Antagonistic effect of nickel on the fermentative growth of Escherichia coli K-12 and comparison of nickel and cobalt toxicity on the aerobic and anaerobic growth.

The facultative anaerobic enterobacterium Escherichia coli requires the activity of nickel-containing hydrogenase for its anaerobic growth. Deficiency of the specific nickel transport system led to a hydrogenase-minus phenotype and slowed down the fermentative growth in the nik mutant. Addition of 300 microM nickel to the growth medium could restore the hydrogenase activity. This restoration resulted in the recovery of anaerobic growth. A further increase of nickel concentration inhibited growth. Thus nickel shows an antagonistic effect on the anaerobic growth of E. coli. To study the mechanism of nickel toxicity, two classes of nickel-resistant mutants were isolated. The nkr mutant was obtained by selecting colonies grown on nickel-containing minimal plate. It acquired simultaneously the resistance to cobalt. A nonspecific magnesium transport mutant corA was isolated on cobalt-containing plate. The corA mutant was also resistant to nickel. When analyzing the influence of nickel and cobalt on the bacterial growth, we obtained two interesting observations. First, anaerobic growth was less sensitive than aerobic growth to cobalt toxicity. In contrast, nickel toxicity did not vary from the growth conditions. Second, cobalt seems to abolish the growth, while nickel appears to slow down the growth rate under the condition used.


Introduction
Nickel has been known for a long time to act as a toxic heavy metal to eukaryotic cells (1). Epidemiologic studies have identified nickel as potentially carcinogenic and allergenic to humans (2,3). In the carcinogenic processes, particulate nickel compounds are more offensive than water-soluble nickel salts (4). Nickel-generated formation of oxygen radicals plays a very important role in nickel carcinogenesis. Regarding its toxicity to prokaryotic cells, nickel can severely inhibit the aerobic growth of various microorganisms (5). This toxic effect depends on many biotic and abiotic environmental factors. This paper was presented at the Second International Meeting on Molecular Mechanisms of Metal Toxicity and Carcinogenicity held 10-17 January 1993 in Madonna di Campiglio, Italy.
We are greatly indebted to L. MacWalter for critically reading the manuscript. We thank M.C. Pascal for strain CGSC6403. This work was supported by grants from the Centre National de la Recherche Scientifique to URA 1486 and from the Ministere de la Recherche et de l'Espace (Action ' However, the relationship between the toxicity and oxygen was not analyzed. Nickel is also an essential trace element for many microorganisms. It forms the active center of four metalloenzymes and plays an important role in at least four biological processes: hydrolysis of urea, uptake and production of hydrogen, methanogenesis, and acetogenesis (6). These bioprocesses have an important influence on the environment and human health. The implication of nickel-containing bacterial urease in human pathogenesis has been reviewed (7). In microorganisms, there are at least three classes of proteins involved into nickel metabolism: high affinity nickel-specific transporters participating in both the sensing and transport of nickel; accessory proteins fulfilling the role of nickel incorporation into proteins; and nickel-containing metalloenzymes (8). Knowledge obtained from studies using microbes as a model system should provide fundamental insight into important aspects of nickel toxicity, carcinogenicity, and allergenicity.
The facultative anaerobic enterobacterium Escherichia coli K-12 has three nickel-containing hydrogenase isoenzymes. The hydrogenases I and II catalyze the oxidation of hydrogen coupled with the pro-duction of energy for its anaerobic growth (9). The hydrogenase III participates in the degradation of formate into hydrogen and carbon dioxide. This pathway maintains the intracellular redox potential and keeps pH constant during fermentative growth (10). We have isolated a class of nik mutants defective in the specific nickel transport system. The intracellular nickel content in nik mutants is about 10% the level in a wild-type parental strain. This deficiency results in hydrogenase-minus phenotype (11). Addition of 500 pM NiCl2 to the growth medium can restore the nickel content as well as hydrogenase activity. This restoration is specific to nickel and depends on the protein biosynthesis. The nonspecific magnesium transport system(s) seems to be required for the restoration (12). We have cloned the nik operon. It contains five genes coding for five proteins with molecular weights ranging from 28 to 57 kDa (13). Sequence analysis shows that these proteins are homologous to the five components of periplasmic binding proteindependent oligopeptide transporter family (14). In this article we demonstrate, by using the nik mutant, an antagonistic effect of nickel on fermentative growth of E. coli.

