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Microbiology (2004), 150, 335–340
DOI 10.1099/mic.0.26731-0
Physiological implications of class IIa bacteriocin
resistance in Listeria monocytogenes strains
Viveka Vadyvaloo, Jacky L. Snoep, John W. Hastings
and Marina Rautenbach
Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland,
South Africa
Correspondence
Jacky L. Snoep
[email protected]
Received 25 August 2003
Revised 17 October 2003
Accepted 5 November 2003
High-level resistance to class IIa bacteriocins has been directly associated with the
absent EIIABMan (MptA) subunit of the mannose-specific phosphoenolpyruvate-dependent
phosphotransferase system (PTS) (EIIMan
) in Listeria monocytogenes strains. Class IIa
t
bacteriocin-resistant strains used in this study were a spontaneous resistant, L. monocytogenes
B73-MR1, and a defined mutant, L. monocytogenes EGDe-mptA. Both strains were previously
reported to have the EIIABMan PTS component missing. This study shows that these class IIa
bacteriocin-resistant strains have significantly decreased specific growth and glucose
consumption rates, but they also have a significantly higher growth yield than their corresponding
wild-type strains, L. monocytogenes B73 and L. monocytogenes EGDe, respectively. In the
presence of glucose, the strains showed a shift from a predominantly lactic-acid to a mixed-acid
fermentation. It is here proposed that elimination of the EIIABMan in the resistant strains has
caused a reduced glucose consumption rate and a reduced specific growth rate. The lower
glucose consumption rate can be correlated to a shift in metabolism to a more efficient pathway
with respect to ATP production per glucose, leading to a higher biomass yield. Thus, the cost
involved in obtaining bacteriocin resistance, i.e. losing substrate transport capacity leading to a
lower growth rate, is compensated for by a higher biomass yield.
INTRODUCTION
Food-associated strains of lactic acid bacteria frequently
produce antimicrobial compounds referred to as class IIa
bacteriocins (Ennahar et al., 2000a). Class IIa bacteriocins
have been grouped based on their high homology, conserved N-terminal YGNGV motif and effective antilisterial activity (Klaenhammer, 1993; Ennahar et al., 2000b;
Héchard & Sahl, 2002). The potential application of class
IIa bacteriocins as food preservatives has been extensively
studied in the search for safe, non-toxic, antimicrobial food
additives. However, the frequent occurrence of resistance
has become an increasingly important concern, since it
reduces the value of adding class IIa bacteriocins to foods
(Gravesen et al., 2002a; Ennahar et al., 2000b).
Investigations into the mechanism of class IIa bacteriocin
resistance shows strong evidence for one prevalent mechanism among various listerial strains and Enterococcus faecalis
(Gravesen et al., 2002b; Héchard et al., 2001). This mechanism involves the absence of the EIIAB subunit of a mannosespecific phosphoenolpyruvate-dependent phosphotransferase
system (PTS) (Gravesen et al., 2002b). PTS is a group
Abbreviation: PTS, phosphoenolpyruvate-dependent phosphotransferase system.
0002-6731 G 2004 SGM
translocation sugar transport system where the sugar concomitant with transport is phosphorylated. The phosphate
group is transferred via a number of enzymes from phosphoenolpyruvate to the sugar (Lengeler et al., 1994; Postma
et al., 1993; Tchieu et al., 2001; Siebold et al., 2001). An
insertional inactivation of the mptA gene encoding the
EIIAB subunit that resides on the tricistronic mptACD
in L. monocytogenes resulted in a high
operon of EIIMan
t
level of resistance to class IIa bacteriocins (Dalet et al.,
2001; Gravesen et al., 2002b). It has been suggested that the
PTS membrane component or permease (MptD)
EIIMan
t
could play a role as a possible target for class IIa bacteriocins
(Dalet et al., 2001; Héchard & Sahl, 2002; Gravesen et al.,
2002a).
