FEMS Microbiology Letters 113 (1993) 197-200 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier

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FEMSLE 05654

Induction of cadmium tolerance in Escherichia coli K-12

Ori Inbar and El iora Z. R o n *

Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-AviL,, Israel

(Received 15 June 1993; revision received 6 August 1993; accepted 10 August 1993)

Abstract: Cadmium ions are bacteriocidal, resulting in exponential killing that starts immediately after exposure. We have shown that pretreatment with sublethal concentrations of cadmium induces cadmium tolerance. Protection against cadmium killing can also be obtained by preincubation at elevated temperatures, known to induce the heat-shock response. However, in contrast to pretreatment at elevated temperatures, exposure to sublethal cadmium concentrations does not induce thermotolerance.

Key words." Cadmium; Thermotolerance; Heat-shock; Escherichia coli

Introduction

Exposure to cadmium salts results in killing of all living cells, with survival decreasing exponen- tially. In eukaryotes exposure to cadmium and other heavy metals induces the synthesis of spe- cific peptides (such as metallothioneins, poly (gamma-glutamyl-cysteinyl)-glycine and poly gamma glutamyl-phosphates) that bind the metal ions, thus reducing its lethal effect (for reviews see [1,2]). So far, similar compounds have not been found in Escherichia coli, although treat- ment with cadmium brings about the induction of

* Corresponding author. Phone (3) 6409379 or 6414138, Fax (3) 6409407.

about 40 proteins. The proteins induced by cad- mium include many that have previously been shown to belong to other regulons - the SOS, oxidation stress or heat-shock response [3-5].

The heat-shock response is a universal global control mechanism that causes a reduction in the synthesis of most proteins, coupled to the induc- tion of specific proteins (at least 17 in E. coli) called the heat-shock proteins [6]. The heat-shock response is induced by sublethal elevated temper- atures, a treatment also shown to increase the resistance against heat killing at higher tempera- tures - thermotolerance [6]. The molecular basis for the thermotolerance is not fully understood: it has been shown that several heat-shock proteins, especially the chaperons, are essential but proba- bly not sufficient for thermotolerance [7,8].

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The heat-shock response is also a stress re- sponse as it can be induced by several other environmental stress agents, such as ethanol and heavy metals [5]. Some of these environmental stress agents are involved in the induction of other global regulons, and may play a role in the acquisition of cross protection [9].

We have been studying the induction of cad- mium tolerance, thermototerance and the possi- bility of cross protection. The results presented in this paper indicate that cadmium tolerance can be achieved by pretreatment of E. coli with mild cadmium concentrations or by mild heat shock. However, the pretreatment with cadmium dif- fered from mild heat shock, as it did not induce thermotolerance.

Materials and Methods

Strains and growth conditions Bacterial strain used was Escherichia coil K-12

strain CGSC 4503 (metE, thi) grown in MOPS minimal medium [10] containing 0.2% glucose and supplemented with 2 0 / z g / m l of methionine and 1 / zg /ml of thiamine. Cultures were grown with aeration in a shaking water bath, at 32°C, unless otherwise stated. Colony forming units (cfu) were determined by plating on nutrient agar plates (Difco Laboratories, Detroit, MI) and in- cubating at 32°C.

Pretreatments and determinations of survival Cells were grown exponentially to about 108

ceils per ml, pretreated at 42°C for 30 rain or by the addition of cadmium chloride as specified (20 /xM for 30 rain or 600 /~M for 15 min), and treated by exposure to 50°C or by the addition of cadmium chloride to a final concentration of 1 mM. Samples (in duplicate) were removed for determination of cfu. Time 0 is the beginning of exposure to the lethal conditions. Survival was determined as the fraction of cfu at any given time point divided by the cfu at time 0 ( × 100 = % survival).

Results

Induction of cadmium tolerance by exposure to low cadmium concentrations and to elevated tempera- tures

Ceils were pretreated with 20 /zM cadmium chloride for 30 min before exposure to 1 mM cadmium chloride. The results indicated that this pretreatment protected against the lethal effect of the higher concentration of cadmium. Essen- tially the same degree of cadmium tolerance could also be obtained by pretreating at elevated tem- peratures, conditions known to induce the heat- shock response and thermotolerance (Fig. 1).