Baceria
Bacterial strains used are listed in Table 1.

Media and Growth Conditions
Luria broth (LB) medium and minimal N medium are as described by Miller (15) and Park et al. (16). Minimal N-glucose medium is N medium supplemented with 0.4% glucose, 0.02% methionine, 0.0012% thiamine and 0.1 mM MgSO4. Fermentative growth was carried out in a 2 1 fermenter containing 1 1 LB plus 30 mM formate saturated with nitrogen (50 l/hr) and continuously stirred (60 rotations/min). Samples were removed at different times for measuring optical density (OD) at 600 nm and assaying hydrogenase activity. For the metal resistance study, cells were grown in 5 ml minimal N-glucose medium. Either nickel or cobalt chloride was added at the concentrations indicated. OD was measured after a 15-hr or 3-day incubation. Aerobic growth was performed with vigorous shaking. Anaerobic growth was achieved in GasPak anaerobic jars (BBL Microbiology System, Becton Dickinson, Cockeysville, MD). All growths were carried out at 37'C.

Construction ofNickll-resistant Mutant
CorA mutant was isolated from minimal N-glucose medium containing 0.2 mM cobalt chloride as described by Park et al. (16). To select nickel-resistant mutants, we spread out 108 P4X cells on minimal Nglucose plate. Colonies grown in the inhibitory zone, around a 6-mm-diameter filter paper disc impregnated with 10 p1 of 2 M NiCl2, were selected and analyzed.
Genetic Techniques P1 cml-mediated transduction and Hfr conjugation experiments were performed as described by Miller (15).

Results and Discussion
Antagonistic Effect of Nickel on the Fermentative Growth of E. Coli Under anaerobic conditions, the wild-type strain P4X showed a biphasic growth curve with a mean generation time (MGT) of 30 and 140 min for the first and the second growth phases, respectively (Figure 1, open squares; Table 2). Addition of nickel at 300 and 600 pM increased by approximately 30% the MGT of both the first and the second growth phases. The maximum absorbance at 600 nm of the culture was reduced from 0.6 to 0.5 OD. When nickel was added at 2 mM, the lag phase was extended 2-fold and the MGT of the first growth phase was increased to such an extent that the two phases became almost indistinguishable (Figure 1, open circles). The hydrogenase activity was reduced to about 60% of the full level ( Table 2).
The nik mutant HPX72 contained less than 1% hydrogenase activity compared with the wild-type strain (Table 2; Figure  2). It showed a monophasic growth curve with a MGT= 150 min. The maximum cell density was 60% the level of the wild-type strain P4X (Figure 2). Addition of 300 pM nickel to the growth medium restored the hydrogenase activity and the fermentative biphasic growth (Table 2). With nickel present at 2 mM, the lag phase was extended four fold and the biphasic growth of HPX72 was abolished (Figure 2, filled circles). The MGT was two times shorter than that obtained in the absence of nickel and two times longer than that of the first growth phase with 300 mM nickel ( Table  2). Thus nickel shows an antagonistic effect on fermentative growth.
Nickel-containing hydrogenases are essential for the anaerobic growth of E coli since hydrogenase mutants could not grow in a minimal medium with hydrogen as the only source of energy (our unpublished results). In agreement with the inhibition of fermentative growth, 2 mM nickel reduced the hydrogenase activity to 60 and less than 50% in the wild-type and the nik mutant, respectively (Table 2). However, we cannot determine whether the reduction of activity results in or from the inhibition of growth.

