Molecular Analysis of the Essential Fatty Acid Biosynthetic Gene Cluster of Escherichia Coli

172 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.The genes encoding ACP and several key fatty acid biosynthetic enzymes are clustered at min 24 of the E. coli chromosome in the order of plsX, fabH, fabD, fabG, acpP, and fabF. I demonstrated that this cluster of genes was transcribed as a non-conventional operon. Multiple promoters were present and each gene was cotranscribed with at least one other gene. Transcripts specific for single genes were also present. I developed an approach called polar allele duplication to determine the physiological importance of various chromosomal transcripts within the cluster. I demonstrated that the promoter located immediately upstream of the coding sequence of the acpP gene was sufficient for synthesis of this very abundant protein. However, a difficulty in analysis of the fab cluster is that several of these genes are essential for growth of E. coli. This complication was overcome by use of the analogous fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide the missing functions. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and (as expected) was found to encode proteins with $>$90% homology to those of E. coli. However, differences in the DNA sequences of the two gene clusters were sufficient to prevent recombination of the E. coli sequences needed to direct polar allele duplication with the S. typhimurium sequences. Using this approach, I found that although the plsX gene was cotranscribed with the upstream rpmF ribosomal protein gene and approximately 60% of the plsX transcripts initiated at promoters located far upstream, the proximal promoter of the plsX gene was sufficient to express this protein to levels allowing normal growth. Insertion of a transcription terminator cassette (the $\Omega$-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and was lethal to the cell, thus providing the first indication that fabG is an essential gene. (Abstract shortened by UMI.).U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD