Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Simon J. Foster. Tel: +44 114 2224411. Fax: +44 114 2728697. e-mail: s.foster{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: stress resistance, nutrient limitation, SigB, PrfA
Abbreviations: SSR, starvation survival response
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INTRODUCTION |
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During starvation, most of the population of S. aureus dies; however, the survivors undergo a differentiation process resulting in smaller cells (Watson et al., 1998b ). Associated with long-term starvation, many species show an increase in resistance to a number of environmental stresses such as low pH, heat and oxidative stress (Hartke et al., 1994
; Watson et al., 1998a
). Development of the SSR is dependent on differential gene expression (Morton & Oliver, 1994
; Reeve et al., 1984
), and a number of components important in its development and maintenance have been characterized (Cashel et al., 1996
; Hengge-Aronis, 1996
; Watson et al., 1998b
). Important in the instigation of general stress resistance in Gram-positive bacteria such as Bacillus subtilis, Listeria monocytogenes and S. aureus is the alternative sigma factor SigB, although its role in the SSR is unclear (Becker et al., 1998
; Chan et al., 1998
; Hecker & Völker, 1998
).
L. monocytogenes is well known as a food-borne pathogen, particularly associated with outbreaks involving dairy and meat products (Farber & Peterkin, 1991 ). Infection in humans can occur as bacteraemia, meningitis or disseminated infection, and is especially notable for the high mortality rate it causes in neonates (1050%) (Farber & Peterkin,1991
). The regulation of virulence gene expression is known to occur in response to environmental signals, and is governed by the transcriptional activator PrfA (Behari & Youngman, 1998
; Böckmann et al., 1996
; Leimeister-Wächter et al., 1992
; Mengaud et al., 1991
). The bacterium is highly versatile, and can be found in a remarkably wide range of environments, including water, soils, silage, plant surfaces and the human intestinal tract. It is likely that this organism commonly encounters conditions of starvation, and thus the SSR of L. monocytogenes has implications for survival of the bacterium in the environment, in the colonization of food production sites, in the subsequent colonization of food products and in the ability to establish infections. Survival of L. monocytogenes has been studied in foodstuffs; however this is typically in relation to food preservation methods (Bolton & Frank, 1999
; Casadei et al., 1998
; Gahan et al., 1996
). In this work we have characterized the SSR of L. monocytogenes EGD, and have identified the roles of two novel starvation survival loci.
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METHODS |
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Glucose- or amino-acid-limited CDM was made by decreasing the glucose concentration from 0·2% (w/v) to 0·1% or reducing the total amino acid concentration from 0·12% (w/v) to 0·0016%, respectively. Glucose-minus medium was prepared by omitting glucose from the medium.
Starvation cultures were inoculated to an OD600 of 0·01, prepared as previously described (Watson et al., 1998b ), incubated with shaking (250 r.p.m.) at 37 °C to stationary phase (18 h), then incubated statically at 25 or 37 °C. All cultures reached an OD600 of 0·650·75, which corresponded to approximately 1x109 c.f.u. ml-1. Multiple-nutrient-limited cells were prepared by harvesting stationary-phase CDM cultures (37 °C, 18 h) by centrifugation (4000 g for 10 min at room temperature). The cells were washed twice by resuspension and centrifugation with an equal volume of PBS (Sambrook et al., 1989
) or distilled water, before being resuspended in PBS or distilled H2O, respectively, to the original culture volume. Viable counts were determined by serial dilution in PBS, plating on TSB agar and incubation overnight at 37 °C. The minimum detection level was 100 c.f.u. ml-1. Results are mean values of at least two separate experiments, for which the SD was less than ±40% of the mean.
Electron microscopy.
Samples were centrifuged (10000 g for 5 min at 4 °C) and the supernatant removed. The cells were fixed and stained by standard means (Watson et al., 1998b ), and sections studied with a Phillips CM-10 transmission electron microscope. Mean values±SD were calculated from at least 60 individual cells viewed within a randomly selected area.
Penicillin G.
Penicillin G to a final concentration of 20 µg ml-1 (20xMIC) was added to starved cultures incubated at 37 °C. Penicillin G was still biologically active to greater than MIC level after 15 d incubation at 37 °C (data not shown). Glucose-limited CDM was used for all penicillin G experiments.
Chloramphenicol.
