, phase 1)
and the Eh7 ceased to decrease and remained stable
when the maximal amount of thiol groups was reached
(Fig 5A, phase 2) During the reduction phase, the
amount of the exofacial thiol groups was correlated
with growth, with a value of 7.7 attomolÆcell)1
(Fig 5B, phase 1) When the final Eh7 was reached,
growth had not yet finished and the amount of
exofa-cial thiol groups per cell decreased to 5.1 attomolÆcell)1 (Fig 5B, phase 2) These results confirm that the decrease in Eh7 was directly related to exofacial thiol groups
Exofacial protein thiols Bacterial cells of L lactis were labeled with a biotiny-lated cell impermeable thiol reagent (BIAM) [19,20] targeting only thiol groups on the external face of the membrane The membrane protein fraction of the sam-ples treated with BIAM only or the samsam-ples pre-trea-ted with NEM and then treapre-trea-ted with BIAM were loaded (20 lg) onto each lane After western blotting, BIAM-labeled SH groups were mainly detected in the lanes loaded with BIAM-treated samples only (Fig 6),
in contrast to the lanes loaded with NEM-pre-treated samples These results confirmed that the thiol groups were located on the external face of L lactis, on pro-teins The thiol groups were mainly on cysteine resi-dues in proteins Consequently, the results obtained suggest that exoproteins (i.e membrane proteins or cell wall proteins) were involved in the decrease in redox potential
Discussion
The capacity of L lactis to decrease Eh to a reducing value is known, although the mechanism involved is not understood [3,16] An interesting redox property
of L lactis is the final reducing redox value of
Eh7)200 mV, regardless of the culture medium and the initial Eh7 value [3] Moreover, this reducing Eh7 value remained very stable until the end of fermenta-tion External reducing Eh stability suggests that reversible redox systems might be involved [21] We showed that thiol-reactive reagents were able to cancel this reducing Eh stability under anaerobic conditions; therefore, a thiol–disulfide couple (Eqn 1) is likely to play a major role in maintaining the external reducing
–300
–200
–100
0
100
200
300
Eh7
E
E
E
h7 f,n,a
a a
Fig 3 Effect of thiol-reactive reagents and filtration on the
decrease in E h7 by Lactococcus lactis TIL 46 in MRS media.
Eh7i = Eh7in sterile MRS media (n = 12); Eh7r = Eh7after reduction,
(n = 12); Eh7f, n, a = Eh7after filtration (f) or the addition of NEM
(n) or AMdiS (a) Each different treatment (filtration, NEM or
AMdiS) was performed in a separate experiment and each
experi-ment was carried out in triplicate ANOVA (P = 0.05) was used for
the statistical analysis and significant differences are shown by an
‘a’ above the column.
Fig 4 Labeling with Oregon Green 488
maleimide of surface thiol groups on the
bacterial cell surface of Lactococcus lactis
TIL 46 Images were realized using bright
field (A), or laser excitation at 480 nm (B) to
confirm that all the bacterial cells were
fluorescent.
Trang 5Eh [glutathione (GSH); E0¢ =)240 mV and cysteine;
E0¢ =)340 mV] [22,23]
RSSR + 2Hþ + 2e$ 2RSH ð1Þ
It could be suggested that the Eh was stabilized when the concentration of thiol molecules compared to the bacterial cell density was sufficient to establish a more intense current between the thiol–disulfide redox couple and the Pt electrode than other redox couples
in the culture medium [24] The thiol concentration was directly related to the bacterial cell concentration, which means that a minimum cell density around the
Pt electrode surface was required for an optimal exchange current between the thiol–disulfide molecules and the Pt electrode This might explain the same final
Ehvalues in different complex media (MRS⁄ milk)
In aerated E coli and Bacillus subtilis cultures, a sharp decrease in Eh during the transition from the active growth phase to the stationary phase was observed and was related to a transitory increase in thiol groups in both the culture medium and on the cell surface [15] The Eh of the periplasm of E coli
Fig 6 Labeling of membrane protein fraction with selective
bioti-nylated thiol reagent (BIAM) Twenty micrograms of proteins were
loaded in each lane and transferred to a nitrocellulose membrane
for western blotting Lane A, membrane protein fraction from
NEM-untreated sample; lane B, membrane protein fraction from
NEM-treated sample.
0 2 4 6 8 10 12 14 16 18 20 22 24
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
A
B
8 cells.mL
Time (h)
6
8
10
12
14
16
18
Eh7
Eh7
0
2
4
0 2 4 6 8 10 12 14 16 18 20 22 24
Cell concentration (10 11 cells L –1 )
y = 0.77
2 = 0.9896)
Fig 5 Evolution in the concentration of the exofacial thiol groups during reduction by Lactococcus lactis TIL 46 (A) Time course evolution of growth ( ) and time course evolution of concentration of exofacial thiol groups ( ) (B) Evolution according to the amount of exofacial thiol groups per cell during growth of L lactis Phase 1 is the reduction phase and phase 2 is the phase when the Eh7is stabilized at )200 mV Exofacial thiol groups were measured using Ellman’s method.
