Characterization of an operon containing a ribosomal protein gene and lipid biosynthetic genes in Escherichia coli K-12

The plsX50 mutation is required together with plsX26 (encoding a K_m- defective glycerol 3-phosphate acyltransferase) for the conferral of a glycerol 3- phosphate auxotrophic phenotype. A 4.9 kb segment of DNA complementing the plsX50 mutation have been cloned and sequenced. Six open reading frames (ORF’s) were found with five reading in the same direction and one in the opposite direction relative to the plsX gene. Each ORF encoded a protein, as demonstrated by radiolabeling in maxicells. ORF1 (orfY) encodes a protein of unknown function. ORF2 and ORF3 (rpmF) were sequenced prior to this study and encode a protein called G30k of unknown function and L32, a protein of the large ribosomal subunit, respectively. ORF4 complemented the p/sX50 mutation. ORFS was identified as fabH encoding 3-ketoacyl-ACP synthase III. ORF6 was identified as fabD encoding malonyl-CoA/ACP transacylase. The fabG gene encoding 3- ketoacyl-ACP reductase and the acpP gene encoding acy] carrier protein are located just downstream of the fabD gene. Northern and promoter activity analysis demonstrated that the rpmF-plsX-fabH-fabD-fabG-acpP genes comprise an operon suggesting a coordinate control of the synthesis of a ribosomal protein (L32), PlsX protein, and fatty acid biosynthetic enzymes. However, several features were identified that are likely to be important for differential expression of the individual genes. These include the presence of multiple promoters, an internal terminator (attenuator), differential degradation of transcripts, and differential efficiency of translation initiation.

Portions of transcripts arising upstream of rpmF terminate at the attenuator located just downstream of the plsX initiation codon, and some of the transcripts continue into the plsX-jab genes. The fabH-fabD-fabG-acpP genes are also cotranscribed from a promoter located upstream of the fabH gene, within the pisX structural gene. There are additional cotranscripts responsible for the expression of the fabD-fabG-acpP genes. The acpP gene is encoded by several more transcripts. Transcription initiation sites upstream of rpmF were identified by primer extension analysis and the attenuator site was identified by S1 mapping analysis. N-terminal amino acid sequence analysis identified the translation initiation codons of orfY, plsX, fabH, fabD and fabG. Short intergenic distances (15 and 12 bp) found between fabH and fabD and between fabD and fabG implicate translational coupling as a mechanism for coordinate control of fabHDG expression. The p/sX50 mutation was identified as deletion of a single nucleotide from the 6th codon of plsX resulting in a frame shift nonsense mutation.