Copper Adsorption by Escherichia coli

System. Appl. Microbiology. 10, 313-317 (1988)

Copper Adsorption by Escherichia coli C. COTTER and

J. T. TREVORS*

Department of Environmental Biology, Chemistry-Microbiology Bldg., University of Guelph, Guelph, Ontario, Canada NlG 2Wl Received December 28, 1987

Summary Adsorption of Cu2+ by Escherichia coli RtSI and E. coli W677 was investigated under different assay conditions using a Cu2+ specific ion electrode. Cu2+ adsorption at 37°C was very similar in both organisms, in the presence of glucose as a carbon source. Incubation at 4°C caused a reduction in Cu 2 + adsorption. However, the metabolic inhibitors, dinitrophenol (DNP) and antimycin A did not markedly reduce Cu2+ adsorption. Killed cell suspensions of E. coli RtSI and W677 adsorbed Cu2+ at rates very similar to viable resting cells. The results suggested that Cu2+ adsorption is rapid and concentration dependent. However, there was no conclusive evidence that Cu2+ was accumulated in bacterial cells by an active transport mechanism.

Key words: Escherichia coli - Copper uptake

Introduction Copper is an essential trace metal for many organisms (Belliveau et al., 1987; Trevors, 1987). Nevertheless, it is still a highly toxic metal that can exert an inhibitory effect on bacteria and fungi (Bitton and Freihofer, 1978; Goodson and Rowbury, 1986; Zevenhuizen etal., 1979; Yamamoto et al., 1985). Copper forms intranuclear complexes in algae (Silverberg et al., 1976); is absorbed by certain yeast species (Wakatsuki et al., 1979); forms complexes with micribiological media (Bird et al., 1985), and is taken up via energy dependent transport in Penicillium ochra chloron (Gadd and White, 1985) and Aureobasidium pullulans (Gadd and Mowll, 1985). For recent reviews on Cu2+ toxicity and accumulation in bacteria, articles by Belliveau et al. (1987) and Trevors (1987) are available. Cu2+ -resistance has been described in several bacterial species (Bender and Cooksey, 1986; Ishihara et al., 1978; Terawaki and Rownd, 1972; Tetaz and Luke, 1983; Rouch et al., 1985; Stall et al., 1986; Lutkenhaus, 1977 and Cotter et al., 1987). Terawaki et al. (1967) described a Proteus vulgaris strain harboring a thermo sensitive conjugative plasmid (RtSI) that encoded for kanamycin resistance. Ishihara et al. (1978) later reported that the RtSI plasmid also carried Cu 2 + resistance as a second marker. The exact mechanism for the resistance was not described. * Corresponding author.

However, reduced 64CU2+ accumulation has been observed in another Cu2+ resistant strain of Escherichia coli (Rouch et al., 1985). The gurpose of this paper is to describe some properties of Cu + adsorption in Escherichia RtSI and in E. coli (W677), using a Cu2+ specific ion electrode and ion meter. In addition, the influence of metabolic inhibitors (dinitrophenol and antimycin A), absence of an energy source (glucose) and incubation at 4°C were used to determine if Cu2+ uptake was influenced by different incubation conditions. The use of the Cu2+ specific electrode (or other specific ion electrodes for metals like Hi+, Ag+, Cd2+) may prove useful in situations where isotopes of these metals are not readily available or lack of facilities prevent their use in uptake or adsorption experiments. Materials and Methods Bacterial Strains and Growth Conditions Escherichia coli RtSI is a Cu2 + and kanamycin (Km) resistant strain originally described by Terawaki et al. (1967), Terawaki and Rownd (1972) and Ishihara et al. (1978 ). Both resistances are conferred by the RtSI plasmid. E. coli W677 does not contain the RtSI plasmid and is sensitive to Cu2+ and kanamycin (Ishihara et al., 1978). Cultures were grown in LB broth (gil distilled water: 10 tryptone, 5 yeast extract, 10 NaCl, pH 7.2.). Filter-sterilized CUS04

314

C. Cotter and J. T. Trevors

was added aseptically to give the desired concentration. After CUS04 addition, the pH was readjusted to 7.2 using sterile 1 N NaOH. E. coli RtSI was maintained on LB agar and broth containing 250 Ilglml CUS04 and 50 Ilglml kanamycin.