Carbohydrates are required by L. monocytogenes as the
primary free-energy source for growth, with glucose being
the preferred source (Pine et al., 1989; Premaratne et al.,
1991). There is evidence for the presence of two glucose
transport systems in L. monocytogenes, a high-affinity
PTS and a low-affinity proton-motive-force-driven system
(Parker & Hutkins, 1997). There are indications that the
mannose PTS transports glucose in L. monocytogenes (Dalet
et al., 2001) and this PTS is also known to transport
mannose and 2-deoxyglucose (Chaillou et al., 2001; Romick
et al., 1996). In many lactic acid bacteria and streptococci,
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335
V. Vadyvaloo and others
transport and phosphorylation of glucose occurs mainly
via a mannose PTS (Chaillou et al., 2001; Vadeboncoeur &
Pelletier, 1997), which may be similar for L. monocytogenes.
Schott bottle cultures, at regular intervals from early-exponential
phase through to stationary phase, for monitoring of glucose
consumption.
The aim of this study was to investigate the effect of the
on glucose metabolism
missing MptA subunit of the EIIMan
t
in class IIa bacteriocin-resistant L. monocytogenes strains.
We focused on glucose consumption rates and analysis
of the end products of glucose metabolism. The growth
patterns of L. monocytogenes were also analysed in this
study in brain–heart infusion (BHI) culture medium with or
without added glucose as a free-energy source. All studies
were done on two wild-type, sensitive L. monocytogenes
strains, and their corresponding class IIa-resistant variant,
of which one was a spontaneous mutant and the other a
genetically defined mutant.
Quantification of glucose and fermentation end products.
METHODS
Bacterial strains and growth conditions. All strains were grown
in glucose-free BHI broth (Difco), supplemented, or not supplemented, with 10 mM glucose (Associated Chemical Enterprises,
Glenvista, South Africa), according to the requirements of the study.
The strains were cultivated at 37 uC without shaking, in tightly
capped Spectronic tubes or in Schott bottles. The L. monocytogenes
strains used were the following: wild-type food isolate L. monocytogenes B73, and corresponding class IIa bacteriocin spontaneous
mutant L. monocytogenes B73-MR1; wild-type clinical isolate L.
monocytogenes EGDe, and corresponding insertionally inactivated
mptA mutant L. monocytogenes EGK54 (Gravesen et al., 2002b;
Héchard et al., 2001), referred to as L. monocytogenes EGDe-mptA,
which displays resistance to class IIa bacteriocins. Media used to
grow L. monocytogenes EGDe-mptA were supplemented with 5 mg
erythromycin ml21.
Growth analysis. Bacterial growth was monitored using optical
density (OD) at 600 nm. Dry weight measurements were calibrated
against OD600 measurements. An OD600 value of 1?0 corresponds
to 0?64 g dry weight l21. Specific growth rates were calculated from
the growth absorbance data collected from Spectronic tube cultures,
and the same cultures were sampled for analysis of end products
of fermentation. In a separate experiment, samples were taken from
Samples collected for HPLC analysis were prepared and analysed
as described in Ward et al. (2000). Samples were analysed for
glucose, lactate, pyruvate, acetate, formate and ethanol. Glucose
was also determined enzymically using a linked hexokinase/glucose6-phosphate dehydrogenase assay. The buffer for the assay contained
890 mM Tris/HCl buffer pH 7?6, 2?38 U hexokinase, 1?19 U glucose6-phosphate dehydrogenase, 8?26 mM ATP, 1?27 mM NADP and
10 mM MgSO4. Product analysis allowed the calculation of carbon
recovery, glucose yields, ATP yields and glucose consumption rates.
Calculations and statistical analysis. Calculations of specific
growth rates and Student’s t-tests were done using GRAPHPAD PRISM
3.0 (GRAPHPAD software, San Diego, CA, USA) and MATHEMATICA
(Wolfram Research; http://www.wolfram.com).
RESULTS
Growth in the presence or absence of glucose
To investigate the physiological implications of acquiring
class IIa bacteriocin resistance, growth was followed in
wild-type and class IIa bacteriocin-resistant strains grown
on BHI in the absence or presence of glucose (Fig. 1).
Comparing the specific growth rate of the wild-type strains
and the respective resistant strains, we see that in the
presence of glucose the wild-type strains have a higher
growth rate, while in the absence of glucose the resistant
strains grow faster (Table 1). Biomass concentrations of
resistant strains and their respective wild-type strains were
comparable in the absence of glucose, while in the presence
of glucose the resistant strains resulted in a higher final
biomass than the respective wild-type strains (Table 1).
