Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa
Correspondence
Jacky L. Snoep
jls{at}sun.ac.za
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ABSTRACT |
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INTRODUCTION |
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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 mannose-specific phosphoenolpyruvate-dependent phosphotransferase system system (PTS) (Gravesen et al., 2002b
). PTS is a group 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 operon of
in L. monocytogenes resulted in a high 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) 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, 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.
The aim of this study was to investigate the effect of the missing MptA subunit of the on glucose metabolism 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 brainheart 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.
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METHODS |
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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 l-1. 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 Schott bottle cultures, at regular intervals from early-exponential phase through to stationary phase, for monitoring of glucose consumption.
Quantification of glucose and fermentation end products.
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/glucose-6-phosphate dehydrogenase assay. The buffer for the assay contained 890 mM Tris/HCl buffer pH 7·6, 2·38 U hexokinase, 1·19 U glucose-6-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).
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RESULTS |
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DISCUSSION |
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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 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 -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)-1; B73-MR1, 8·9 g dry weight (mol ATP)-1; EGDe, 13·0 g dry weight (mol ATP)-1; and EGDe-mptA, 14·8 g dry weight (mol ATP)-1. 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 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 10-6 to 10-8 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 been shown that expression of the in a normally 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 August 2003;
revised 17 October 2003;
accepted 5 November 2003.
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