Trang 6depends on the presence of thiol–disulfide proteins
(Dsb) and GSH, and was maintained at)165 mV [25]
Moreover, the standard redox state (E0¢) of
thioredox-in superfamily protethioredox-ins characterized by two active-site
cysteine residues separated by two amino-acids
(CX1X2C) was in the range )125 mV (DsbA) to
)270 mV (thioredoxin ⁄ DsbB) [26] A method based on
the protein–protein redox equilibrium enabled the E0¢
of two thiol–disulfide oxidoreductases of E coli:
glut-aredoxin 1 and 3, to be determined ()233 and
)198 mV, respectively) [27] It was strongly suggested
that the release of extracellular GSH participates in
modulating the thiol–disulfide ratio in the medium and
on the cell surface in response to a variation in the
intracellular pH [15] GSH is not synthesized in
L lactis[28] and a decrease in Ehis mainly associated
with an increase in accessible thiol groups on the cell
surface
The results obtained in the present study clearly
identify the implication of exofacial thiol groups in the
decrease in Eh and suggest that these thiol groups are
located on proteins (exoproteins: membrane proteins,
cell wall-bound proteins) Thiol groups are known to
play a central role in protection against oxidative stress
and contribute to detoxifying the reactive oxygen
spe-cies by reversible thiol oxidation to bound disulfide
[29] One or several proteins might be implicated; for
example, an arginine–ornithine antiporter in L lactis
was characterized by reactive exofacial thiol groups
displayed on the outer surface of the cytoplasmic
membrane [30] The identification of proteins located
on the extracellular surface and involved in the
decrease in Ehwould be of interest for increasing our
understanding of the mechanisms involved as well as
the reducing activity of L lactis
A Gram-positive bacterium such as L lactis has a
thick cell wall composed of mainly peptidoglycan and
teichoic acids Proteins present on the external surface
are mainly anchored to the cytoplasmic membrane, in
contrast to Gram-negative bacteria Despite these
major structural differences, thiol–disulfide
oxidoreduc-tases characterized by thioredoxin-like sequence motifs
(CXXC) that form the core of the active site [31], and
with a similar Dsb function, are present in vegetative
forms of B subtilis The latter and L lactis are
mem-bers of the same phylogenetic class and the analysis of
the L lactis genome revealed that the conserved
thio-redoxin-like motifs are present in numerous ORFs
encoding repair or stress response proteins [32]
Homo-log disulfide bond formation proteins in B subtilis
such as BdbB, BdbC or CcdA may also be involved in
the display of exofacial thiol groups and their role in
decreasing the Ehcan be implied
Bacteria are able to sense the extra or intracellular environmental redox state with redox sensing mecha-nisms related to the thiol–disulfide balance and adapt their cell activity [29] Two genes encoding transcrip-tion factor fumarate nitrate reductase (FNR)-like pro-teins (flpA and flpB) with a potential for mediating the dithiol–disulfide regulatory switch, were discovered in
L lactis [33] In E coli, the FNR protein plays a major role in altering gene expression under aerobic and anaerobic conditions [29] Thereby, as demon-strated in Bacillus cereus, FNR-like proteins can act coordinately with another redox response regu-lator such as ResDE, which is composed of a mem-brane sensor and a cytoplasmic regulator [34,35] Thiols might be used as ligands to coordinate such redox-responsive clusters [29]
In conclusion, the present study has shown that a decrease in anaerobiosis and the reduction phenomenon are not coupled to an accumulation of reducing end-products in the environment or the consumption of oxidizing compounds, as is mainly observed for other bacterial species The exofacial thiol groups play a cen-tral role in decreasing the Eh, and this Eh reduction appears to be linked to the density of cells around the
Pt electrode Thiol groups displayed on proteins on the bacterial cell surface could establish a reducing microen-vironment around the cell Maintaining a low reducing potential was not directly related to metabolic activity, whereas reducing equivalents such as NADH or thio-redoxin are likely to be involved in the formation of exofacial thiol groups during the reducing phase
Materials and methods
Chemicals
5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB), N-acetyl-l-cys-teine, phenylmethylsulfonyl fluoride (PMSF) nigericin and valinomycin were purchased from Sigma (St Quentin
France)
Bacterial strains and culture conditions
The L lactis subsp cremoris TIL46 derived from L lactis NCDO763 cured of its 2 kb plasmid (National Collection
of Food Bacteria, Shinfield, Reading, UK) and SK11 pro-vided from the CNRZ collection of INRA, as used in the present study, were kindly provided by Dr M Yvon The
the collection of the French Association for Research in the
Trang 7Dairy Industry (Paris, France) A concentrated stock cell
suspension was stored in MRS media supplemented with
Cultures were grown in static conditions in MRS media
harvested and concentrated by centrifugation (3500 g for
15 min) and resuspended in buffer 7 (0.1 m potassium
phos-phate, pH 7) for inoculation
General methods: L lactis growth and data
acquisition
