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Title: Metal Biosensors: Development and Environmental Testing
Investigators: Anne J. Anderson, Charles D. Miller and Joan E. McLean

Abstract of Talk:
Environmental and health risks of toxic metals are based on total metal concentration assayed chemically. However, chelation and sorption may influence metal bioavailabilty. Our goal is to use metabolic changes induced in microbes on contact with the toxic metals to detect bioavailable Cu and Cd. The deliverables will be two types of arrays, one based on live cell response and the second based on gene sequences, to be employed in the specific detection of low doses of bioavailable metals in solution. Metal detection using the cell array is based on increased production of light because the cells contain fusions of a luxAB cassette, encoding light production, with promoters that increase in activity when they detect the metal. Thus, one cell line in the array will emit light only when Cu is present and another only for Cd. The gene array will be used by preparing RNA from the biosensor after metal exposure and assessing which genes show changes in transcript level. The pattern of transcript abundance will be metal specific.

The microbe used in the construction of these molecular-based sensors is Pseudomonas putida strains KT2440, selected because its genome is completely sequenced. Previous work with mutants of P. putida strain Corvallis detected less 1 mg/L free Cu. These mutants possessed fusions of the promoters of genes involved in protecting the cells against oxidative stress with a luxAB cassette. Light emission from these constructs decreased in the presence of toxic metals, but the responses had little metal specificity presumably because of induced cell death in the compromised mutants. However, the literature suggests that other promoters, for example for genes encoding proteins involved in efflux, may respond with greater specificity and with increased expression to low doses of the metals.

To understand the issue of toxicity and its impact on the biosensors, we determined the threshold for toxicity upon Cu exposure for wild type KT2440. Loss in culturability occurred at about 10 mg/L Cu for both KT2440 and Corvallis, a value over ten fold higher than the free Cu concentrations that we aim to detect.

To initiate the production of the cell and gene arrays we have generated random promoter fusions in KT2440 with inserts of the luxAB cassette and measured how light emission changed after exposure to metal. In trials examining 18 members of an insertion library with exposure to the lesser-toxic Fe3+, six of these mutants responded with increased rather than decreased light emission. By examining the site of the luxAB insertion, we found one of the genes that increased in gene expression was in GTP pyrophosphokinase. Its product, guanosine 3',5'-bispyrophosphate (ppGpp), accumulates in bacteria in response to either amino acid or energy source starvation. Consequently, to our knowledge this is a novel response of the bacterial cell to metal exposure. Studies of responses to Cu and Cd are underway to determine whether this gene is another general response gene or one with specificity. We propose that the cell arrays will consist of promoter fusions that respond with increased light emission after Cu or Cd exposure. The genes identified through determining the site on the KT2440 genome bearing the luxAB insertion will be used in the gene array.

Our second approach to identify genes that would be likely candidates for promoter fusions and in the construction of a gene array is based on reverse genetics. This method relies on the identification of the KT2440 proteins that change in accumulation in response to metals. 2-D gel electrophoresis demonstrates changes in levels of several distinct peptides after exposure of KT2440 to Cu and Fe, showing degrees of specificity in response to these different metals. By MALDITOF analysis, two of the peptides that changed in response to 10 mg/L Cu were shown to correspond to a flagellin, which decreased three fold, and the lipoamide dehydrogenase component of 2-oxoglutarate dehydrogenase (OGDH), which increased three fold. These proteins are not amongst a published list of genes in KT2440 that are predicted to be involved in metal responses. This finding illustrates the value in this reverse genomics approach. However, decreased flagellin production has been correlated by other workers with water stress in the cell for P. putida. The increase in the OGDH suggests that carbon flow through the TCA cycle may be enhanced by Cu exposure as anticipated from activated metabolism to overcome stress. The availability of the complete genomic sequence of KT2440 permits us to synthesize the promoters for these genes to directly construct the luxAB fusions.

These findings confirm that specific sequences in the genome of KT2440 can be harnessed for the detection of toxic metals. Such detection is a prelude to the use of the constructs to probe the bioavailability of different metal complexes occurring in the biological and geological world.

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