Abstracts. P. De Spiegeleer

Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Journal of Applied Microbiology, July 2004, Vol 97, No. 1, pp. 124-133

Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli

P. De Spiegeleer, J. Sermon, A. Lietaert, A. Aertsen and C.W. Michiels

ABSTRACT

Aims: To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress.

Methods and Results: Cultures of Escherichia coli MG1655 were grown in Luria-Bertani (LB) medium at 37°C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress. A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures. Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy). By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using 2-22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx.

Conclusions: Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media. Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress.

Significance and Impact of the Study: This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology.

INTRODUCTION

It is well known that the formulation of culture media for counting viable cells of microbial populations that have been exposed to stress is very important. Media that support good growth of unstressed cells do not always support the growth of stressed cells of the same organism (Rahman et al. 1996; Wang and Doyle 1998; Wuytack et al. 2003). This is because stress can cause sublethal injury in cells, and injured cells may be sensitive to conditions that do not affect uninjured cells. Injured cells can resume growth only after the injury has been repaired, and, depending on the type and severity of the injury, this repair process may require more specific and narrow nutritional and environmental conditions than those that support growth of uninjured cells. In the field of food microbiology, specific recovery media and procedures that allow maximal recovery of sublethally injured cells have been developed. A factor that seems to be particularly important during recovery is oxidative stress (Aldsworth et al. 1998; Dodd and Aldsworth 2002). Oxidative stress in bacterial cultures is caused by reactive oxygen species (ROS), which include hydrogen peroxide (H₂O₂), the superoxide radical (O₂^-), the hydroxyl radical (OH°) and singlet oxygen (¹O2). Recovery of injured cells can be dramatically enhanced by supplementing recovery media with enzymic or nonenzymic components that deplete oxygen or ROS such as oxyrase (Baylis et al. 2000; Stephens et al. 2000; Reissbrodt et al. 2002), catalase, pyruvate or -keto-glutaric acid (Mizunoe et al. 1999), and by providing anaerobic conditions (Knabel and Thielen 1995; Bromberg et al. 1998). ROS can be generated by specific reactions involving culture media components such as riboflavin, reducing sugars, transition metal ions, thiols, flavonoids and many others, and that are stimulated by light and heat (autoclaving) (Stephens et al. 2000; Grzelak et al. 2001; Halliwel 2003). Further, ROS are also generated by bacterial metabolism, for example by escape of electrons from the electron transport chain. It is estimated that 0·1-1·0% of the electrons that are transferred to oxygen produce (O₂^-) under normal conditions (Fridovich 1999). An even higher proportion of electrons participate in H₂O₂ formation. To cope with this exogenous and endogenous oxidative burden, cells are harnessed with several defence systems, such as catalase and superoxide dismutase enzymes, which detoxify H₂O₂ and O₂^-, respectively, but also low molecular weight antioxidants such as glutathione. To explain the observation that recovery of stressed cells is enhanced by relief of oxidative stress, the hypothesis has been raised that stress-induced sublethal injury causes a metabolic imbalance resulting in an increased endogenous generation of ROS, so that the cellular defence systems become overwhelmed and the cells become extremely sensitive to oxidative stress (Bloomfield et al. 1998). In addition, sensitivity to oxidative stress may also result from the direct inactivation of protecting enzymes by stresses such as heat treatment or high hydrostatic pressure treatment.

Exogenous oxidative stress in bacteriological culture media is generally considered to be relevant only when stressed cells have to be cultured. Therefore, in bacterial stress research, dedicated media and procedures that reduce oxidative stress are only used for growing cells after exposure to stress, while standard broths are used for growing the primary cultures. Cells cultured in these standard broths are considered to be unstressed. However, for in vitro cultured mammalian cells, there is increasing evidence that oxidative stress from medium components can cause significant physiological changes (Halliwel 2003). In the current work, we report that Escherichia coli cells harvested from Luria-Bertani (LB) broth containing tryptone from two different sources, exhibited strongly different sensitivities to growth inhibition by the lactoperoxidase system, and we present evidence linking this observation to medium-dependent oxidative stress. In an earlier study, Stephens et al. (2000) already demonstrated that the source of peptone affected recovery of heat-injured Salmonella in buffered peptone water, and this was also ascribed to differences in formation of ROS. However, to our knowledge, our study is the first to relate such subtle differences in medium composition to the oxidative stress resistance of unstressed bacterial cells.