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0 20 40 60 80 1 0 0 1 2 0 1 4 0

Time (min) Fig. 1. Induction of cadmium tolerance by elevated tempera- tures or by exposure to low concentrations of cadmium. An exponentially growing culture was divided into three parts upon reaching a turbidity equivalent to about 108 cells per ml. One part was left untreated (control, triangles) the other two parts were preincubated for 30 min at 42°C (open squares), or with 20 /zM cadmium chloride (filled squares). All three cultures were then treated with 1 mM of cadmium chloride and samples were removed at various time intervals in dupli-

cate for determination of colony forming units per ml.

199

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0 20 40 60 80 100

Time (min) Fig. 2. Effect of elevated temperatures or exposure to low concentrat ions of cadmium on induction of thermotolerance. The experiment was carried out as described in Fig. 1, except that the cultures were diluted 1:1000 following pretreatment , in order to dilute out the cadmium. Killing was by exposure to 50°C. Pre t rea tment was with 20 /xM of cadmium for 30 rain (filled squares), with 600 ~ M of cadmium for 15 min (filled triangles), at 42°C for 30 rain (open squares) or no t reatment

(open triangles).

Effect of exposure to elevated temperatures or to cadmium on subsequent induction of thermotoler- ance

The results presented in Fig. 1 indicated that conditions known to induce thermotolerance also induce cadmium tolerance. The experiment pre- sented in Fig. 2 was designed to examine whether pretreatment with cadmium can induce thermo- tolerance. Cultures were exposed to two concen- trations of cadmium chloride before incubation at 50°C. As a control, part of the culture was pre- treated by exposure to 42°C. The results show that pretreatment at 42°C induced thermotoler- ance, while following pretreatment with cad- mium, killing was essentially similar to the un- treated control.

Discussion

The results presented in this communication indicate that it is possible to induce protection against killing by cadmium in E. coli. This cad- mium tolerance can be achieved by exposure to low cadmium concentrations. In eukaryotes sev- eral polypeptides are induced as a response to cadmium and these interact directly with the toxic ion and remove it from the system [1,2]. The existence of such polypeptides has not been shown in E. coil, but treatment with cadmium has been shown to induce about 40 proteins, whose role in cadmium tolerance are not known [3].

The results presented here also indicate that cadmium tolerance can also be induced by expo- sure to sublethal temperatures, a treatment that induces the group of heat-shock proteins. Six of these proteins are also induced by cadmium [4,5] and may, therefore, be the ones that are impor- tant for acquisition of cadmium tolerance. Molec- ular identification of these proteins is important for understanding the basis for cadmium toler- ance.

Pretreatment with sublethal temperatures has previously been shown to induce thermotolerance [4]. It is the same treatment that induced protec- tion against cadmium killing in our experiments. However, we could not find similar cross-protec- tion by exposure to mild concentrations of cad- mium. Thus, in our experiments, pretreatment with sublethal cadmium concentrations resulted in cadmium tolerance but did not bring about thermotolerance as well. This result is in contra- diction to a previous experiment, in which pre- treatment with 600 lzM of cadmium for 15 min protected against subsequent exposure to lethal temperatures [4]. Our experiment was conducted in a similar way to the previously reported experi- ment, and was performed with three different E. coli K-12 strains.

The results presented here indicate that pre- treatment at sublethal temperatures induces global protection against a variety of stress treat- ments, such as exposure to cadmium. A similar situation has been shown in Bacillus subtilis where induction of heat-shock protected against salt stress as well as pretreatment with low salt con-

200

cent ra t ions [9]. In this case, also, the p re t rea t -

men t with low salt concen t ra t ions was less effec-

tive than hea t shock in inducing the rmoto le rance .

These results suggest that stress agents such as

heavy meta ls or salts induce factors that are

mainly essent ial for specific pro tec t ion , while

t r e a t m e n t at e leva ted t e m p e r a t u r e s induces broad

protec t ion . This t r e a t m e n t induces p ro tec t ion not

only against killing by heavy meta ls and salt, but

also against damages caused by ul t raviole t irradi-

ation, oxidat ion and high osmolar i ty [11,12]. This

b road p ro tec t ion can be due to the induct ion of a

wide spec t rum of pro tec t ive factors, or to induc-

tion o f one or more factors that effect ively pro-

tect against a variety of stress si tuations. In addi-

tion, exposure to e leva ted t e m p e r a t u r e s may br ing

about global p ro tec t ion by reduc ing the expres-

sion of a large n u m b e r of vege ta t ive genes -

down-regula t ion . In this respect , sublethal tem-

pe ra tu res may act as carbon s tarvat ion that has

previously been shown to result in e leva ted toler-

ance to a large n u m b e r of env i ronmen ta l condi-

t ions - t empera tu re , oxidative stress condi t ions

and osmot ic stress [13-15].

References

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