Molecular and Microbial Ecology - Research Projects

Metal Resistance in Caulobacter crescentus

Potentially hazardous levels of heavy metals have dispersed into subsurface sediment and groundwater in a number of metal contaminated DOE sites and represent a challenge for environmental restoration.  Effective bioremediation of these sites requires knowledge of genetic pathways for resistance and biotransformation by component organisms within a microbial community. The aquatic bacterium Caulobacter crescentus is a ubiquitous organism with a distinctive ability to survive in low nutrient environments. It has been selected for extensive study by DOE because of its ability to survive in broad environmental habitats where contamination may be present. The recently completed sequence of the strain CB15 has provided the information to study genome wide response to heavy metal stress. A customized 500,000-probe Affymetrix array has been designed by the McAdams laboratory at Stanford University to measure transcription levels of all 3763 putative ORFs, both strands of hypothetical proteins as well as the intervening intergenic regions. This study used this microarray to study transcriptional response to heavy metal stress.

We are studying the toxic effect of six heavy metals (seven compound: methylmercury chloride, cadmium sulfate, sodium selenite, lead nitrate, potassium chromate, potassium dichromate and uranyl nitrate) on growth, survival and cell morphology. We unexpectedly found that strain CB15N was not significantly affected for growth at 1 mM uranium concentration. The highest level of uranium currently observed in ground water at the Oakridge FRC is 200 mM. Under the same conditions in our laboratory, growth of E. coli K-12 was completely stopped and the growth of Pseudomonas putida (Pseudomonas spp. has been reported to accumulate uranium) was drastically reduced.  We believe this is the first study to identify C. crescentus as a uranium-resistant bacterium. Whole genome transcriptional analysis using the Affymetrix C. crescentus microarray revealed groups of genes, operons and pathways, which were up regulated under different heavy metal stresses. Some of the up-regulated pathways (such as DNA repair, removal of superoxide radicals, thio-group protection) confirmed what is known about heavy metal stress on other organisms. Nine transcripts were commonly up-regulated when the cells were stressed with four different toxic metals. We also observed the up-regulation of specific regulatory genes as well as genes and operons of unknown function in response to specific metal stresses. In cells stressed with uranium we observed the up-regulation of four proteins that belong to two different two-component signal transduction systems. Their involvement in uranium stress was confirmed in phenotypic studies by deletion mutants of one signaling pathway. We also identified groups of genes and operons of unknown functions, including transcripts from antisense strand of a predicted gene. Further studies may elucidate function of these transcripts and, ultimately, the mechanism used by C. crescentus to overcome uranium toxicity. Whole genome transcriptional analysis provides a powerful tool for the detection of candidate genes, with no prior knowledge, that may be involved in metal stress survival.  Such analysis will be increasingly necessary as more microbial genome sequences are completed with only computational annotation to suggest function.