INTRODUCTION

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It is well established that heavy metals, such as Cd²⁺, are toxic for microorganisms, although the underlying mechanism is not clear. In most cases, heavy metals must enter the cells to be toxic (21, 23). They can be accumulated by cells via uptake systems responsible for essential cations. In Escherichia coli, Cd²⁺ enters cells via a Zn²⁺ transport system (16). In gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, or Lactobacillus plantarum, Cd²⁺ competes for transport with Mn²⁺ (5, 8, 15, 22). Of all the reported Mn²⁺ and Cd²⁺ uptake systems, the one in L. plantarum appears to have the highest affinity. L. plantarum contains high concentrations of Mn²⁺ (30 to 35 mM), which acts as a scavenger of toxic oxygen species, such as superoxide radical anions (O₂), replacing the micromolar level of superoxide dismutase which functions in other oxygen-tolerant organisms (1-4). A highly active Mn²⁺ uptake system has been identified in L. plantarum; this uptake system maintains its high Mn²⁺ content (5). This Mn²⁺ uptake system also takes up Cd²⁺. Several Cd²⁺-resistant mutants of B. subtilis have been isolated (8, 15, 17). Surprisingly, only Cd²⁺ transport was reduced in these mutants; their Mn²⁺ transport was unaffected.

In an attempt to understand Cd²⁺ uptake by bacterial Mn²⁺ transport systems and to explore the possible applications of uptake systems in the bioremediation of heavy metal-contaminated environments (9, 10), we further characterized the Cd²⁺ uptake activity of L. plantarum and isolated Mn²⁺-dependent mutant strains by chemical mutagenesis and ampicillin enrichment. The mutant strains were found to have reduced Mn²⁺ and Cd²⁺ uptake activities.

	MATERIALS AND METHODS

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Organisms and culture conditions. L. plantarum ATCC 14917 and ATCC 8014 were obtained from the American Type Culture Collection, Manassas, Va. L. plantarum CCM 1904 and NCIMB 7220 were kind gifts from Francoise Bringel (7). Cells were grown in APT complex medium as described previously (5), except that Tween 80 was omitted. Omission of MnSO₄ produced low-Mn²⁺ APT medium, containing 1.0 to 1.8 µM Mn²⁺ derived from the tryptone and yeast extract. Modified APT medium containing 100 mM MnSO₄ was used to grow Mn²⁺-dependent mutants. It has the same composition as APT medium, except that it contains only 1 g of K₂HPO₄ per liter and the pH is adjusted to 5.6. Cells were grown at 37°C without shaking.

Cd²⁺ transport assay. Cd²⁺ uptake experiments were performed with Mn²⁺-starved or Mn²⁺-sufficient cells of L. plantarum. To obtain Mn²⁺-starved cells, cells from an Mn²⁺-sufficient culture were washed with low-Mn²⁺ APT medium, diluted 1:100 in the same medium, and incubated overnight at 37°C. One milliliter of this culture was inoculated into 100 ml of fresh low-Mn²⁺ APT medium; after several hours at 37°C, growth tapered off at an optical density at 600 nm (OD₆₀₀) of about 0.5 due to Mn²⁺ limitation. The cells were washed with fresh low-Mn²⁺ APT medium, resuspended in transport medium (see below), and placed on ice. Mn²⁺-sufficient cells were harvested at an OD₆₀₀ of about 0.5 and washed and resuspended as described above. Mn²⁺-starved mutant cells were obtained by harvesting log-phase cells from modified APT medium containing 100 mM MnSO₄, washing the cells with fresh low-Mn²⁺ APT medium, resuspending the cells in low-Mn²⁺ APT medium, and incubating the cells at 37°C. They only divided two or three times due to Mn²⁺ limitation. Cells were harvested, washed, and resuspended as described above. The transport medium was low-Mn²⁺ APT medium, except that in some experiments, morpholinepropanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), or phosphate buffer with glucose was used.

