Impact/Purpose:

Metals, such as Cu and Cd, threaten the environment and human health because they impair cellular function when present above threshold levels. Currently, assessments of risk are based on chemical techniques that determine only total levels of the metal. These total levels may differ from those that are bioavailable and having an impact on living cells. Bioavailability is influenced by the association of the metal with solids, colloids or low molecular weight ligands. Ligands include inorganic cations (chloride, nitrate and phosphate) as well as the organic structures (di- and tri-carboxylic acids and humic acids). Our objectives are to develop and test biosensors and DNA arrays that will detect Cu and Cd specifically and indicate the bioavailability of these metals to a bacterium.

Description:

Proteomic and Transcriptional Findings

P. putida cells responded differentially to Cd and Cu exposures at the proteomic and transcriptome levels. The cells displayed different stress responses that correlated with a more intense oxidative stress imposed by Cd than by Cu.

The proteomic studies involved two-dimensional (2-D) gel electrophoresis of extracts of cells exposed in minimal medium for 6 hours and demonstrated that some peptides increased in accumulation in response to both metals, whereas others were metal-specific (Figure 1).

Figure 1. Representation of Proteome Changes in Response to Cu or Cd Treatments (10 mg/L) in Minimal Medium for 6 Hours

We performed studies to determine transcript levels of the genes encoding the responsive proteins to determine whether the increases were due to transcriptional changes. For most of the 26 genes examined, transcript abundance was enhanced by 1 and/or 4 hours of exposure to 10 mg/L of metal in minimal medium, and the specificity of response was the same as that observed by the proteomics study. This finding suggests that a sensitive and specific detection system could be achieved using a gene chip array with selected responsive genes to detect altered gene expression in exposed P. putida cells.

Analysis by Geochem modeling showed that, in minimal medium, most of the Cd or Cu would exist as citrate, phosphate, and sulfate complexes (Table 1).

Table 1. Effect of the Composition of Minimal Medium on Complexation of Cu and Cd Ions

Thus, the cells showing the changes in transcription and protein accumulation occurred under conditions where the metals were not present as free ions but as complexes. This situation related directly to the problem for the U.S. Environmental Protection Agency (EPA) of how to assess the risk levels of Cd and Cu in aqueous solutions from the environment. The fact that complexes were bioavailable to the P. putida cell conflicts with the Biotic Ligand Model now used in risk assessment of Cu; the Biotic Ligand Model is built on the premise that only free ions would be perceived and active within cells.

Luminescent Biosensor Studies

To explore bioavailability further, we utilized biosensors constructed with fusions of promoters with the luxAB cassette to endow light production when the promoter was activated. One set of fusions were constructed with promoters of genes involved in protection against activated oxygen stress, because oxidative stress is one of the consequences of Cu and Cd exposures (see Figure 1).

Dose-dependent decreases in Lux output, compared with nonexposed control cells, was observed with the exception of Cu-treatment at 0.01 mg/L of the FeSOD mutant (Figure 2). For Cu, decreased Lux at 10 mg/L corresponded to cell death, but with Cd, the cells only underwent a temporary stasis in cell growth at this exposure dose.

Figure 2A. Cd response

Figure 2B. Cu response

Figure 2. Effect on Light Output (Lux) of Exposure to Cd (A) and Cu (B) of Logarithmic Phase Cells with Promoter Fusions With the bfr Gene (Encoding an Iron Binding Protein), catA (Encoding a Major Catalase Gene), and sodB (Encoding the Major Fe Superoxide Dismutase)

To derive biosensors with more specific and sensitive responses, we generated fusions with the promoters identified as being metal responsive in our RNA assays with isolate KT2440. We housed these fusions in a stable plasmid rather than creating a knockout mutant as in the cells used in Figure 2. Representative data for these biosensors are shown in Figure 3. Unexpectedly, we found that the constructs showed no little specificity in response to Cu or Cd; rather a rapid decline in Lux (instead of the anticipated increase) was observed. The promoter in construct Pp_0588 was for a Cu-specific gene, yet both Cu and Cd decreased Lux output to the same extent as a fusion with a nonmetal responsive promoter (data not shown). We postulate that the metal ion exposure reduced the supply of FMNH2 so that the luciferase could not function. This is an intriguing finding because similar constructs in Escherichia coli function well as biosensors, demonstrating increased promoter activity. We suspect that there may be fundamental differences between the metabolism of these two cell types.

Figure 3. Response of Promoter Fusion in P. putida KT2440 to Cu and Cd Free Ions

Although the biosensors lacked specificity, we have used them to examine the effect of competing metal background ions and complexation.

1. We find that a background of K⁺ does not affect the metal response, but that Ca²⁺ protects cells more against Cd than Cu exposure. This finding suggests that modification of risk assessments for hardness factors, as presently in place for Cu evaluation, may have to be tailored to the metal.
2. We also see that complexation with sulfate and citrate for Cu and Cd exposure or phosphate and chloride for Cd exposure reduced the toxicity, but that each of these complexes was bioactive. This finding for pseudomonad cells questions how Biotic Ligand modeling should be involved in risk assessment for heavy metals.

Cell Sorbtion Studies

The relevance of the Biotic Ligand Model to interactions of the pseudomonad cells with Cu and Cd was examined directly using chemical methods to assess how the metal was associated with the cell (at exchangeable surface sites, at nonexchangeable sites [periplasmic] and internal). As shown in Figure 4, partitioning between Cd and Cu was different with Cu mainly being held in the periplasm and Cd at exchangeable surface sites. These findings agree with the ability of Ca to protect KT2440 cells from Cd²⁺ but not Cu²⁺.

Figure 4. Partitioning of Cu and Cd Within P. putida Cells

Testing the experimental sorbtion data with the Langmuir isotherm showed that free metal exposure correlated well with the Biotic Ligand Model. However, when the same analyses were performed with the data from use of the metal complexes, none of the associations could be well explained by the Biotic Ligand Model. Increases in internal levels of Cd were especially notable for the citrate complexes. These observations demonstrate with a fourth technique that heavy metal complexes are bioavailable to the pseudomonad cells. The findings also emphasize how the Biotic Ligand Model should be used in the EPA assessment of Cu and Cd risk levels.

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The biosensors will be constructed with a root-colonizing bacterium, Pseudomonas putida, KT2440. This bacterium is robust, offers no environmental threat and its genomic sequence, almost completed, will be released in 2003. Fusions will be generated between the promoters of P. putida genes responding with increased transcription when exposed to Cu or Cd and the open reading frame of a reporter gene. We will use a reporter encoding for light production to screen for constructs showing increased light emission when exposed to low non-lethal levels of the metals. These promoter-fusion biosensors will be used singly for quantitative assays of Cu/Cd concentration. The response to the metals associated with the different ligands will be assayed to determine how the response relates to total metal concentration and free metal availability. In the laboratory, we will use in situ detection for real-time imaging of the promoter fusion activities while these bacterial sensors are colonizing roots of plants growing in metal-contaminated soils. The fusions will be deployed in plate arrays to permit rapid assessment of Cu and Cd bioavailability. A second detector system will array the KT2440 genes that react to both metals. Differential hybridization of the gene arrays to RNAs extracted from control and sample-exposed P. putida cells will indicate the level of impact of any metals in the samples. We will compare the responses from the promoter fusion constructs to Cu and Cd complexed with different ligands to their responses at the transcriptional level.

We will test the sensitivity and specificity of the biosensor plate arrays and the gene arrays using metal-contaminated soils and water from mine and government sources that are undergoing a remediation process.

Cost :$336,000.00

Research Component :Nanotechnology

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