MATERIALS AND METHODS

Growth media and chemicals

LB broth (10 g l¹ tryptone, 5 g l¹ yeast extract and 5 g l¹ NaCl) was used to grow E. coli cultures. Two versions of LB medium that were identical, except for the brand of tryptone, were used in this study, and designated as LBx and LBy. Throughout the course of this work, ingredients from a single lot number were used to avoid lot to lot variability. Media were sterilized at 121°C for 15 min, taking care that for each experiment LBx and LBy were autoclaved in the same cycle to avoid differences in sterilization conditions. Media were stored in the dark and used within 2 days. Ampicillin (100 g ml¹) (ICN Biomedicals, Asse-Relegem, Belgium) was added to the growth medium for plasmid containing strains. Tryptone soya agar (TSA) (Oxoid, Basingstoke, UK) was used as plating medium to enumerate viable counts of E. coli, and tryptone soya broth (TSB) (Oxoid) was used as growth medium to evaluate the bacteriostatic activity of the lactoperoxidase-H₂O₂-SCN(LPS) system. Stock solutions (10 mg ml¹) of lactoperoxidase (Sigma-Aldrich, Bornem, Belgium) and glucose oxidase (100 units ml¹) (Sigma-Aldrich) were stored at 18°C in a 50% glycerol solution in phosphate-buffered saline (2·87 mmol l¹KH₂PO₄, 7·12 mmol l¹ K₂HPO₄, 0·151 mol l¹ NaCl, pH 6·0). Potassium thiocyanate (KSCN) (Acros Organics, Geel, Belgium) was stored at 4°C as a 25 mmol l¹ stock solution. Plumbagin (5-hydroxy-2-methyl-1,4-naphtoquinone) (Sigma-Aldrich) was stored at 18°C as a 0·1-mol l¹ stock solution in dimethyl sulphoxide, and t-butyl-hydroperoxide as a 7·3-mol l¹ stock solution in water at 4°C.

Bacterial strains and plasmids

The E. coli strains and plasmids used in this study are listed in Table 1.

DNA manipulations

Escherichia coli recA, grxA, trxA, katG, katE, zwf, dnaK and sodA promoters were PCR amplified using pfx polymerase (Invitrogen, Merelbeke, Belgium) according to manufacturers' standard protocol and with appropriate primers (Eurogentec, Seraing, Belgium) (Table 2). BamHI and XbaI digested PCR reaction products were cloned into the pFPV25 vector cleaved with the same enzymes and dephosphorylated. Clones were verified by restriction analysis and PCR amplification using tac polymerase (Roche Diagnostics, Vilvoorde, Belgium) and using a primer pair consisting of the same upstream primer as used in the initial amplification step, and a primer based on the gfp sequence (gfp-pR, Table 2). Finally, functionality of the cloned promoters was confirmed by induction of GFP fluorescence by heat, u.v. (254 nm), plumbagin or H₂O₂.

Alkaline phosphatase assay

Escherichia coli strains were grown in microplates on a rotary shaker (200 rev min¹) at 37°C in LBx or LBy containing 100 g ml¹ ampicillin and 5 mmol l¹ isopropyl-1-thio-d-galactopyranoside (IPTG). At different intervals during growth (16, 21 and 24 h), cells were collected by centrifugation and resuspended in 1·0 mol l¹ Tris-HCl (pH 8·0) and O.D. (600 nm) was measured using an optical density microplate reader (Multiskan RC; Thermo Electron Corporation, Vantaa, Finland). Alkaline phosphatase activity was determined as described (Prinz et al. 1997), using PNPP (p-nitrophenyl-phosphate) as a substrate. Units of alkaline phosphatase were calculated as described earlier (Brickman and Beckwith 1975):

The O.D. at 550 nm provides a correction factor for cell absorption at 420 nm.

Monitoring of oxidative stress gene promoter activity

Eight E. coli MG1655 transformants, each containing a different cloned oxidative stress gene promoter fused to the gfp reporter gene (Table 1), were grown in LBx and in LBy in a microplate in a fluorescence microplate reader (Fluoroskan Ascent FL; Thermo Electron Corporation) at 37°C. Fluorescence was measured every 15 min and the ratio of the fluorescent signals obtained in both growth media (RFU_LBy/RFU_LBx) was calculated for each clone.

Growth curves

Bacterial growth curves in LBx and LBy were recorded using the Bioscreen C Automatic Growth Analyser (Thermo Electron Corporation). An overnight culture (21 h at 37°C) in LBx or LBy was diluted 10⁵-fold in LBx or LBy, respectively, and 300 l was transferred to the wells of a honeycomb microplate (Thermo Electron Corporation) and incubated at 37°C in the Bioscreen C apparatus. Optical density was measured using a wide band filter (405-600 nm) every 15 min after shaking at medium intensity for 60 s.