Mn²⁺ or Cd²⁺ accumulation assay. To measure long-term Mn²⁺ or Cd²⁺ uptake, cells were grown, harvested, washed, and resuspended as described above. After recovery at 37°C for 10 min, MnCl₂ or CdCl₂ was added to the culture to various final concentrations, and the mixture was incubated in a 37°C shaking water bath for 1 h. Cells were harvested and washed three times at 4°C with low-Mn²⁺ APT medium by centrifugation. The cell pellets were lyophilized, and the dried cells were digested overnight in 70% nitric acid at 45°C. The digestion mixture was diluted with water to a final nitric acid concentration of 10 to 15%. The total Mn²⁺ or Cd²⁺ content of the cells was measured with a Perkin-Elmer model 2380 atomic absorption spectrophotometer.

Isolation of Mn²⁺-dependent mutants. L. plantarum ATCC 8014 cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and mutants were selected in BK-4 medium (19) containing ampicillin as described below. An overnight culture in APT medium was diluted 1:100 and grown to an OD₆₀₀ of 0.1. The culture (10 ml) was harvested by centrifugation at 3,000 × g for 10 min, washed, and concentrated 15-fold in 0.1 M potassium phosphate buffer (pH 7). The cell suspension was treated with MNNG at a concentration of 170 µg/ml at 37°C for 15 min, harvested immediately at 3,000 × g for 2 min, washed in the same buffer twice, and resuspended in 100 ml of modified APT medium containing 100 mM MnSO₄. This culture was incubated at 30°C for 9 h to allow the growth of mutants.

	RESULTS

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Kinetics of Cd²⁺ uptake. L. plantarum ATCC 14917 cells were assayed for their initial Cd²⁺ uptake rate in low-Mn²⁺ APT medium containing CdCl₂ at concentrations of 0.01 to 200 µM (Fig. 1A and B). In Mn²⁺-starved cells, the uptake rates followed Michaelis-Menten kinetics (Fig. 1). The calculated K_m was 0.44 µM, and the V_max was 3.6 µmol g of dry cell¹ min¹. In Mn²⁺-sufficient cells, transport was not saturated, even at 200 µM Cd²⁺, so that the K_m could not be calculated. The dramatically different level of transport between Mn²⁺-starved and Mn²⁺-sufficient cells indicated that Mn²⁺ starvation induced a high-affinity, high-velocity Cd²⁺ uptake system in this organism. Furthermore, the difference in kinetics indicated that Cd²⁺ uptake in Mn²⁺-starved cells was due to the induction of an uptake system which was repressed in Mn²⁺-sufficient cells. This result was observed for all L. plantarum strains tested: ATCC 8014, NCIMB 7220, and CCM 1904. The Cd²⁺ uptake activity of all these strains was comparable to that of ATCC 14917 in both Mn²⁺-starved and Mn²⁺-sufficient cells (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Kinetics of initial Cd2+ uptake by Mn2+-starved (A) or Mn2+-sufficient (B) L. plantarum ATCC 14917. Cells were prepared and preincubated for 10 min at 37°C in low-Mn2+ APT medium as described in Materials and Methods. The initial Cd2+ uptake rate was determined with duplicate 0.2-ml samples harvested 15 s after radioactive Cd2+ was added. The solid line in panel A results from fitting the data according to the Michaelis-Menten equation to produce a Km of 0.44 ± 0.07 µM and a Vmax of 3.57 ± 0.13 µmol of Cd2+ g of dry cell1 min1.

Specificity of Cd²⁺ uptake. The cation specificity of Cd²⁺ uptake was investigated with both Mn²⁺-starved and Mn²⁺-sufficient cells by exposing them to 0.1 µM ¹⁰⁹Cd²⁺ combined with 20 µM each of a series of other metal ions, including Mn²⁺ (Fig. 2A and B) and Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, and Ni²⁺ (data not shown). Only Mn²⁺ significantly inhibited the rate of Cd²⁺ uptake in Mn²⁺-starved cells (Fig. 2A), while none of the cations inhibited Cd²⁺ uptake in Mn²⁺-sufficient cells (Fig. 2B). The difference in cation specificity is further evidence that there are at least two different Cd²⁺ uptake systems in L. plantarum: a low-affinity, nonsaturable Cd²⁺ uptake system which is independent of Mn²⁺ starvation and is not inhibited by Mn²⁺ and a high-affinity Cd²⁺ uptake system which is induced by Mn²⁺ starvation and is inhibited by Mn²⁺.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Cation specificity of Cd2+ uptake by Mn2+-starved (A) or Mn2+-sufficient (B) L. plantarum ATCC 14017. Cells were prepared and preincubated for 10 min at 37°C in low-Mn2+ APT medium as described in Materials and Methods. The initial Cd2+ uptake rate was determined with duplicate 0.2-ml samples harvested 15 s after radioactive Cd2+ was added to a 0.1 µM final concentration together with 20 µM MnCl2.