Biomass yield on glucose, calculated as the difference in
biomass concentration in the presence and absence of
glucose divided by the glucose used, indicated higher values
for the resistant strains [respectively, 29?1 and 45?1 g dry
Fig. 1. Growth of L. monocytogenes strains in BHI supplemented with 10 mM glucose (a) and BHI without glucose (b).
Growth studies were carried out at least in duplicate for all the strains. &, Wild-type B73; m, class IIa-resistant mutant
B73-MR1; ., wild-type EGDe; X, class IIa-resistant, insertionally inactivated mptA mutant EGDe-mptA.
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Microbiology 150
Bacteriocin resistance in L. monocytogenes
Table 1. Maximum specific growth rate and biomass of wild-type and class IIa bacteriocin-resistant L. monocytogenes strains
in BHI broth supplemented, or not supplemented, with glucose
Experimental values represent a mean of at least two independent measurements, and standard deviations are shown in parentheses.
L. monocytogenes
strain
Specific growth rate (h”1)
BHI with 10 mM glucose
B73
B73-MR1*
EGDe
EGDe-mptA*
0?68
0?46
0?84
0?70
(0?018)
(0?008)D
(0?028)
(0?015)D
Biomass (OD600)
BHI without glucose
0?70
0?83
0?52
0?67
(0?014)
(0?007)D
(0?027)
(0?011)
BHI with 10 mM glucose
0?68
0?76
0?79
0?91
(0?003)
(0?019)
(0?004)
(0?001)D
BHI without glucose
0?28
0?28
0?33
0?36
(0?006)
(0?003)
(0?023)
(0?009)
*Indicates the class IIa-resistant L. monocytogenes strains.
DRepresents a significantly different (P<0?05) growth rate or growth yield (biomass) of the resistant strain compared to the corresponding
wild-type strain.
weight (mol glucose)21 for B73-MR1 and EGDe-mptA]
than the wild-type strains [respectively, 24?1 and 27?9 g dry
weight (mol glucose)21 for B73 and EGDe].
Fermentation analysis
The difference in biomass yields in the different cultures
was further investigated by analysing the fermentation
products. BHI is a rich medium and, even without an
additional free-energy source added to the medium, significant growth was observed. However, the final biomass
concentrations were much lower as compared to the
cultures to which 10 mM of glucose was added. Glucose
consumed by the bacteria was completely converted to
fermentation products, indicating that it served as freeenergy source (not as carbon source) and that biomass
formation in BHI was limited by the availability of a freeenergy source. Only very low concentrations of fermentation products were formed during growth on BHI (without
glucose), typically 3 mM of formate and 2 mM of acetate
(data not shown). Lactate, acetate, ethanol and formate
were typical fermentation products observed when the
bacterial strains were grown in the presence of glucose
(Table 2). A marked change in fermentation pattern was
observed between the wild-type strains and the bacteriocinresistant strains. The wild-type strains showed a homolactic
type of fermentation (i.e. 83 and 94 % of all product carbon
was present in lactate for B73 and EGDe, respectively),
while the fermentation pattern in the resistant strains was
shifted more towards a mixed-acid type of fermentation
(47 and 50 % of all product carbon was present in lactate
for, respectively, B73-MR1 and EGDe-mptA). Since a mixedacid fermentation has a higher ATP per glucose yield as
compared to homolactic fermentation, the change in
metabolism in the resistant strains is at least qualitatively
in agreement with the observed higher biomass yields in
these strains.
Glucose consumption rates
Realizing that the EIIABMan is used for glucose transport
in L. monocytogenes, we investigated whether the reduced
Table 2. Fermentation product analysis and carbon recovery of L. monocytogenes strains grown in BHI supplemented with
10 mM glucose
The concentration values represent a mean of at least three independent measurements, and standard deviations are shown in parentheses.