Synchronous glucose limitation of cultures was achieved by harvesting mid-exponential (OD600 0·3, 37 °C) cells by centrifugation and resuspension in glucose-minus CDM. Chloramphenicol (100 µg ml-1) was added after 0, 1, 2, 4, 8 and 24 h incubation at 37 °C. Control cultures with no chloramphenicol added were also monitored. Viability was determined by plating on TSB agar.
Stress resistance.
Mid-exponential-phase and 6 h post-exponential-phase cells grown at 37 °C were harvested by centrifugation (5000 g, 3 min, room temperature). Challenge with heat, acid or oxidative stress was carried out as described by Watson et al. (1998a) , except that acid resistance was examined in CDM adjusted to pH 3·5 with HCl. Viability was determined by plating on TSB agar.
Phage transduction of mutations into wild-type EGD background.
Lysates of L. monocytogenes LMA2B carrying a null mutation of the sigB gene (Becker et al., 1998 ) and starvation-survival-defective mutants were prepared using bacteriophage
LMUP35 (Hodgson, 2000
). Exponential-phase cells of L. monocytogenes EGD (1x108 c.f.u.) were incubated for 40 min at room temperature with 1x108 p.f.u. of phage. The mixture was overlaid onto BHI agar (Difco) plates containing sodium citrate (10 mM, pH 7·5) and kanamycin (40 µg ml-1) using 3 ml BHI with 0·75% (w/v) agar. For selection using erythromycin, the mixture was overlaid onto BHI agar plates containing sodium citrate (10 mM, pH 7·5) and erythromycin (1·5 µg ml-1) and incubated for 2 h at 37 °C. The plates were then overlaid with 3 ml BHI with 0·75% (w/v) agar and 15 µg erythromycin ml-1. Transductants were selected after the plates had been incubated overnight at 37 °C.
Catalase activity during growth of L. monocytogenes EGD.
Cells from a 1 ml sample of culture were harvested by centrifugation (14000 g, 2 min), and incubated in 2·5 mg lysozyme ml-1 dissolved in Kpi buffer (0·615 M K2HPO4 and 0·385 M KH2PO4 in distilled H2O) at 37 °C for 30 min. Catalase activity was determined by decrease in H2O2 concentration (Beers & Sizer, 1952 ) in Kpi buffer (pH 7·0, 25 °C) using a Shimadzu UV-2401 PC spectrophotometer. Protein concentration was determined colorimetrically (Bio-Rad protein assay reagent), using BSA as standard.
Tn917-LTV3 insertion libraries.
Two libraries of Tn917-LTV3 transposon insertional mutants were created using the temperature-sensitive vector pLTV3 as previously described by Camilli et al. (1990) , with the exception that the cells were grown through three subcultures at 40·5 °C in BHI containing 5 µg erythromycin ml-1. The libraries both contained approximately 2x1010 c.f.u. ml-1 and over 90% of the cells contained transposon insertions.
Starvation survival mutant selection.
A library of transposon mutants was screened as described by Watson et al. (1998b ), with the exceptions that the glucose-limited CDM agar described above was used and the screen was conducted at 37 °C over 12 d.
Molecular biological methods.
All molecular biological methods were performed as described by Sambrook et al. (1989) . Chromosomal DNA was isolated using the QIAGEN 100/G kit as directed in the manufacturers manual.
Nucleotide sequence analysis.
DNA sequence was obtained directly from purified chromosomal DNA using an ABI BigDye sequencing kit and an ABI 373A DNA sequencer (Applied Biosystems). A 19 bp oligonucleotide (5'-CTCACAATAGAGAGATGTCACCGTC-3') complementary to the lacZ-proximal end of the transposon was used as sequencing primer. Homology searches were performed using the BLAST search program at the National Centre for Biotechnology Information database (Bethesda, USA).
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RESULTS |
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Role of cell wall biosynthesis during starvation survival
Penicillin G kills actively growing and dividing cells and so can be used to determine the division status of the population. The addition of penicillin G to 6 h post-exponential-phase cultures increased the rate of loss of viability compared to the control cultures (Fig. 3). After an initial fall of 99% in viability over the first 2 d, similar to that in the controls, viability continued to fall steadily past the point where that of the control cultures stabilized at about 0·2% of the original population (Fig. 3
). When penicillin G was added to 7 d glucose-limited cultures, no effect was seen compared to the untreated control (Fig. 3
).
