Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster.
The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis 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 growth of E. coli. We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide functions necessary for growth. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and was found to encode proteins with > 94% homology to those of E. coli. However, the S. typhimurium sequences cannot recombine with the E. coli sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth. We have also found that the fabG gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (omega-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene. Insertion of the omega-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s).
Zhang
Y
Y
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Cronan
J E
JE
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005227:Fatty Acids / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007700:Kinetics; D005810:Multigene Family; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005227:Fatty Acids; C490246:plsX protein, E coli; C088150:plsX protein, bacteria