Cu 2 + Adsorption One hundred mL of mid-log phase cells previously grown in the absence or presence of 2 mM CUS04 in LB broth were centrifuged at 10 000 x g for 10 min at 4°C. The cells were resuspended in 1 mM PIPES (piperazine-N, N-bis[2-ethanesulfonic acid]) buffer, pH 6.5. The cell suspension was recentrifuged, and resuspended in 8 mL of buffer, followed by 2 h incubation in PIPES buffer at with shaking to starve the cells. Killed cell suspensions were prepared by exposing washed cells to a 240 uW/cm2 ultraviolet lamp (254 nm) for 30 min. Cu2+ adsorption in cell suspensions was measured continuously using in Orion 407A meter (Orion Research Incorporated, Cambridge, Mass., USA) and a Cu2+ specific ion electrode. Cu2+ displays negligible binding to 1 mM PIPES. Therefore, the copper was present as Cu2+. A single junction reference electrode (Orion) was used in conjunction with the Cu2+ electrode. When the Cu2+ electrode contacts the solution containing Cu2+, an electrode potential is created across the membrane, which is dependent on the concentration of free Cu 2 + in solution. The electrode responds to free Cu2+ over a wide range (l00 to 10-7 M). CUS04 standards in distilled water and PIPES buffer were used to standardize the ion meter. Glucose and CUS04 were added to a final amount of 20 mM and 0.1 mM, respectively. However, in some experiments, the CuS04levels were changed to investigate the influence of the metal concentration on adsorption kinetics. Cu 2 + uptake was me.asured at both 37 and 4°C. The assay mixture consisting of PIPES buffer, CUS04, glucose and either inhibitor (dinitrophenol or antimycin A) were preincubated at 37°C in a plastic beaker prior to initiation of measurements, at which time 1 ml of the cell suspension was added. Dinitrophenol and antimycin A were used as metabolic inhibitors to ascertain if Cu 2 + uptake was an energy dependent process (Gottschalk, 1985). The ion probe was connected to a chart recorder for recording of data. Reproducibility to less than 5% difference was obtained with frequent probe calibration and constant temperature control. De Rome and Gadd (1987) have reported a very similar value (3%). Adsorption was expressed as Cu2 + uptake/mg dry weight cells. Dry weights were obtained by drying cell suspensions overnight at 105°C.

3rc

Organism

E. coli RtSI E. coli RtSI E. coli W677 E. coli W677 E. coli RtSI (killed cells) E. coli W677 (killed cells) E. coli RtSI E. coli W677

Results and Discussion

Initial and final Cu2+ adsorption at 37°C by E. coli RtSl and E. coli W677 in the presence of glucose was very similar over the 15 min incubation period (Table 1). Measurements extended beyond 15 min showed very little increase in Cu2 + adsorption. In the absence of glucose, Cu2+ adsorption was 4.38 !lmoUmg in E. coli W677 cell suspensions and 7.50 !lmoUmg in E. coli RtSl assays after 15 min. These differences may simply be due to differences in binding sites andlor differences in intracellular accumulation between the two organisms. In addition, killed cell suspensions of E. coli RtSl and E. coli W677 adsorbed 7.78 and 8.10 !lmoUmg cell biomass, resp,ectively after 15 min. This does not mean that Cu + removal from the assay mixture was entirely due to passive adsorption to bacterial cells. Since Cu2+ is a required trace element, traces of Cu2+ must enter the cells to meet normal requirements (Trevors, 1987). However, it is possible that the actual concentration of Cu2 + required is extremely small. Therefore, if metabolic dependent uptake was occurring, it is possible that the system was saturated at extremely low Cu 2 + concentration(s), not detectable by the ion probe. While the metabolic dependent Cu 2 + transport system was being saturated, passive binding to the cell wall, outer and cytoplasmic membrane could also occur. Decreased Cu2+ adsorption was observed at 4°C for both organisms in the presence of glucose (Table 1). E. coli W677 adsorbed 3.6 !lmol Cu2 +/mg during a 15 min period. This was equivalent to a 43% decrease when compared to the 37°C result. However, E. coli RtSI only exhibited a 10.8% decrease under identical assay conditions. Decreased uptake at 4°C in the presence of a carbon sOurce like glucose, suggested that an energy dependent process was responsible for Cu2+ removal from the assay medium. To further investigate this observation, Cu2+ adsorption was measured in the presence of dinitrophenol (DNP) and antimycin A. DNP is a commonly used uncoupling agent (Gottschalk, 1985). Antimycin A also is an uncoupling agent that exerts its inhibitory activity between cytochrome b and cytochrome 0 or d in E. coli

Cu2+ adsorption (Ilffiollmg)

Incubation conditions 2

4

Time (min) 6

8

15

37°C no glucose glucose no glucose glucose glucose

4.58 4.15 3.09 4.03 5.08

5.29 4.96 3.65 4.65 5.78

6.19 5.80 3.82 5.31 6.48

6.49 6.31 4.04 5.67 6.91

6.89 6.66 4.16 5.95 7.29

7.50 7.36 4.38 6.55 7.78

glucose

5.63

6.30

6.92

7.32

7.54

810

4°C glucose glucose

3.30 2.08

4.04 2.19

4.81 2.92

5.20 3.15

5.46 3.26

5.88 3.60

Table 1. Cu2 + adsorption by viable resting and killed cell suspensions of E. coli RtSI and W677 at 4 °C and 37°C in the absence and presence of 20 mM glucose