Energy dependence of Cd²⁺ uptake. In order to determine the energy requirement of Cd²⁺ uptake by L. plantarum, the uncouplers 2,4-dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added to Mn²⁺-starved or Mn²⁺-sufficient cells in low-Mn²⁺ APT medium. The effect of low temperature was also studied by carrying out the uptake assay at 4°C. As shown in Fig. 3, when cells were poisoned or kept at 4°C, there was no significant uptake of Cd²⁺ by either Mn²⁺-starved or Mn²⁺-sufficient cells. Although low levels of ¹⁰⁹Cd²⁺ were present at all the time points, the amount of radioactivity did not increase with time. These results indicated that Cd²⁺ uptake by both the saturable and the nonsaturable Cd²⁺ uptake systems in L. plantarum is energy dependent. These properties had been observed for Mn²⁺ uptake by Mn²⁺-starved cells (5).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Effect of low temperature or ionophores on Cd2+ uptake by Mn2+-starved (A) or Mn2+-sufficient (B) L. plantarum ATCC 14917. Cells were prepared as described in Materials and Methods. Cd2+ uptake was assayed with low-Mn2+ APT medium containing 0.1 µM Cd2+ at either 37°C (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol) or 4°C.

Isolation and characterization of Mn²⁺-dependent mutants. L. plantarum strains with mutations eliminating the high-affinity Mn²⁺ and Cd²⁺ uptake system should require a high concentration of Mn²⁺ for growth. Mutants of L. plantarum requiring such supplementation were isolated after MNNG mutagenesis and ampicillin enrichment. Two Mn²⁺-dependent strains, designated mnd11-06 and mnd15-26, were obtained after screening of 3,500 survivors of the enrichment process. Both strains failed to grow in normal or modified APT medium containing up to 10 mM Mn²⁺, and growth started only when the Mn²⁺ concentration was over 20 mM (Fig. 4). In contrast, the wild-type strain required only 2 µM Mn²⁺ for growth. The growth rate of the mutant strains did not reach that of the wild-type strain even at 100 mM Mn²⁺. However, anaerobic growth of the mutant strains started at a lower level of Mn²⁺ (1 to 5 mM). This result was expected because a high intracellular Mn²⁺ level (30 to 35 mM) is required for L. plantarum cells to grow under aerobic conditions, as Mn²⁺ acts as a scavenger of toxic oxygen species. These results suggested that the high Mn²⁺ requirement of the mutants was due to an intracellular limitation of Mn²⁺. The Mn²⁺ accumulation assay confirmed that the mutant strains accumulated a much lower level of Mn²⁺ than the wild-type strain (Table 1).

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Mn2+-dependent growth of mutants isolated from L. plantarum ATCC 8014. A liquid culture of the mutant or parental strains was harvested, washed with low-Mn2+ APT medium, and diluted in modified APT medium containing different levels of Mn2+ to an OD600 of 0.01. After 24 h of incubation at 37°C, the OD600 was determined.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Cd2+ uptake by Mn2+-dependent mutants of L. plantarum ATCC 8014. Mn2+-starved cells were prepared and assayed with low-Mn2+ APT medium containing 0.1 µM Cd2+ as described in the text.

	DISCUSSION

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Cd²⁺ uptake in L. plantarum was first reported by Archibald and Duong (5) in their study of Mn²⁺ uptake by L. plantarum ATCC 14917. However, the kinetics that they measured were different from what was seen in this study, as they observed high-affinity, high-velocity, nonsaturable Cd²⁺ uptake in Mn²⁺-starved cells and did not measure Cd²⁺ uptake in Mn²⁺-sufficient cells. Our kinetic parameters were determined from samples taken from 15 s to 5 min. The rapid initial uptake phase (within 15 s), which increased with increasing Cd²⁺, was subtracted, as it most likely represented cell surface binding. The resulting linear rate was used for the analysis of kinetics.

