The genes encoding several key fatty acid biosynthetic enzymes (called thefabcluster) are clustered in the orderplsX-fabH-fabD-fabG-acpP-fabFat min 24 of theEscherichia colichromosome. A difficulty in analysis of thefabcluster 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 growth ofE. coli. We overcame this complication by use of thefabgene cluster ofSalmonella typhimurium, a close relative ofE. coli, to provide functions necessary for growth. TheS. typhimurium fabcluster was isolated by complementation of anE. coli fabDmutant and was found to encode proteins with >94% homology to those ofE. coli. However, theS. typhimuriumsequences cannot recombine with theE. colisequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of theplsXtranscripts initiate at promoters located far upstream and include the upstreamrpmFribosomal protein gene, a promoter located upstream of theplsXcoding sequence (probably within the upstream gene,rpmF) is sufficient for normal growth. We have also found that thefabGgene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (Ω-Cm cassette) between thefabDandfabGgenes of theE. colichromosome abolishedfabGtranscription and blocked cell growth, thus providing the first indication thatfabGis an essential gene. Insertion of the Ω-Cm cassette betweenfabHandfabDcaused greatly decreased transcription of thefabDandfabGgenes and slower cellular growth, indicating thatfabDhas only a weak promoter(s).