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Starvation-induced stress resistance
The effects of exposure to heat (55 °C), low pH (pH 3·5) and oxidative stress (7·5 mM H2O2) were tested on cells at different growth phases in glucose-limited cultures (exponential phase, 6 h post-exponential phase and 7-d-starved) (Fig. 4). As the age of the cultures increased, the cells developed a much greater level of resistance to acid (Fig. 4a
) and heat stress (Fig. 4c
). In fact 7-d-starved cells were over 1000-fold more resistant after exposure to acid or heat (for 60 and 12 min, respectively) compared to exponential-phase cells. H2O2 resistance increased during post-exponential phase, and then fell during long-term starvation (Fig. 4b
). Resistance to oxidative stress was significantly increased if the cells were retained in the original culture supernatant. Approximately 10% of exponential-phase cells remained viable after 90 min, whilst cells from 6 h post-exponential-phase and 7 d glucose-limited cultures were totally resistant (data not shown).
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Catalase activity during growth of L. monocytogenes EGD
Catalase is a major factor involved in the resistance of cells to oxidative stress, via degradation of hydrogen peroxide. Catalase activity increased steadily during growth of L. monocytogenes EGD to a maximum level in stationary phase [1x105 U (mg protein)-1] (Fig. 7). The pattern of catalase activity development in the sigB mutant during growth was the same as in EGD; however the levels observed were consistently reduced by up to 64% (8 h, Fig. 7
). During mid- to late-exponential phase, catalase activity in the prfA mutant increased to over 3·5 times that seen in EGD, reaching a peak after 8 h [1·2x105 U (mg protein)-1]. Activity then fell on entry to stationary phase to levels approximately 50% of wild-type levels (Fig. 7
). In all three strains, during long-term starvation the cells retain relatively low levels of catalase activity [4·9x1031·5x104 U (mg protein)-1] (data not shown).
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Analysis of Tn917-LTV3 insertion in starvation survival mutants
Southern hybridization analysis of the mutants revealed that all contained a single transposon (data not shown). DNA sequencing directly from genomic DNA of the mutants revealed that the four clones were two pairs of siblings. The transposon insertion in DES028 and DES029 was found to be located 129 bp downstream of the putative start site of the coding sequence for a homologue of the B. subtilis gene yulB [D70014; 39% identity (17/43 amino acids)]. DES035 and DES045 were found to have the transposon insertion 69 bp downstream of the putative start site in the coding sequence for a gene (ORF1) whose product had no significant homology to any proteins in the database.
Stress resistance of starvation survival mutants
DES028 and DES045 were chosen as representative of the sibling pairs. When DES028 was incubated at 55 °C, long-term (7 d) glucose-starved cells exhibited a 40-fold reduction in resistance compared to EGD (12 min, data not shown), whilst the same cells were over 30-fold less resistant to 7·5 mM H2O2 than EGD (50 min, data not shown). DES045 cells showed no significant change in starvation-associated stress resistance compared to wild-type cells.
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DISCUSSION |
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The development of the SSR is characterized by a rapid fall in viability of 9099·9%, prior to the long-term maintenance of cell viability. This phenomenon occurs in S. aureus, E. coli and Salmonella typhimurium (Siegele et al., 1993 ; Spector & Cubitt, 1992
; Watson et al., 1998a
), and is proposed to be the result of cryptic growth the maintenance of a small population of cells via the release of utilizable nutrients from the death and lysis of the majority. Survival kinetics similar to those seen with multiple-nutrient limitation in this report have been found in L. monocytogenes Scott A when stored in phosphate buffer (Lou & Yousef, 1996
). Establishment of the SSR was found to be cell-density-dependent and so cell death be it via nutrient limitation and/or osmotic stress and the subsequent release of nutrients, and possibly also signalling molecules, is required for the maintenance of the long-term viability of the remaining cells. This supports the idea of cryptic growth in these cultures. Further evidence is provided by the fact that spent culture supernatant allows the regrowth of cells resuspended at low cell densities to a consistent concentration.
During prolonged glucose starvation, cells of a number of species undergo a series of morphological changes. In E. coli, the RpoS-regulated bolA gene is responsible for the starvation-induced reduction in cell length (Aldea et al., 1998 ; Lange & Hengge-Aronis, 1991
). Here, we have shown that L. monocytogenes most likely undergoes reductive division during starvation, resulting in shorter cells. Similar reduction in cell dimensions is also witnessed in S. aureus and Micrococcus luteus (Mukamolova et al., 1995
; Watson et al., 1998a
); however the molecular basis for this in the Gram-positive bacteria is unknown.