Isolation of Nickel-resistant Mutants
With nickel in the culture, growth of the nik mutant was slightly faster than that of the wild-type strain (Table 2; Figures 1, 2). The mutant HPX72 is defective in the highaffinity nickel-specific transport system. This mutation might account for the slightly higher tolerance to nickel of the mutant than the wild-type strain. It also suggests that penetration of nickel into the cell is a crucial aGrowth was carried out in a two-liter fermenter as described in Materials and Methods. bSemi-log graph was drawn by using the OD data in log scale. Two points in a straight line region were taken and the mean generation time (MGT) was calculated by using the formula MGT = (time 2-time 1 )/(log OD2-log OD1)/3.32. Hydrogenase activity increased in the early exponential growth phase. It reached the maximum level in the middle of exponential growth phase, then remained stable. The results presented here are the average of the data once the activity reached its maximum level. The specific activity is expressed as pmoles benzyl viologen reduced per minute per milligram bacterial dry weight. Enzyme assay was performed as described previously (11).
Environmental Health Perspectives 298 NICKEL EFFECTS ON THE GROWrH OFE. COLI step in its toxic process. To study the mechanism of nickel toxicity, we attempted to isolate new nickel-resistant mutants.
When present at high concentration, nickel can penetrate the cells via the magnesium transport system (17). According to the protocol described by Park et al. (16), we isolated a corA mutant, defective in the nonspecific transport of magnesium. The cotransduction frequency between the ilvC (85 min) and metE (86 min) markers and the cobalt resistance phenotype confirmed the correct location of the mutation at 86 min on the chromosome map of E. coli. The corA mutant has simultaneously acquired the resistance to nickel (Table 3).
On the other hand, we isolated nickel resistant mutants by spreading P4X cells on minimal N-glucose plates as described in Materials and Methods. One of them, PNV1, was characterized and is presented in Table 3. By conjugation experiment, we have localized the nkr (nickel resistance) mutation on the chromosome map of E. coli. When PVN1 was used as donor and PA309 was used as receiver, the coinheritance of nickel resistance with thr (O min) was 100% and that with xyl (80 min) was 70%. Therefore, this mutation is likely to be located close to 0 min on the E. coli chromosome and thus is different from the corA mutation.
In bacteria, inorganic ion transport systems can be coded by either chromosomal or plasmid DNA. Chromosomally based systems are usually responsible for the uptake of essential ions, while plasmid-based systems play an important role in the toxic ion efflux (18,19). In this study, we demonstrate that deficiency in specific nickel transport slighdly increases the tolerance to nickel in the nik mutant. Further, our results show that chromosomal mutations corA, which is impaired in the nonspecific magnesium transport system, and nkr also can confer to cells a resistance to both nickel and cobalt (Table 3). Further characterization of these mutations is currently in progress. aGenotype of the strains: P4X: Hfr, met8, CN20: same as P4X, but corA; PNV1: same as P4X but resistant to nickel. bThe corA mutation was localized by P1 cm/-mediated transduction as described by Miller (15). CN20 was transduced to tetracycline resistance with phage P1 cml grown on the strain CGSC6403 (metE165.:Tn 10, ivC7). The Tn 10 insertion was found to be 45% linked to cobalt resistance and 20% to llvC. CThe mutation was localized by conjugation using PVN1 as donor and PA309 (thr-1, xy/7J as receiver. Coconjugation frequencies of nickel resistance were 100% with thr(O min) and 70% with xy/(80 min). Cells were grown in minimal N-glucose medium (see Materials and Methods).
Nickel or cobalt chloride was added at the concentrations indicated. Optical density (OD) was measured after 15 hr or after 3 days (presented in parentheses) incubation. Aerobic growth (+02) and anaerobic growth (-02) were performed as described in Materials and Methods.

Comparison ofNickel and Cobalt Toxicities on Aerobic and Anaerobic Growth
The toxic effect of nickel and cobalt on anaerobic and aerobic growth of the wild-type and mutant strains was analyzed and is presented in Table 3. Almost no aerobic or anaerobic growth was observed for the wild-type strain P4X after 15 hr of incubation with 50 jM nickel in the minimal N-glucose medium. Similarly, 25 and 50 IM cobalt were able to achieve an equivalent inhibition of the aerobic and the anaerobic growth, respectively. Therefore, aerobically grown cells can tolerate twice as high a concentration of nickel than cobalt. In addition, only cobalt appears more toxic in the presence of oxygen than in its absence. After three days incubation the OD of the aerobic and the anaerobic cultures of P4X with 200 sM nickel reached 100 and 57% of those without nickel. In contrast, cobalt still totally abolished growth (Table 3). These results suggest that the mechanism and targets of nickel toxicity are different from those of cobalt.