Mapping Chromosome Accessability to Transposition Using Microarrays

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Two related methods have been developed to study chromosome structure inside living bacteria. One technique called Muprinting uses PCR reactions to generate high resolution maps of phage Mu insertions in chromosomes. By comparing in vitro and in vivo Muprints of specific DNA regions, the binding of repressors, chromosome partitioning proteins, and RNA polymerase can be monitored. A new method exploits DNA microarrays and bioinformatics to generate genome-wide scans of chromosome structure. Microarray "snapshots" identify chromosomal locations with very high and very low frequencies of transposon insertions. Three goals of the current project are: (i) Make genome snapshots of chromosomes for cells growing with 90 min. doubling time (minimal glucose medium), 125 min doubling time (minimal alanine) , and 300 min. doubling time (minimal succinate medium). When combined with snapshots from cells growing at maximal speed, this information shows how chromosome structure changes in response to cellular metabolic needs. (ii) Define molecular mechanisms that enhance and cloak DNA accessibility to Mu transposition. DNA sites where cis- and trans-regulatory factors are implicated in genetic control will be studied using high resolution in vitro and in vivo Muprint reactions. (iii) E. coli and S. typhimurium transposition patterns will be compared to learn how evolution has altered transposon in the 63% of genes that have been conserved since divergence of these two bacteria from common ancestors. These studies provide a new biochemical model to understand chromosome structure and allow students from high school to graduate school to study evolutionary differences in two genetically related bacteria.