Copper Adsorption by Escherichia coli Organism

E. coli RtSI E. coli RtSI E. coli W677

No cells No cells

Incubation conditions

1 mMDNP antimycin A DNP DNP antimycin A

Cu2+ adsorption (!1mollmg) 1

2

4

5.69 4.89 3.71 1.06 0

6.48 5.62 4.38 1.06 0.79

7.67 6.75 4.94 1.19 0.79

Time (min) 6 8 8.20 7.54 5.39 1.45 0.66

8.60 7.94 5.73 2.12 0.66

10

15

8.86 8.33 6.18 2.38 0.53

9.39 8.86 ND 2.65 0.66

315

Table 2. Effect of 1 mM dinitrophenol (DNP) and 0.02 mg/ml antimycin A on Cu2 + adsorption by resting cell suspensions of E. coli RtSI and E. coli W677 at 3 rc in the presence of 20 mM glucose

ND - not determined

(Gottschalk, 1985). These agents make the cytoplasmic membrane permeable to protons. As a result, a !:!. pH cannot be established, and adenosine triphosphate (ATP) is not synthesized by oxidative phosphorylation. DNP (4 rnM) did not decrease Cu2+ adsorption in either E. coli RtSI or E. coli W677 (Table 2). It is also noteworthy that in the absence of viable cells, DNP-Cu2+ binding occurred during the assay (Table 2). After 10 min, about 2.65 Ilmol Cu2+ was removed from the assay mixture by complexation with DNP (Table 2). Bauda et al. (1987) have also reported cadmium binding to DNP and N,N1-dicyclohexylcarbodiimide (DCCD). It was observed that 60% of the metal of a 1 ppm Cd2+ solution was complexed by a 1 mmol DNP solution. It was suggested that the uptake of a complexed form of cadmium was different from free ion uptake. This would offer an explanation for the lower metal ion uptake observed in the presence of DNP (Bauda et aI., 1987). Care must therefore be exercised when interpreting metal uptake data under similar conditions. Antimycin A also displayed a low degree of complexation to Cu2+ (0.66 Ilmol/mg) in the absence of resting cell suspensions (Table 2). Resting cells that were previously grown in LB broth amended with 2.0 mM CUS04 (non-inhibitory concentration) were also used in Cu2+ adsorption studies (Table 3). Cu2+ adsorption by resting cell suspensions was very similar to the rates and final amounts of Cu2+ adsorbed by cell suspensions previously grown in LB broth without additional Cu2+. This suggested that the binding sites for Cu2+ on the cells previously grown in the presence of Cu2+ were not saturated and/or the Cu2+ was reversibly bound to the cells was removed from the binding sites during centrifugation and washing in buffer. It is difficult to interpret the effect of pre-exposure to Cu2+ on subsequent Cu2+ adsorption. One problem is the high degree of complexation

Organism

E. E. E. E.

coli RtSI coli RtSI coli W677 coli W677

Incubation conditions

no clucose glucose no glucose glucose

of Cu2+ ions with growth media components (Bird et aI., 1985), phosphates and glassware. The degree of complexation would significantly reduce the free Cu2+ ion concentration. Therefor~, the actual Cu2+ available for adsorption to bacterial cell components may be relatively small even in the presence of high concentrations of added Cu2+ (Bird et al. (1985). However, in the present experiment, this problem was avoided by using the Cu2+ electrode to measure free Cu2+ concentrations. Cu2+ adsorption was also dependent on the initial Cu2+ concentration present in the assay mixture (Table 4). Both E. coli RtSI and E. coli W677 exhibited very similar adsorption kinetics after 15 min in the presence of glucose as a carbon source. The adsorption rates were linear (Y = 54.52 x + 1.31 for E. coli RtSI and Y = 56.76 x + 0.67 for E. coli W677) and almost identical, suggesting that Cu2+ adsorption was via passive binding in both organisms. In addition, the correlation coefficients for both adsorption curves was 0.99. Saturation kinetics are usually observed for an energy-dependent process (Gadd and Mowll, 1985; Gadd and White, 1985). However, this was not observed in the present investigation. The adsorption kinetics observed in this study indicated an initial rapid phase (less than 1 min) of initial Cu2+ adsorption, follower by a more gradual adsorption (and possibly accumulation) of the metal. Cotter et al. (1987) have reported on reduced Cu2+ uptake in E. coli RtSI as the mechanism for Cu2+ resistance. However, Cu2+ uptake was measured using an atomic absorption spectrophotometer. In the present study, a Cu2+ s~ecific ion electrode was used to continuously measure Cu + adsorption in the same sample. It is possible that the electrode does not respond to very small differences in free Cu2+ concentrations, thus not permitting very small differences in Cu2+ adsorption to be measured. However, the method

