Biography: Dr. Trun received her Ph.D. from Princeton University and was a Jane Coffin Childs Postdoctoral Fellow at NCI. She is also an Instructor in the Advanced Bacterial Genetics course at Cold Spring Harbor Labs and holds an Adjunct Assistant Professorship at the University of Maryland at College Park.
Research: Many cellular processes require spatial and temporal coordination of a complex series of events. To understand how cells coordinate many events without making fatal errors, we have been studying the cell cycle of Escherichia coli. Using the compound camphor, we have identified nine new mutants that miscount their chromosomes and by flow cytometry appear to be diploid.
We have been focusing on mbrA. The mbrA mutant contains more DNA and replicates this DNA using oriC. It dies when cell cycle independent origins are activated. We have suggested that mbrA may define a novel coupling between DNA replication and cell elongation that functions after initiation of replication and serves to keep chromosome number tied to growth rate. Fine structure mapping and plasmids were used to show that mbrA4 is allelic to the previously identified rep gene and that mbrA4 is dominant to wild-type. rep encodes a 5' to 3' DNA helicase that is used for replication of the chromosome.
Our rep mutants suggest that unwinding of the double helix could play an important role in determining the number of chromosomes per cell. This would suggest that other proteins are involved in the process and that they should communicate with rep. To identify such proteins, we have isolated suppressors of mbrA4. One suppressor mapped to the cya gene of E. coli. Cya encodes adenylcyclase, which synthesizes cAMP, and is necessary for Crp to activate genes. Further experiments indicated that mbrA4 is regulated by catabolite repression.
In the original suppressor selection, we were identifying many nonspecific suppressors. To avoid these, we changed the selection conditions to make them more stringent and identified 63 new suppressors of mbrA4. One phenotype we unexpectedly encountered was sensitivity to minimal media. Ten of the mutants, when they became able to grow on rich media, simultaneously lost the ability to grow on minimal media. One of the suppressors is near but not in the rep gene. Thirteen mutants map near 91 minutes. The other 49 mutants have not yet been localized. The initial characterizations identifies them as candidates for other genes whose products either function with the MbrA helicase or regulate the synthesis of it.
We have been using camphor to study cell division. The loci we have identified appear to have affected different parts of the cell cycle to lead to diploidy. This suggests that camphor has a unique affect on cells, altering some parameter of chromosome biology that is used to monitor DNA content per cell. Determination of the mechanism of action of camphor would allow us to understand what parameter is being measured.
To characterize the mechanism of action of camphor, we looked for any gene which when overproduced would result in camphor resistance. One fragment of chromosomal DNA, that maps to 14.2 min., when present in the plasmid vector, pBR322, gives this phenotype. While the plasmid confers camphor resistance, we have determined that it does not result in diploidy. Deletion analysis indicates that the genes responsible for the phenotype resides within a 2.3 kb fragment. Sequence from the insert indicates that it contains cspE, a cold shock gene homologue and two other ORF's. All three genes are required to confer camphor resistance. Additionally, these three genes also suppress a temperature sensitive defect in the chromosomal partition gene, mukB. Elucidation of the mechanism of action of these three genes may provide insights into unexplored areas of bacterial chromosome biology.