The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784 -3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by ϳ75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.Undecaprenyl phosphate (C 55 -P) 1 is a key lipid intermediate involved in the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1-5). This carrier lipid enables the transport of hydrophilic precursors through the hydrophobic environment of the membrane to the externally located sites of polymerization. Because a single lipid participates in the synthesis of various wall polymers, C 55 -P has been considered previously to be a potential site of control over the synthesis of these polymers that prevents an imbalance in the formation of the cell envelope as a whole (6). However, our knowledge of C 55 -P metabolism remains very limited and based on fragmentary data obtained from various bacterial species (Scheme 1). The precursor for C 55 -P, undecaprenyl pyrophosphate (C 55 -PP), is synthesized by addition of isoprene units onto farnesyl pyrophosphate (Scheme 1, step 1). This reaction is catalyzed by a cis-prenyl pyrophosphate synthase that has been characterized in detail both biochemically and structurally (7-11). The dephosphorylation of C 55 -PP (Scheme 1, step 2) is required before the lipid carrier becomes available for use in the various biosynthetic pathways, and this reaction must also occur at the end of each cycle of polymerization reaction (e.g. of peptidoglycan) where the lipid carrier is released in the pyrophosphate form. This reaction of dephosphorylation, which is the site of action of bacitracin, is catalyzed by a membrane-bound phosphatase (12). The intriguing presen...

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106 -30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant ⌬bacA,⌬ybjG,⌬pgpB in which the expression of bacA is impaired at 42°C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.An essential carrier lipid, undecaprenyl phosphate (C 55 -P), 1 is required for the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1-5) (Scheme 1). It is synthesized as a pyrophosphate precursor (C 55 -PP) by the addition of eight isoprene units to farnesyl pyrophosphate, a reaction catalyzed by the well characterized cis-prenyl-pyrophosphate synthase UppS (6 -10). However, genes and enzymes involved in subsequent steps of C 55 -P synthesis and recycling still remained to be identified. Bacitracin is a dodecapeptide antibiotic known to specifically block this metabolism by forming a specific complex with C 55 -PP. As a result, cell wall biosynthesis is inhibited and cell lysis finally occurs (11)(12)(13)(14). Bacillus licheniformis strains that produce bacitracin are resistant to this antibiotic due to the presence of an appropriate ABC transporter efflux system (15, 16). Several mutations leading to bacitracin resistance were identified in Escherichia coli and other Gram-negative bacteria. Interestingly, all these mutations were shown to block the synthesis of non-essential cell envelope polymers such as osmoregulated periplasmic glycans and capsule polysaccharides that also requ...

Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAcpentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.Colicins are plasmid-encoded toxins, synthesized and released in the growth medium by Escherichia coli, which kill susceptible E. coli strains and closely related bacterial species (1-3). Strains are protected against the colicin they produce by concomitant expression of a highly specific immunity protein.The lethal action of colicins can be divided into three steps:binding to a specific outer membrane receptor protein, translocation through the cell envelope, and finally interaction with the target and killing effect. To each of these steps corresponds a specific protein domain, the different colicins showing a similar three-domain structural organization. Depending on the import pathway they parasitize to enter the cells, the Tol system or the TonB system, colicins are classified into two groups A and B, respectively (3). The mode of action of colicins is variable: formation of voltage-gated pores in the cytoplasmic membrane (e.g. colicins A, B, E1, Ia, N), inhibition of protein synthesis (e.g. colicins D and E3), enzymatic degradation of cellular DNA or 16S rRNA (e.g. colicin E2) and inhibition of cell envelope biosynthesis (colicin M, see below).Various bacterial cell envelope polysaccharides (peptidoglycan, O-antigen, teichoic acid, capsular polysaccharide) in both Gram-negative and Gram-positive bacteria have a lipid-linked intermediary stage in their biosynthesis that is dependent on the essential carrier lipid undecaprenyl phosphate (C 55 -P) 4 (4 -15) (Scheme 1). In the peptidoglycan pathway, this lipid is needed for the synthesis and trans...

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.