L. monocytogenes strain
BHI+10 mM glucose control*
B73
B73-MR1
EGDe
EGDe-mptA
[Glucose]
(mM)
10?6
0?03 (0?007)
0?04 (0?04)
0?04 (0?007)
2?8 (0?33)
Concentration of product (mM)
Lactate
5?0
20?9 (0?13)
13?6 (0?59)
22?0 (1?30)
10?6 (0?38)
Formate
Acetate
Ethanol
Carbon recovery
(%)
9?3D
6?4
11?5
3?4
9?3
(0?57)
(1?72)
(1?97)
(1?46)
2?9
6?6
0?3
4?2
(0?34)
(0?96)
(0?48)
(0?36)
7?9 (0?77)
14?0 (2?90)
94?4
104?6
86?8
93?8
NB: Media concentrations of lactate, glucose and ethanol (only for EGDe-mptA) have not been subtracted from product concentrations values
shown here, but are subtracted for the calculation of the carbon recovery.
*Represents the medium without bacterial inoculum.
DIndicates ethanol from the erythromycin stock used to supplement the growth of L. monocytogenes EGDE-mptA.
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V. Vadyvaloo and others
growth rate in the resistant strains when grown in the
presence of glucose could be related to a lower glucose
consumption rate. Clearly, EIIABMan is not the only enzyme
capable of transporting glucose since the resistant strains
also consume glucose. However, this does not exclude that
EIIABMan has a control on growth rate and its elimination
could affect the glucose consumption rate and specific
growth rate. We took samples for glucose analysis at regular
time intervals during the exponential growth phase and
from a non-linear fit to the decreasing glucose concentrations with time we calculated the glucose consumption rate.
The specific glucose consumption rate was subsequently
calculated by normalizing the glucose consumption rate for
biomass. The following specific glucose consumption rates
in mmol glucose (g dry weight)21 h21 were determined at
mid-exponential growth phase for the various L. monocytogenes strains: B73, 215?51; B73-MR1, 26?7; EGDe,
210?73; EGDe-mptA, 23?3. Thus, a significant reduction
in the glucose consumption rate was observed for the class
IIa-resistant strains in comparison to their corresponding
wild-type strains.
DISCUSSION
The observation that the specific growth rate in class IIa
bacteriocin-resistant strains, B73-MR1 and EGDe-mptA,
on media containing glucose is lower than that of the
corresponding wild-type strains has also been described
for another class IIa bacteriocin-resistant L. monocytogenes
strain, 412P, also showing loss of MptA expression
(Gravesen et al., 2002a, b). The decreased growth rate in
412P, and in other class IIa bacteriocin-resistant B73
strains (Dykes & Hastings, 1998), has been interpreted
as a fitness cost associated with class IIa bacteriocin resistance. This fitness cost was thought to be due to energyexpensive metabolic pathways in resistant strains (Dykes &
Hastings, 1998). By taking a closer look at the physiology
of these resistant strains, we can suggest a more straightforward explanation for the reduction in specific growth
rate, namely the reduced consumption rate of glucose. It
may be the major transporter of gluappears that EIIMan
t
cose for L. monocytogenes considering the greater than
50 % decrease in glucose consumption rate observed for
the resistant strains lacking MptA and evidence for the
existence of only the glucose-specific enzyme IIA component and no other functional components of the glucosespecific PTS in L. monocytogenes EGDe (Glaser et al., 2001).
Furthermore, we suggest that the lower activity of glucosetransporting enzymes causing this extensive decrease in the
glucose consumption rate is responsible for the decrease
in specific growth rate.
In contrast to the results obtained in media containing
glucose, we observed an increased specific growth rate for
the resistant strains compared to the wild-type strains in
the absence of glucose. We do not have a straightforward
explanation for this result. It might be that, due to the
missing glucose transporter, an up-regulation of metabolic
338
routes for other substrates has occurred which gives these
cells an advantage in the absence of glucose. An example
of such an up-regulation exists for two enzymes associated with b-glucoside-specific PTSs in class IIa-resistant
L. monocytogenes strains (Gravesen et al., 2002b). Such an
up-regulation can explain that the specific growth rate
of EGDe-mptA is largely unaffected by the availability of
glucose. However, the marked decrease in specific growth
rate of B73-MR1 in the presence of glucose as compared to
growth on BHI without added glucose would indicate that
the regulation is repressed in the presence of glucose or that
glucose has an otherwise inhibitory effect on growth rate
in this resistant strain.
