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Project Progress

The overall objective of this project is to identify genes and proteins that enable bacteria to inhabit the permafrost environment. An initial goal is to determine the complete genome sequence — the “informational content” — of two bacterial permafrost isolates. Exciting progress has been made. In collaboration with the DOE Joint Genome Institute (JGI), we have obtained nearly the complete genome sequence of Psychrobacter 273-4, a bacterial strain that we previously isolated from 20-40 thousand year old Siberian permafrost soil. In addition, progress is being made on determining the genome sequence of Exiguobacterium 255-15, a bacterial strain that we isolated from 2-3 million year old permafrost soils in Siberia. Preliminary analysis of the Psychrobacter genome, in collaboration with JGI and the bioinformatics group at Oak Ridge National Laboratory, indicates that the isolate encodes approximately 2,140 predicted open reading frames (ORFs), with 11% of the ORFs being unique to Psychrobacter. Furthermore, 42 % of the ORFs had a “hypothetical protein” as the best “hit” in the GenBank database, indicating that the function of a major portion of the Psychrobacter genome is yet unknown. A search of the Psychrobacter ORFs indicated the presence of potential orthologs for proteins previously shown to be involved in cold adaptation, including four Csp cold shock proteins, six proteins with known cold-induced expression and 12 stress-associated proteins. However, a number of genes known to be involved in bacterial growth at low temperature, including polynucleotide phosphorylase and CsdA, were not found, suggesting that novel cold adaptation genes remain to be discovered in the Psychrobacter genome. Interestingly, 40 transposases from other microorganisms were identified in the Psychrobacter isolate, suggesting that horizontal gene transfer may have played a significant role in the evolution of its genome.

An integral component of this project is to determine how expression of the Psychrobacter and Exiguobacterium genomes is affected by low temperature and other conditions associated with the permafrost environment. Work to date with the Psychrobacter isolate has demonstrated that the proteome responds to changes in temperature. In particular, using two-dimensional polyacrylamide gel electrophoresis, we found that certain proteins were only present at either cold (4°C) or warm (24°C) temperature. Analysis of these polypeptides by trypsin digestion followed by liquid chromatography and tandem elecrospray mass spectrometry indicated that three of the low temperature specific proteins were orthologs of the Escherichia coli Csp cold shock proteins and that another was novel. In addition, one high temperature specific protein was found to belong to the diene lactone hydrolase protein family while two others have not been previously described. To obtain higher resolution of the Psychrobacter proteome, we have been developing a novel 2-dimensional liquid fractionation method that uses a pH column-based separation in the first dimension followed by separation of the proteins in each pH fraction using nonporous silica (NPS) reversed phase high-performance liquid chromatography (HPLC). The result is a 2-dimensional image of the proteins in the cell as a function of pH versus hydrophobicity. After considerable effort to develop effective cell lysis conditions and appropriate temperatures for protein fractionation, we now have a powerful set of procedures and methods to produce a high resolution protein “map” for the Psychrobacter isolate.