Earth Sciences Division Berkeley Lab
Environmental Remediation Program
Research Summaries 2004-2005
WHOLE GENOME TRANSCRIPTIONAL ANALYSIS OF TOXIC METAL STRESSES IN CAULOBACTER CRESCENTUS
RESEARCH OBJECTIVES
Gary L. Andersen, Ping Hu, and Eoin L. Brodie Contact: Gary Andersen, 510/495-2795, GLAndersen@lbl.gov
Caulobacter crescentus is an extremely ubiquitous organism with a distinctive ability to survive in low nutrient environments. In this study, we exposed the Caulobacter crescentus cells to five heavy metals (chromium, cadmium, selenium, lead, and uranium) and analyzed genome-wide transcriptional activities. The understanding of resistance pathways can provide important insight and knowledge for environmental restoration.
APPROACH
Transcriptional regulation was measured using a Caulobacter Affymetrix GeneChip array custom designed by the McAdams Lab (Stanford, California). In addition to the multiple probes for all predicted coding regions, probes were tiled for both strands, encoding all hypothetical proteins plus all intergenic regions. This feature makes it possible to detect all transcripts without prior knowledge and bias, including untranslated regulatory RNAs and antisense transcripts (transcripts from the opposite strand of a predicted open reading frame).
Figure 1. Extracellular uranium-bearing precipitates. The EDX showed the precipitates contained uranium, phosphate, and calcium.
We have also used electron microscopy (EM) to visualize Caulobacter crescentus cells under 200 µM uranium stress for 30 minutes and found that uranium was associated with cells extracellularly. Energy Dispersive X-ray (EDX) analysis was carried out to determine elemental compositions of uraniumbearing solid phases associated with cells.
RESULTS
In addition to the unexpected finding that C. crescentus CB15N is tolerant to high levels of uranium, our studies—combining physiology observation, transcriptional measurement, and imaging analysis—clearly showed that Caulobacter formed a calcium-uranium-phosphate precipitate extracellularly (Figure 1). This observation was consistent with a limited response to
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oxidative stress such as that seen with other metals and also with the up-regulation of a secreted calcium-dependent phytase domain protein, which may serve as a nucleation site for uranium precipitation. The strategy of lowering intracellular metal concentration was also present in cadmium and chromium response. Efflux pumps were up-regulated under cadmium stress. C. crescentus does not seem to have a specific extrusion mechanism for chromium; however, the cells down-regulated a sulfate transporter, which may reduce the uptake of chromate. Our data have also clearly demonstrated the importance of interrogating the whole genome on both strands. We have identified at least six antisense transcripts that are differentially regulated specific to metals, which, as either proteins or RNAs, may play an import part in the response model. Using knockout mutants, we also confirmed regulatory roles played by a pair of two-component signal transduction proteins under uranium stress.
SIGNIFICANCE OF FINDINGS
The principal response to most metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase, sodA), while glutathione S-transferase, thioredoxin, glutaredoxins and DNA repair enzymes responded specifically to cadmium and chromate. Cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps being employed to remove cadmium while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. The function of a two-component signal transduction system involved in uranium response was confirmed by knockout mutants. In addition, several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the importance of evaluating the transcriptome using the whole genome.
RELATED PUBLICATION
Hu, P., E.L. Brodie, Y. Suzuki, H.H. McAdams, and G.L. Andersen, Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. Journal of Bacteriology, 187(24), 8437-49, December 2005. Berkeley Lab Report LBNL-59011.
ACKNOWLEDGMENTS
This study was funded by Department of Energy, Genomes to Life: Microbial Cell Program. This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Berkeley National Laboratory, under Contract No. DEAC03-76SF00098.