Evaluation of bacteriostatic activity of the lactoperoxidase (LPS) enzyme system

Cultures grown for 21 h at 37°C in LBx or LBy were diluted 10⁵-fold in TSB, supplemented with the components of the LPS system (lactoperoxidase, 5 g ml¹; KSCN, 0·25 mmol l¹; glucose oxidase, 0·1 units ml¹; and glucose, 0·4%, w/v), and further incubated in the Bioscreen C apparatus for 44 h at 25°C. The function of the glucose oxidase enzyme and glucose is to generate H₂O₂ in situ, which can subsequently serve as substrate for the lactoperoxidase enzyme.

Inactivation of E. coli by ROS

Escherichia coli MG1655 was grown in 20 ml LBx or LBy in 100 ml Erlenmeyer flasks incubated on a rotary shaker (200 rev min¹) at 37°C for 21 h. Cells were collected by centrifugation, resuspended in the same volume of 0·1 mol l¹ HEPES [4-(2-hydroxyethyl)-piperazine-1-ethanesulphonic acid] (pH 7·0) supplemented with 1·5 mmol l¹ plumbagin (superoxide generator) or 0·22 mol l¹ t-butyl-hydroperoxide, and incubated with shaking (200 rev min¹) at 20°C. At regular intervals samples were taken, diluted and plated on TSA to count survivors.

RESULTS

Growth inhibition of E. coli by the lactoperoxidase system (LPS)

Growth inhibition of E. coli MG1655 by the LPS system was analysed in TSB medium inoculated with cells grown for 21 h at 37°C either in LBx or in LBy. The growth inhibition test was initially performed at 37 and 25°C, but the results at 37°C are not shown because they were more difficult to reproduce for reasons that were not further investigated. At 25°C, the LPS had a complete bacteriostatic effect during at least 44 h when the inoculum had been grown in LBx (Fig. 1a), but caused only a partial growth inhibition when the inoculum was from LBy (Fig. 1b). This partial growth inhibition was characterized by an increased apparent lag phase, reduced exponential growth rate and reduced stationary phase cell density. In absence of LPS, the growth curves obtained with inoculum from LBx and LBy were identical. In TSB containing only glucose and glucose oxidase (generating H₂O₂), partial growth inhibition was observed both with an inoculum from LBx and from LBy, and the inhibition was only slightly stronger for the former. Cells originating from LBy were about equally sensitive to the glucose oxidase enzyme system alone as to the complete LPS.

Inactivation of E. coli by ROS

Cells from MG1655 cultures grown at 37°C for 21 h in LBx or LBy were treated with bactericidal concentrations of plumbagin and t-butyl-hydroperoxide. t-Butyl-hydroperoxide was used to generate peroxide stress rather than H₂O₂ because the latter is progressively degraded by the E. coli catalases. Plumbagin is a redox-cycling agent, that diverts electrons from the electron transport system to O₂, thereby causing the formation of superoxide radicals (Farr et al. 1985). Figure 2 shows that cells grown in LBx were more rapidly inactivated by superoxide and t-butyl-hydroperoxide than cells grown in LBy.

Taken together, the results from Figs 1 and 2 indicate that cells grown in LBx are more sensitive to different forms of oxidative stress than cells grown in LBy. At this stage of the work, a possible trivial explanation for this difference could be that the cultures produced in both media differed in cell density and/or growth phase due to a difference in growth rate. Therefore, growth curves of MG1655 in LBx and LBy at 37°C were compared in the next experiment.