Cd²⁺ uptake has been reported for other gram-positive as well as gram-negative bacteria. In gram-positive organisms, such as B. subtilis (15) and S. aureus (22), Cd²⁺ competes for transport with Mn²⁺, while in E. coli, Cd²⁺ competes with Zn²⁺ (16). The reported K_m values are 2.1, 1.8, 5.4, and 820 µM for Cd²⁺ uptake in E. coli (16), B. subtilis (15), S. aureus (22), and Pseudomonas putida (14), respectively. These values are higher than that for L. plantarum (0.44 µM), while the rate of uptake for L. plantarum (3.6 µmol g of dry cell¹ min¹) is comparable to those for other studied bacteria (reported in the same units: E. coli, 0.83; B. subtilis, 1.5; S. aureus, 2.6; and P. putida, 8.2).

L. plantarum and related lactic acid bacteria contain millimolar levels of Mn²⁺ for protection against oxygen radical damage (1-4). A high-affinity, high-velocity Mn²⁺ uptake system that creates a high Mn²⁺ content has been identified in L. plantarum. Interestingly, the Mn²⁺ uptake system also takes up Cd²⁺ and appears to have a higher affinity for Cd²⁺. A higher affinity of an Mn²⁺ uptake system for Cd²⁺ has also been found in S. aureus (20, 23). In terms of its crystal and hydrated radii, electron shell configuration, and citrate complex stability constants, Mn²⁺ is more similar to Co²⁺ or Fe²⁺ than to Cd²⁺. However, only Cd²⁺ inhibited Mn²⁺ uptake and only Mn²⁺ inhibited Cd²⁺ uptake by Mn²⁺-starved L. plantarum cells. It is possible that the form of Mn²⁺ or Cd²⁺ recognized by the uptake system may be other than the free ions. Archibald and Duong (5) found that an Mn²⁺ ion did not appear to be as available to the uptake system as Mn²⁺ complexed with an anion, such as citrate or acetate. They demonstrated that at pH 6.7, a variety of buffered organic acids provided good Mn²⁺ availability to the uptake system, and that at pH 5.5, Mn²⁺-starved L. plantarum cells could efficiently take up Mn²⁺ only when buffers contained citrate or related tricarboxylic acids. In this study, Cd²⁺ uptake was weakly inhibited by the presence of 20 mM citrate, suggesting that the forms of Mn²⁺ and Cd²⁺ recognized by the uptake system are different. It is likely that the Cd²⁺ form recognized by the uptake system is the free or hydrated ion instead of one complexed with an anion.

While Mn²⁺ inhibited Cd²⁺ uptake by the high-affinity Cd²⁺ uptake system in L. plantarum, none of the divalent cations tested, i.e., Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, and Ni²⁺, inhibited Cd²⁺ uptake by the low-affinity Cd²⁺ uptake system expressed in Mn²⁺-sufficient cells. It is unlikely that cells evolved this uptake system for Cd²⁺, and its physiological function is not known. It could be a transport system similar to the tetracycline-resistant systems from pBR322 and S. aureus (11, 13). Both of these systems mediate potassium transport in E. coli. The pBR322 tetracycline-resistant element also confers on E. coli cells an increased sensitivity to a variety of organic compounds and cadmium (6, 12, 18).

Two mutants which grew only in medium supplemented with high levels of Mn²⁺ were isolated. Their growth requirement for Mn²⁺ was more than 5,000 times higher than that of the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The results of an analysis of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also indicated that the mutations in these strains eliminated the high-affinity Mn²⁺ and Cd²⁺ uptake system. Cd²⁺-resistant mutants of B. subtilis were isolated by growth of the parental strain in high concentrations of Cd²⁺ (8, 16, 17). The mutants had reduced Cd²⁺ transport, but Mn²⁺ transport was unaffected.