Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations

Abstract

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdx4, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdrA, pdxB, and pdxH but not in pdr. This observation suggested that pd4, like pdx4, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxj in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxj-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxj, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth ofpdrJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

“…Both proteins also share 38% identities with their counterparts in E. coli. The pI value of S. pneumoniae AcpS was estimated to be 6.5, which is much lower than 9.98, the pI value of E. coli AcpS (16,17). Therefore, S. pneumoniae AcpS is significantly less basic than E. coli AcpS.…”

“…The pCZA342 clone was digested with NdeI and BamHI. The NdeI-BamHI DNA fragment containing acpP was subcloned into pET-11a (Novagen), resulting in pRBP- 16.…”

“…Thus, the acpS operon appears to consist of the genes that are required in aromatic amino acid biosynthesis (aroF and aroG encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases), cell wall biosynthesis (alr encoding D-alanine racemase), and DNA recombination (recG). In this regard, the genomic organization of acpS in S. pneumoniae is quite different from that of acpS in E. coli, since acpS in E. coli consists of an operon with its upstream pdxJ gene that is required for vitamin B 6 biosynthesis (16,17).…”

“…HT253 contains a mini-Tn10 insertion in the pdxJ gene, which is upstream of and forms an operon with acpS (16,17). The mini-Tn10 carries two divergent tetracycline-inducible promoters (17).…”

“…The acpS gene from E. coli forms an operon with the upstream gene, pdxJ, whose function is required for vitamin B 6 biosynthesis (16,17). The acpS gene was originally identified as dpj (downstream of pdxJ) whose function, although unknown, was required for the growth of E. coli (16,17). Later, the landmark biochemical study by Lambalot and Walsh (5) led to the identification of Dpj as AcpS.…”