Growth of E. coli in LB medium at 37°C

Eight replicate MG1655 cultures grown in TSB overnight at 37°C in a microplate on a rotary shaker (200 rev min¹), were diluted 10⁵ times in LBx and LBy to ca 10⁴ CFU ml¹ and incubated in the Bioscreen C reader at 37°C for 44 h. Growth curves in both media exhibited a lag phase, a rapid and a slow exponential growth phase, and a stationary phase (Fig. 3a). The lag phase consists of the true lag phase and the time in exponential growth needed to reach a detectable optical density, corresponding to ca 10⁷ CFU ml¹. Therefore, it should be denoted as apparent lag phase. As this apparent lag phase was the same for both media, we must assume that exponential growth in both media was also identical, except for the last cell doublings in the first exponential phase, where growth of the LBx culture seems to slow down sooner. The growth rate during the second phase of exponential growth was the same in LBx and LBy, but the LBx culture entered stationary growth phase more rapidly than the LBy culture. Cultures grown for 21 h, as had been used in the previous oxidative stress resistance experiments, were in early stationary phase in case LBx was used as growth medium, but still in (second) exponential growth phase in the case of LBy. As a consequence, it could be argued that the incubation period of 21 h, as used in the previous experiments, was an unfortunate choice. However, for these experiments cells were grown under conditions that are likely to support better growth than in the Bioscreen C due to better aeration (20 ml cultures in a 100-ml Erlenmeyer on a rotary shaker at 200 rev min¹). This assumption was experimentally confirmed, and it was found that LBx and LBy cultures grown exactly as described for the oxidative stress sensitivity experiments were in stationary phase already after 15 h of growth (data not shown). Therefore, the difference in growth rate seen in the Bioscreen C does not explain the different oxidative stress sensitivity of cells grown in LBx and LBy. To further investigate the differential effect of both tryptones, we compared the growth curves of MG1655 in regular LB and in LB with 20% excess of tryptone (Fig. 3b). Addition of extra tryptone (source x) did not change growth in LBx; however, addition of extra tryptone (source y) to LBy extended the second exponential growth phase, resulting in a higher stationary phase cell density. As stationary phase growth arrest in E. coli has been related to oxidative stress (Dukan and Nyström 1998), and in view of the difference in oxidative stress resistance between cells grown in either medium (see above), the next experiments were intended to compare the levels of cytoplasmic oxidative stress in E. coli grown in LBx and in LBy.

Cytoplasmic protein oxidation in E. coli

Escherichia coli strain WP551 is a K12 strain like MG1655, from which the chromosomal copy of the alkaline phosphatase (AP) gene has been deleted, and which carries a plasmid-encoded IPTG-inducible gene encoding a leaderless alkaline phosphatase (2-22 AP) (Prinz et al. 1997). The leader peptide serves to target wild-type AP to the periplasm, where AP monomers are linked by disulphide bonds to form an active enzyme dimer. The 2-22 AP lacks the leader peptide and therefore remains in the cytoplasm, where disulphide bond formation and consequently activation of the enzyme does not normally occur (Prinz et al. 1997). However, the cytoplasmic 2-22 AP can be activated upon exposure of the cells to oxidative stress, and based on this property the truncated enzyme has been used as a probe to measure intracellular oxidative stress (Prinz et al. 1997; Dukan and Nyström 1998).

This system was applied to evaluate cytoplasmic oxidative stress in WP551 during growth in LBx and LBy (Fig. 4). The results indicate that oxidative stress in the cytoplasm increases with increasing culture age, as reported previously (Dukan and Nyström 1998), but also that cells experienced a significantly higher level of oxidative stress in LBy than in LBx throughout late exponential (16 h) and early stationary phase (21 and 24 h). It was reported that oxidation-reduction potential (ORP) of the growth medium influences the thermal resistance of Salmonella (Komitopoulou et al. 2004). To check whether a similar explanation could be found for the differences in oxidative stress resistance the ORP of both growth media was measured at different points during growth. No significant difference was found (results not shown).

Expression of oxidative stress genes in E. coli

Fusions of eight stress gene promoters to a gfp reporter gene were constructed (Table 1), and used to measure stress gene expression in MG1655 during growth in LBx and LBy. Cells were grown overnight at 37°C in TSB, then diluted 10⁵ times in LBx or LBy and further incubated at 37°C in the fluorescence reader. The promoters of katG, sodA, zwf and trxA showed a higher expression (P < 0·05) in LBy than in LBx, while expression of the other promoters was the same in both media (Table 3).

Together, the results obtained with the 2-22 AP and from the analysis of stress gene expression indicate that cells are exposed to a higher level of intracellular oxidative stress during growth in LBy than in LBx.

FIGURES

Fig. 1 Growth inhibition of Escherichia coli MG1655 in TSB by the lactoperoxidase enzyme system.

Fig. 2 Survival of Escherichia coli MG1655 in 0·1 m HEPES buffer (pH 7·0) containing 1·5 mmol l¹ plumbagin (a...

Fig. 3 Growth curves of Escherichia coli MG1655 in LBx (--) and LBy (--) with standard quantities of t...

Fig. 4 Alkaline phosphatase activity in Escherichia coli WP551 producing truncated 2-22 alkaline phosph...

Table 1 Escherichia coli strains and plasmids used in this study

Table 2 PCR primers used in this work

Table 3 Differential expression of stress gene promoters in Escherichia coli MG1655 after 16, 21 and 2...