If the population is undergoing cell division then active peptidoglycan biosynthesis must be occurring, rendering them sensitive to the action of penicillin G. In the early stages of starvation, penicillin G has a deleterious effect on viability, which suggests active division is still occurring. However, after 7 d no further effects are seen, and so perhaps by this stage the population has reached stasis with no further cell division. In L. monocytogenes, starvation survival is dependent on gross protein synthesis. This correlates with E. coli and S. aureus (Reeve et al., 1984 ; Watson et al., 1998a
) as the cells synthesize novel proteins in response to starvation conditions. Interestingly, it appears that L. monocytogenes no longer synthesizes proteins by 8 h into starvation as the addition of chloramphenicol has no effect on viability. However, the possibility cannot be ruled out that by this stage the organism has a phenotypic tolerance to chloramphenicol.
In other organisms, many of the differentially expressed proteins have been identified, and several have been found to regulate or confer resistance to environmental stresses (Matin, 1991 ). An increase in cross-resistance to several stresses has been found to occur as cells become starved (Jenkins et al., 1988
; Seymour et al., 1996
; Watson et al., 1998a
). L. monocytogenes EGD also shows a starvation-associated increase in resistance to environmental stress. Resistance to H2O2 is probably primarily mediated by an extracellular catalase, which is produced throughout growth. Oxidative damage is a major contributor to cell death and ageing in starved cultures (Dukan & Nyström, 1998
; Nyström, 1999
), and may also occur as a result of treatment with acid (Clements et al., 1999
). The effect of culture supernatant from aged cultures in increasing the apparent resistance of L. monocytogenes EGD cells to H2O2 is most likely via the protective action of a catalase secreted into the medium, an effect also seen in both S. aureus and B. subtilis (Leimeister-Wächter et al., 1992
; Watson et al., 1998a
).
The increase in heat resistance during starvation supports previous findings on the effect of nutrient stress on thermotolerance (Lou & Yousef, 1996 ). It is known that exposure of L. monocytogenes to heat shock increases the expression of at least 14 proteins, including GroEL and DnaK (Hanawa et al., 1995
). B. subtilis possesses at least four distinct classes of heat-induced proteins regulated by distinct mechanisms: class I (HrcA repressor-dependent), class II (SigB-dependent), class III (CtsR-dependent) and class IV (whose mode of regulation is unknown) (Derré et al., 1999
). Studies to date provide evidence that L. monocytogenes has the elements of at least three of the classes found in B. subtilis (Derré et al., 1999
; ODriscoll et al., 1996
; Wiedmann et al., 1998
).
The regulation of the SSR is complex and not well understood in Gram-positive bacteria. In E. coli and other Gram-negatives, the alternative sigma factor RpoS has a central role; however this component is absent in Gram-positives. In B. subtilis, the alternative sigma factor SigB has been recognized as a general stress response regulator (Völker et al., 1999 ), controlling a regulon of over 50 genes (Antelmann et al., 1997
; Bernhardt et al., 1997
). SigB has also been found in other Gram-positives, including S. aureus and L. monocytogenes (Kullik & Giachino, 1997
; Wiedmann et al., 1998
). Expression of sigB in L. monocytogenes was found to increase in response to hydrogen peroxide, acid and temperature shock, increased osmolarity and addition of EDTA (Becker et al., 1998
), and a sigB mutant had reduced acid resistance in stationary phase, but unchanged virulence (Wiedmann et al., 1998
). Previous work in our laboratory has shown SigB not to have a major role in starvation survival in S. aureus (Chan et al., 1998
). In L. monocytogenes EGD, however, SigB seems to contribute to starvation survival as the mutant DES011 shows a 10-fold reduction in number of survivors. Low pH is unlikely to be the principal cause of the reduced survival of DES011, though the possibility remains that the cumulative effect of the slightly reduced pH over a prolonged period could be sufficient to produce the phenotype seen. Although SigB is partially responsible for acid resistance during exponential and post-exponential phase, it has no role in the high-level resistance seen in long-term-starved cells. This is equivalent to that seen in S. aureus (Chan et al., 1998
). Therefore, there are at least two mechanisms of acid resistance in L. monocytogenes EGD one SigB-dependent, and one starvation-associated, SigB-independent. Acid tolerance is important for L. monocytogenes as mutants with increased resistance are also more virulent (ODriscoll et al., 1996
). Bacterial acid resistance also has implications in food hygiene, as the aforementioned acid-tolerant mutants show increased survival potential in a variety of foodstuffs (Gahan et al., 1996
). In L. monocytogenes, we have found that a SigB mutation results in increased H2O2 resistance, but only during exponential phase. This seems to be independent of catalase expression, and only occurs in washed cells. The molecular basis for this phenomenon is unknown. In S. aureus, SigB regulates H2O2 resistance during both exponential phase and starvation survival (Chan et al., 1998
).