Cu2+ adsorption (!1mol!mg) 1

2

4

5.43 4.59 3.95 4.65

5.80 5.25 4.18 4.99

6.40 6.04 4.53 5.75

Time (min) 6 8 6.76 6.70 4.76 6.04

7.25 7.13 4.94 6.21

10

15

7.43 7.43 5.11 6.33

7.73 7.97 5.. 34 6.68

Table 3. Cu2+ adsorption by resting cell suspensions of E. coli RtSI and E. coli W677 at 37°C in the absence and presence of 20 mM glucose. Cultures were previously grown in LB broth amended with 2.0 mM CUS04

316

C. Cotter and J. T. Trevors

Organism

Cu2+ adsorption (!ffi1ol!mg) Initial CuH (mM) 0.05 0.08

0

0.03

E. coli RtSI Linear line of best fit Correlation' coefficient

0 Y = 54.52 X 0.99

2.77 + 1.31

3.95

E. coli W677 Linear line of best fit Correlation coefficient

Y = 56.76 X 0.99

0

2.26 +0.67

3.54

a

0.1

0.2

6.18

6.58

12.15

4.97

6.80

11.91

Table 4. Effect of Cu2+ concentrations on Cu+ adsorption by resting cell suspensions of E. coli RtSI and E. coli W677 at 3rC in the presence of 20 mM glucose. Incubation time was 15 min

Lines of best fit and correlation coefficients were calculated using CMA Statistics (Charles Mann and Associates, Santa Fe Trail, California, USA) operated on an Apple II plus microcomputer.

has been previously used by Bitton and Freihofer (1978) and Bauda et al. (1987) to measure free metal ion concentrations in microbial systems, and by de Rome and Cadd (1987) measuring metal adsorption to fungi. It is highly probably that small differences in Cu2+ adsorption/uptake can only be detected with isotopes of Cu2+, that have extremely short half-life values (Cadd and Mowll, 1985; Trevors, 1987). The use of specific ion electrodes may offer an alternative method for adsorption/uptake studies where the objective is to measure metal complexation, adsorption or active uptake (in well characterized uptake systems). To date, the mechanism for Cu2+ entry into bacterial cells is not really known. It is also difficult to ascertain if Cu 2 + enters bacterial cells via energy dependent metabolism. This area of research has recently been reviewed by Trevors (1987). De Rome and Cadd (1987) have studied Cu2+ adsorption in Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum using a Cu2+ -selective electrode. Cu2+ adsorption by C. resinae and P. italicum followed Freundlich and Langmuir isotherms for single-layer adsorption whereas R. arrhizus followed the BrunauerEmmett-Teller (BET) isotherm for multi-layer adsorption (De Rome and Cadd, 1987). The authors suggested that filamentous fungi seem to exhibit low rates of intracellular accumulation. Therefore, in fungi and bacterial strains exhibiting this process, it is likely that metal binding or adsorption is of more interest (De Rome and Cadd, 1987). Also, the use of specific ion electrodes in industrial situations offers an alternative to atomic absorption methods and use of isotopes to study metal accumulation. Acknowledgements. This research was supported by operating grants from Canada Agriculture and the Natural Sciences and Engineering Research Council of Canada (NSERC) to J. T. T. Sincere appreciation is expressed to B. McGavin for typing the manuscript.

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Copper Adsorption by Escherichia coli Terawaki, Y., Takayasu, H., Akiba, T.: Thermosensitive replication of a kanamycin resistance factor. ]. Bact. 94, 687-690 (1967) Tetaz, T. J., Luke, R. K.: Plasmid-controlled resistance to copper in Escherichia coli. J. Bact. 154, 1263-1268 (1983) Trevors, J. T.: Copper resistance in bacteria. Microbiol. Sci. 4, 29-31 (1987) Wakatsuki, T., Imahara, H., Kitamura, T., Hidehiko, T.: On the

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adsorption of copper into yeast cell. Agric. BioI. Chern. 43, 1687-1692 (1979) Yamamoto, H., Tatsuyama, K., Uchiwa, T.: Fungal flora of soil polluted with copper. Soil BioI. Biochem. 17, 785-790 (1985) Zevenhuizen, L. P. T. M., Dolting, J., Eshuis, E. J., ScholtenKoerselman, 1. J.: Inhibitory effects of copper on bacteria related to the free ion concentration. Microbiol. Ecol. 5, 139-146 (1979)

Dr. Jack T. Trevors, Dept. of Environmental Biology, Chemistry-Microbiology Bldg., University of Guelph, Guelph, Ontario, Canada N1G 2W1