During our physiological characterization we noted that,
in addition to the apparent disadvantage of a lower growth
rate in the presence of glucose, the resistant strains had
a higher biomass yield on glucose (Table 1). A product
analysis revealed that the resistant strains have more of a
mixed-acid type of fermentation as compared to the homolactic fermentation in the wild-type strains. In a homolactic
fermentation, 2 mol ATP is formed per mole of glucose
fermented and in a pure mixed-acid fermentation (i.e. no
lactate formed and acetate and ethanol formed in a 1 : 1
ratio), 3 mol ATP is formed per mole of glucose fermented.
The increased biomass observed in media to which glucose
is added and the complete recovery of glucose in fermentation products indicates that biomass formation in our
media and culture conditions is limited by the availability
of the free-energy source. Thus, a shift in metabolism from
a homolactic to a mixed-acid type of fermentation would
result in an increase in the final biomass concentration.
Quantitatively, one can check this hypothesis by calculating the biomass yield per ATP. Taking the difference in
biomass formed in the presence and absence of glucose and
calculating the moles of ATP formed on the basis of the
product concentrations, we calculated the following biomass yields per mole ATP (YATP) for the four strains: B73,
9?5 g dry weight (mol ATP)21; B73-MR1, 8?9 g dry weight
(mol ATP)21; EGDe, 13?0 g dry weight (mol ATP)21; and
EGDe-mptA, 14?8 g dry weight (mol ATP)21. The YATP
values for the clinical isolate appear to be higher than
those of the food isolate, but importantly the values for
the resistant strains are similar to the YATP values of the
corresponding wild-type strains. These results indicate that,
apart from the changes in fermentation type, there is no
apparent change in free-energy metabolism between the
wild-type and the resistant strains.
A detailed mechanistic model of regulation of metabolism
in Listeria is not available at present, but a comparison to
the shift from homolactic to mixed-acid fermentation in
lactic acid bacteria indicates similar correlations. Lower
growth rates, glucose consumption rates and glucose
limitation are directly implicated in the shift from homolactic to mixed-acid fermentation (Andersen et al., 2001;
Cocaign-Bousquet et al., 1996; Garrigues et al., 1997; Yamada
& Carlsson, 1975) in Lactococcus lactis and, although the
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Microbiology 150
Bacteriocin resistance in L. monocytogenes
precise details of regulation might be different, a similar
response has been observed in our studies.
Listeria monocytogenes has been shown to spontaneously
develop resistance to class IIa bacteriocins at high frequencies from 1026 to 1028 in food and laboratory media
(Rekhif et al., 1994; Ennahar et al., 2000b). Our results
indicate that physiological responses, related to the absence
of MptA in class IIa bacteriocin-resistant strains, could
further compromise the potential use of class IIa bacteriocins as biopreservatives. Although resistant strains, in the
presence of glucose, showed a lower specific growth rate
than the wild-type strains, we have shown that the biomass
yield on glucose (and potentially other energy sources) was
significantly increased.
Our second finding is that together with the inactivation
of the MptA a shift in metabolism occurs that could significantly alter the final concentrations of the fermentation
products. Our results therefore also suggest a strong possibility that the end product of metabolism in lactic acid
bacteria starter cultures could change as a result of acquiring this type of resistance to class IIa bacteriocins. It has
in a normally
been shown that expression of the EIIMan
t
insensitive Lactococcus lactis MG1363 strain results in the
induction of sensitivity of this strain to class IIa bacteriocins (M. Ramnath, personal communication). The shift
in metabolism and subsequent change in the end product
would profoundly influence both the organoleptic qualities
and spoilage potential of the food product.
ACKNOWLEDGEMENTS
We would like to thank C. J. Malherbe, M. Mrwebi and A. Arends for
assistance with the analysis of fermentation products using HPLC
and enzymic analysis. We would also like to thank Y. Héchard for
providing the Listeria monocytogenes EGDe and EGK54 strains
used in the experimentation. This study was partially funded by a
National Research Foundation grant to J. W. Hastings, and grants
to M. Rautenbach and J. L. Snoep.
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