Another important regulator of gene expression in L. monocytogenes is PrfA. This transcriptional activator regulates the production of the major virulence gene cluster in L. monocytogenes in response to environmental signals (temperature, growth phase, iron concentration and pH) in association with an unknown cofactor (Behari & Youngman, 1998 ; Böckmann et al., 1996
; Leimeister-Wächter et al., 1992
; Mengaud et al., 1991
). PrfA is well known as a homologue of the global regulators Crp and Fnr (Lampidis et al., 1994
), and recently the potential role of PrfA in regulating other cellular functions has come to light with the discovery that PrfA negatively regulates ClpC expression (Ripio et al., 1998
). ClpC, a member of the Clp ATPase stress protein family, is also known to have a dual role in stress resistance and virulence, with mutants found to be sensitive to iron limitation, increased osmolarity and heat stress, coupled with reduced virulence in vivo (Rouquette et al., 1996
). Here we have shown that PrfA contributes not only to starvation survival but also to stress resistance. In fact PrfA seems to down-regulate stress resistance mechanisms during exponential growth. This is likely to be due to PrfA being a member of the complex interacting hierarchy of gene regulators which control the physiology of L. monocytogenes in response to environmental stimuli.
Catalase activity in L. monocytogenes is known to be induced by stresses, namely heat, hydrogen peroxide and salt (Dallmier & Martin, 1998 , 1990
). Here, catalase activity in L. monocytogenes EGD was shown to be at least partially controlled by SigB, and thus the situation correlates with that in B. subtilis, where SigB-dependent expression of katE forms part of a non-specific oxidative stress response (Engelmann & Hecker, 1996
). The fact that a PrfA deletion mutation changes the temporal expression of catalase activity highlights a further example of stress gene regulation and indicates a possible extended role of PrfA into the basic physiology of L. monocytogenes. It remains to be seen, however, whether this phenomenon results from the direct action of PrfA.
A number of cellular components have been identified with a role in the initiation and maintenance of starvation survival (Watson et al., 1998b ). Here, we have identified two novel loci involved in starvation survival under glucose limitation, to our knowledge the first report of such mutants of L. monocytogenes. The insertional mutation in DES028 (DES029) occurred within a homologue of the yulB gene from B. subtilis, the gene product of which is a member of the DeoR family of transcriptional regulators. Notably, the majority of these proteins act as activators or repressors of genes involved in sugar utilization (Beck von Bodman et al., 1992
). The ability to use alternative carbon sources is of great importance for cell survival when the initial source of glucose is depleted, and mutations within the complex regulatory pathways would be likely to have a deleterious effect on survival. The inability to recycle those carbon sources released during the death and lysis of the majority of cells in the culture may well cause the defect seen in DES028 (DES029). The reduction in resistance to hydrogen peroxide and heat resistance that is also observed during post-exponential phase and long-term starvation may therefore be a result of the mutant being unable to meet the energy requirements needed to resist these stresses. In strain DES035 (DES045), the Tn917 insertion occurred in a gene, the product of which had no significant homology to known proteins. Therefore, a role for this gene in starvation survival cannot yet be determined.
We have shown that L. monocytogenes has an SSR that results in increased resistance to environmental stresses, and that a number of genes play a role in the starvation survival of L. monocytogenes. This highlights the need to further understand the SSR, as in this state L. monocytogenes is highly resistant to environmental stress and so is more difficult to eradicate.
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ACKNOWLEDGEMENTS |
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Received 28 February 2001;
revised 27 April 2001;
accepted 8 May 2001.