As with L lactis, there is an oxygen-responsive FNR-like
transcriptional regulator [33,36,37], and all experiments
were carried out in a specific anaerobic chamber (Bactron I;
Sheldon Manufacturing, Cornelius, OR, USA) to prevent
oxygen having an effect on the medium’s redox properties
and any oxygen-induced oxidative stress MRS media was
pH were measured with a combined autoclavable redox
electrode and a combined autoclavable pH electrode
SARL, Paris, France) The next steps were performed in an
anaerobic chamber Oxygen was degassed by nitrogen
Ilkirch, France)
the latter is different from hydrogen In our case, the
Filtration
Filtration and heat treatment were performed at the end of
poly(vinylidene fluoride) (Millipore, Carrigtwohill, Ireland)
and the redox potential of the filtrate was measured Syn-thetic membrane filters [poly(vinylidene fluoride)], charac-terized by very low protein absorption, were used and were degassed by three nitrogen injections beforehand
Thiol-reactive reagents and energetic inhibitors
reagents (NEM, AMdiS) and inhibitors (DCCD, CCCP, nigericin and valinomycin) were added at the end of the reduction stage As a control, a stock solution of 1 m NEM was prepared in methanol : water (3 : 1) and degassed, and the equivalent volume of a methanol : water mixture was added The final concentration of the NEM batch was
25 mm A 65.2 mm AMdiS stock solution was prepared in water with a final concentration of 9 mm A 0.2 m DCCD stock solution was prepared in acetonitrile, with a final DCCD batch concentration of 9 mm A 140 mm CCCP stock solution was prepared in methanol with a final CCCP batch concentration of 152 lm A nigericin : valinomycin stock solution was prepared in methanol with a concentra-tion of 1.6 and 12 mm, respectively, with a final
respectively For each experiment, controls were carried out using equivalent volumes of the solution used for diluting the chemical compounds (methanol, acetonitrile and water)
Titration of free accessible exofacial thiol groups
Exofacial (accessible) thiol groups were measured using Ellman’s method DTNB is membrane impermeable, and only the thiol groups on the bacterial cell surface can react with the reagent Cells were collected by centrifugation for
15 min at 3500 g, and they were dislocated with 1 mL of buffer 8 (0.1 m potassium phosphate buffers, pH 8) con-taining 10 lL of 6 mm DTNB After 30 min of incubation
in the dark at room temperature, the cell suspension was centrifuged for 15 min at 3500 g The supernatants
the filtrate was measured and the concentration of accessi-ble free thiol groups was calculated using the N-acetyl-l-cysteine standard curves The standard curves were in the range 5–60 lm
Fluorescent thiol labeling on the bacterial cell surface
The bacterial culture was centrifuged for 15 min at 3500 g and the supernatant was eliminated Cell pellets were
times with buffer 7 and mounted in Fluorsave reagent (Calbiochem, San Diego, CA, USA) to avoid rehydration and to reduce fluorescence decay The slides were dried
Trang 8overnight in the dark at room temperature and analysed
using bright field or at a wavelength of 480 nm under an
upright fluorescent microscope (Axioplan 2i; Carl Zeiss,
Jena, Germany) Images were acquired using axiovision
4.8 software and an AxioCam MRm digital camera
(Carl Zeiss)
Protein extraction and blotting analysis
Bacterial cells of L lactis TIL 46 were produced as
previ-ously described Cells were collected by centrifugation for
15 min at 3500 g at room temperature when the minimal
(0.05 m potassium phosphate buffers, pH 7.5) Part of the
cells was incubated with 100 mm NEM for 30 min to block
all free thiol groups NEM-treated and NEM-untreated
cells were incubated with 0.9 mm BIAM for 30 min at
NEM and the cells were washed three times in buffer 7.5
protop-lasts were then centrifuged for 15 min at 21 000 g and the
supernatant was removed The pellet was resuspended in
buffer 7.5 containing 0.2 mm PMSF and glass beads
the final mixture The latter were homogenized with
was centrifuged for 1 min at 800 g to eliminate the glass
beads The membranes were pelleted by centrifugation 1 h
at 300 000 g and resuspended in buffer [Tris-HCL, 5 mm;
EDTA, 20 mm, PMSF, 0.2 mm; n-dodecyl-l-maltoside,
for 1 h at room temperature and lastly the insoluble
mate-rial was removed by centrifugation for 1 h at 21 000 g and
Protein titration was carried out using the Bio-Rad
Twenty micrograms of protein from each sample was
sub-jected to a short SDS-PAGE using 12.5% polyacrylamide
(the samples were boiled for 5 min in Laemmli sample
buffer prior to loading on the gel) The proteins were
trans-ferred to nitrocellulose membranes (Bio-Rad) using humid
Cell; Bio-Rad) and revealed by exposing the blot to
avidin-horseradish peroxidase conjugate (Bio-Rad) followed by
development with 3,3¢-diaminobenzidine Color
Develop-ment Solution (Bio-Rad) and hydrogen peroxide
Statistical analysis
Data were analysed using the statistical analysis software
plots were compared by analysis of variance (anova) with
Acknowledgements
This work was supported by a EUREKA Research grant (R!3562-LABREDOX) We would like to thank Catherine Vergoignan (INRA) for her technical aid as well as all members of the LABREDOX project
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