Contribution of the P(mra) promoter to expression of genes in the Escherichia coli mra cluster of cell envelope biosynthesis and cell division genes

Abstract

Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802-5811, 1997). Disruption of this promoter (P(mra)) on the chromosome and its replacement by the lac promoter (P(mra)::P(lac)) led to isopropyl-β-D-thiogalactopyranoside (IPTG)- dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P(mra) to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P(mra)::P(lac) promoter expression was repressed or fully induced. It was confirmed that the P(mra) promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P(mra)::P(lac) cells. This was correlated with an accumulation of the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP-N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when P(mra) expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the P(mra) promoter.