Transcriptional Analysis of Essential Genes of the Escherichia coli Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous Salmonella typhimurium Gene Cluster

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Vol. 180, Issue 13, 3295-3303, July 1, 1998

Transcriptional Analysis of Essential Genes of the Escherichia coli Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous Salmonella typhimurium Gene Cluster

Yan Zhang¹ and John E. Cronan Jr.¹²

¹ Departments of Microbiology¹ and ² Biochemistry,² University of Illinois, Urbana, Illinois 61801

The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabFat min 24 of the Escherichia coli chromosome. A difficulty inanalysis of the fab cluster by the polar allele duplication approach(Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614-3620,1996) is that several of these genes are essential for the growthof E. coli. We overcame this complication by use of the fab genecluster of Salmonella typhimurium, a close relative of E. coli,to provide functions necessary for growth. The S. typhimuriumfab cluster was isolated by complementation of an E. coli fabDmutant and was found to encode proteins with >94% homology tothose of E. coli. However, the S. typhimurium sequences cannotrecombine with the E. coli sequences required to direct polarallele duplication via homologous recombination. Using this approach,we found that although approximately 60% of the plsX transcriptsinitiate at promoters located far upstream and include the upstreamrpmF ribosomal protein gene, a promoter located upstream of theplsX coding sequence (probably within the upstream gene, rpmF)is sufficient for normal growth. We have also found that the fabGgene is obligatorily cotranscribed with upstream genes. Insertionof a transcription terminator cassette ( $Omega$ -Cm cassette) betweenthe fabD and fabG genes of the E. coli chromosome abolished fabGtranscription and blocked cell growth, thus providing the firstindication that fabG is an essential gene. Insertion of the $Omega$ -Cmcassette between fabH and fabD caused greatly decreased transcriptionof the fabD and fabG genes and slower cellular growth, indicatingthat fabD has only a weak promoter(s).

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