DISCUSSION

In this work, we have demonstrated that the source of tryptone in the LB broth used to grow E. coli MG1655 strongly affected the ability of the organism to withstand subsequent exposure to various forms of oxidative stress. Cells pregrown in LBx (with tryptone of brand x) were more sensitive to inactivation by peroxide and superoxide stress, and particularly to growth inhibition by the lactoperoxidase system than those grown in LBy (with tryptone of brand y). Differences in performance of supposedly identically formulated growth media from different commercial suppliers have been reported previously in the literature (Brun et al. 2001; Kreft et al. 2001). However, the differences among media reported in these studies invariably concern their ability to support growth of certain test strains. In the current study, in contrast, LBx and LBy supported (almost) equal growth of E. coli MG1655 but nevertheless provoked a substantial difference in physiological properties of the cells grown in them. To our knowledge, such an effect caused by different brands of growth media has not been reported before. In this study, we have focussed only on oxidative stress resistance in E. coli, but it cannot be excluded that other physiological properties are also affected, and that similar effects exist in other bacteria. It is known that bacterial stress resistance varies with growth phase, and therefore we compared the growth curves of MG1655 in both media. A small but consistent difference was found when the cultures were grown in the Bioscreen C. However, the LBx and LBy cultures used for testing oxidative stress resistance were grown in well aerated erlenmeyers with constant shaking, and were both in stationary phase at the time of harvesting (21 h); hence their different oxidative stress resistance cannot be explained by a different growth stage. The slightly poorer growth performance of MG1655 in LBx (at least in the Bioscreen C) was attributed to the existence or generation of growth inhibitory components rather than to a lower nutrient availability in this medium, because the addition of 20% excess tryptone did not prolong growth in LBx, as opposed to LBy (Fig. 3). The nature of this inhibitor was however not further investigated.

Further experiments were conducted to probe the oxidative status of the cytoplasm in cells grown in LBx and LBy, using two different in vivo reporter systems of oxidative stress. The first system is based on the formation of intramolecular disulphide bonds in a leaderless alkaline phosphatase enzyme, and therefore reflects the thiol-disulphide balance in the cytoplasm. The second system is based on transcriptional induction of specific stress genes by various types of oxidative stress. Interestingly, both approaches indicated that cells experienced a higher level of oxidative stress during growth in LBy than in LBx, and more specifically, the higher levels of enzymatically active alkaline phosphatase indicate that the cytoplasmic thiol-disulphide balance is shifted more towards oxidizing conditions in LBy grown cells. The elevated expression in LBy of trxA seems to supports this observation because it may reflect an adaptive response of the cells to restore the cytoplasmic thiol-disulphide redox balance by increasing the production of trxA gene product, thioredoxin, which is one of the enzymes involved in maintaining the cytoplasmic protein thiol pool reduced (Prinz et al. 1997). It is known that small doses of oxidative stress can trigger an adaptive response in E. coli and protect the organism from subsequent lethal doses (Demple and Halbrook 1983; Farr et al. 1985). Based on our results, we suggest a similar explanation for the higher oxidative stress resistance of cells grown in LBy compared with LBx: the higher level of cytoplasmic oxidative stress which exists during growth in LBy is expected to induce a stress response, which in turn leads to increased oxidative stress resistance of the cells. The source of oxidative stress in LBy remains unclear. It is possible that both tryptones (source x and y) contain different levels of components that can generate exogenous ROS by (photo)chemical reactions, such as riboflavin. Alternatively, differences in tryptone composition may cause subtle physiological differences in the cells growing on media containing these tryptones, which may in turn affect the rate of endogenous ROS formation. In line with the latter hypothesis, Messner and Imlay (1999) anticipated that the degree of endogenous oxidative stress in aerobically grown E. coli cells may vary with growth conditions because it depends almost entirely on only two cellular enzymes, NADH dehydrogenase II and sulphite oxidase, the expression of which varies up to 100-fold in different environments.

In conclusion, this work shows that brand differences of tryptone may considerably affect the oxidative stress resistance of E. coli grown in LB broth. Evidently, this is highly relevant for studies on oxidative stress in E. coli, as LB is a widely used standard growth medium for laboratory studies in E. coli. More in general, this study clearly illustrates the importance of quality control of growth media for the microbiological research laboratory.

ACKNOWLEDGEMENTS

This work was supported by research grants from the K.U.Leuven Research fund (OT/01/35) and from the Fund for Scientific Research Flanders (F.W.O. G.0195·02). A.A. has a fellowship from the Flemish Institute for the promotion of Scientific Technological Research (I.W.T.).

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