A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW

Abstract

We constructed a null allele of the ftsI gene encoding penicillin-binding protein 3 of Escherichia coli. It caused blockage of septation and loss of viability when expression of an extrachromosomal copy of ftsI was repressed, providing a final proof that ftsI is an essential cell division gene. In order to complement this null allele, the ftsI gene cloned on a single-copy mini-F plasmid required a region 1.9 kb upstream, which was found to contain a promoter sequence that could direct expression of a promoterless lacZ gene on a mini-F plasmid. This promoter sequence lies at the beginning of the mra cluster in the 2 min region of the E. coli chromosome, a cluster of 16 genes which, except for the first 2, are known to be involved in cell division and cell envelope biosynthesis. Disruption of this promoter, named the mra promoter, on the chromosome by inserting the lac promoter led to cell lysis in the absence of a lac inducer. The defect was complemented by a plasmid carrying a chromosomal fragment ranging from the mra promoter to ftsW, the fifth gene downstream of ftsI, but not by a plasmid lacking ftsW. Although several potential promoter sequences in this region of the mra cluster have been reported, we conclude that the promoter identified in this study is required for the first nine genes of the cluster to be fully expressed.Many of the genes involved in the cell division process in Escherichia coli are found localized in a few clusters on the chromosome. The best characterized of the clusters is located at 2 min. It has been designated the mra (for murein A) cluster (11,36,41). It contains at least six cell division genes (fts genes), together with seven murein biosynthetic genes (mur genes, mraY, and ddl) and a lipopolysaccharide biosynthetic gene (envA) (see Fig. 1). Among them is the ftsI (also named pbpB) gene, which codes for penicillin-binding protein 3 (PBP 3). PBP 3 is a cytoplasmic membrane protein that functions as DD-transpeptidase for the formation of a septum of the murein sacculus and is a principal lethal target of ␤-lactam antibiotics (1,16,26). Analyses of thermosensitive cell division mutants showing thermolabile penicillin binding of PBP 3 and their revertants indicated that PBP 3 is an essential cell division component (48), although addition of sucrose suppressed the division defect but not the PBP 3 thermolability. The transpeptidase activity is carried by the C-terminal penicillin-binding domain of PBP 3, while the function of the N-terminal domain is still controversial (16,26,50,57).We initiated an attempt to create a new class of ftsI mutants in the hope of understanding the function of the N-terminal domain of PBP 3, especially in terms of its interaction with other cell division proteins, and realized that a null allele of the ftsI gene on the chromosome would be very useful to characterize mutant alleles newly created on plasmids. To eliminate a possible effect of copy number, we decided to clone the mutant alleles on single-copy mini-F vectors rather than on multicopy vectors. ...

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI

“…Therefore, it can be concluded that ftsI is mainly cotranscribed along with the mraZ, mraW, ftsL and murE genes of the operon probably from the Pmra promoter, as described in E. coli and B. subtilis (Hara et al, 1997), and probably also from a minor promoter (PftsI) as in B. subtilis (Daniel et al, 1996). This result also suggests that the second GTG (at position 2 293 165) most probably is the start codon of ftsI.…”

“…Our results suggest that ftsI is transcribed both from a minor promoter (PftsI), as in B. subtilis (Daniel et al, 1996), and also as a part of the polycistronic mraZ, mraW, ftsL and murE transcript from an upstream promoter (Pmra), as described for E. coli and B. subtilis (Hara et al, 1997;Mengin-Lecreulx et al, 1998). The ftsI gene seems to be essential for the viability of C. glutamicum since gene disruption was possible only in the merodiploid strain C. glutamicum MAPF.…”

“…1b) (Patek et al, 2003) and 99 nt upstream from the C. glutamicum mraZ gene (at the beginning of the mra/dcw cluster of cell division and cell envelope biosynthesis genes) as seen in E. coli (Hara et al, 1997;Mengin-Lecreulx et al, 1998).…”

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli

“…A weak promoter within the ftsQ gene, pA, was reported by Yi et al (1985) and by Flärdh et al (1997), using transcriptional fusions, although the corresponding RNA species was not detected by S1 mapping (Aldea et al, 1990). Evidence was presented that suggested that promoters upstream of pQ2 are necessary to achieve sufficient ftsZ expression for cell viability (Dai and Lutkenhaus, 1991), but recent results have made this possibility unlikely (Hara et al, 1997). To test whether ppGpp affected ftsQAZ expression at an untested promoter or at the post-transcriptional level, we assayed the concentration of FtsZ protein in wild-type cells and in two mecillinam-resistant mutants, argS201 and a p lac -relAЈ-bearing strain, both of which have a twofold higher ppGpp concentration (Joseleau-Petit et al, 1994).…”

RcsA-dependent and -independent growth defects caused by the activated Rcs phosphorelay system in the Escherichia coli pgsA null mutant

“…The E. coli K-12 derivative strains and the plasmids used are listed in Table 1. The rcsA::cam allele, in which the chloramphenicol resistance (cam) gene of pACYC184 was inserted into the EcoRV site within the rcsA gene, was first constructed on the plasmid pHR704, which carries the 4.4-kb BamHI fragment of l clone 344 of the Kohara miniset library (Kohara et al, 1987) containing rcsA, and then crossed into the chromosome as described (Hara et al, 1997).…”