SummaryThe major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo . Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo , and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N-and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.

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...

During the biogenesis of bacterial cell-wall polysaccharides, such as peptidoglycan, cytoplasmic synthesized precursors should be trafficked across the plasma membrane. This essential process requires a dedicated lipid, undecaprenyl-phosphate that is used as a glycan lipid carrier. The sugar is linked to the lipid carrier at the inner face of the membrane and is translocated toward the periplasm, where the glycan moiety is transferred to the growing polymer. Undecaprenyl-phosphate originates from the dephosphorylation of its precursor undecaprenyl-diphosphate, with itself generated by de novo synthesis or by recycling after the final glycan transfer. Undecaprenyl-diphosphate is de novo synthesized by the cytosolic cis-prenyltransferase undecaprenyldiphosphate synthase, which has been structurally and mechanistically characterized in great detail highlighting the condensation process. In contrast, the next step toward the formation of the lipid carrier, the dephosphorylation step, which has been overlooked for many years, has only started revealing surprising features. In contrast to the previous step, two unrelated families of integral membrane proteins exhibit undecaprenyldiphosphate phosphatase activity: BacA and members of the phosphatidic acid phosphatase type 2 super-family, raising the question of the significance of this multiplicity. Moreover, these enzymes establish an unexpected link between the synthesis of bacterial cell-wall polymers and other biological processes. In the present review, the current knowledge in the field of the bacterial lipid carrier, its mechanism of action, biogenesis, recycling, regulation, and future perspective works are presented.

SummaryOne-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species.

Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg 2؉ -dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.Certain Escherichia coli strains produce and release in the growth medium toxins designated colicins in order to kill competitors belonging to the same species or to related species (8,28,29). The various modes of action of colicins include formation of pores in the cytoplasmic membrane, inhibition of protein synthesis, enzymatic degradation of cellular DNA or 16S rRNA, and interference with cell envelope biosynthesis. Colicins and proteins conferring immunity to the producer are generally encoded by plasmids. Depending on the import pathway they use to enter the cells, the Tol or TonB system, the colicins have been classified in group A or B, respectively. Their lethal action occurs in three steps: binding to a specific outer membrane receptor protein, translocation through the cell envelope, and interaction with the target leading to the bactericidal effect. A specific protein domain corresponds to each of the three steps, and the different colicins display a similar three-domain structural organization (8).Colicin M (ColM) exhibits a unique mode of action, as it is the only colicin known to interfere with peptidoglycan biosynthesis and to cause cell lysis (35). This class B colicin is internalized via the TonB translocation machinery and uses the FhuA outer membrane protein as the receptor. ColM lacks peptidoglycan-degrading activity and acts synergistically with ␤-lactam antibiotics, which inhibit the last polymerization step of peptidoglycan synthesis performed by penicillin-binding proteins (34,35). Since ColM inhibited both peptidoglycan synthesis and lipopolysaccharide O-antigen sy...

The synthesis of the lipid carrier undecaprenyl phosphate (C 55 -P) requires the dephosphorylation of its precursor, undecaprenyl pyrophosphate (C 55 -PP). The latter lipid is synthesized de novo in the cytosol and is also regenerated after its release from the C 55 -PP-linked glycans in the periplasm. In Escherichia coli the dephosphorylation of C 55 -PP was shown to involve four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family, PgpB, YbjG, and YeiU. Here, the PgpB protein was purified to homogeneity, and its phosphatase activity was examined. This enzyme was shown to catalyze the dephosphorylation of C 55 -PP with a relatively low efficiency compared with diacylglycerol pyrophosphate and farnesyl pyrophosphate (C 15 -PP) lipid substrates. However, the in vitro C 55 -PP phosphatase activity of PgpB was specifically enhanced by different phospholipids. We hypothesize that the phospholipids are important determinants to ensure proper conformation of the atypical long axis C 55 carrier lipid in membranes. Furthermore, a topological analysis demonstrated that PgpB contains six transmembrane segments, a large periplasmic loop, and the type 2 phosphatidic acid phosphatase signature residues at a periplasmic location.Undecaprenyl phosphate (C 55 -P) 2 is a 55-carbon-long polyprenol (see Fig. 1). It is an essential bacterial lipid required for the synthesis of various cell wall polymers such as peptidoglycan, lipopolysaccharides, teichoic acids, osmo-regulated periplasmic glucans, capsular polysaccharides, and the enterobacterial common antigen (1-10). C 55 -P is utilized as a carrier lipid that allows the transport of the hydrophilic oligosaccharide precursors across the cytoplasmic membrane toward the periplasm where the elongation of the glycan chains takes place. Accordingly, the precursor is linked to the carrier lipid via a pyrophosphate linkage (C 55 -PP-substrate) through the action of a specific glycosyltransferase at the cytosolic side of the inner membrane; thereafter, the complex is translocated through the membrane by a yet unknown mechanism, and finally, the glycosyl moiety is transferred to the appropriate expanding polymer. De novo synthesis of C 55 -P implicates two enzymatic steps (11, 12); it originates from undecaprenyl pyrophosphate (C 55 -PP), itself being synthesized by successive condensations of eight isopentenyl pyrophosphates (C 5 -PP) with farnesyl pyrophosphate (C 15 -PP) (Fig. 1) catalyzed by the cytosolic UppS enzyme, a cis-prenyl-pyrophosphate synthase (13, 14). The C 55 -PP must then be dephosphorylated to yield the active monophosphate form of the carrier lipid (11). C 55 -PP is not solely generated by de novo synthesis, but it is also released and recycled after the transfer of the oligosaccharide unit to the growing polymer in the periplasm. It is yet unclear on which side of the membrane C 55 -PP dephosphorylation occurs and how the carrier lipid is translocated across the membrane before being reused.The enzymatic dephosphoryl...

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.Water availability is primordial for life of all organisms. Bacteria submitted to a severe hyperosmotic stress instantaneously lose a large amount of their intracellular water to balance the osmotic strength between intracellular and extracellular spaces. The subsequent decrease of cellular water activity together with the loss of cell turgor lead to lessen the bacterial cell expansion rate (1). Surviving such injuring conditions implies the reversion of water flux across the cell membrane; this can be achieved by amassing highly soluble compounds termed osmolytes (2, 3). Thus, Escherichia coli cells rapidly take up high amounts of potassium ions (4, 5) and subsequently increase their glutamate content to balance electric charges. To avoid the perturbing effect of elevated ionic strength, K ϩ -glutamate can be progressively replaced by organic osmolytes that behave neutral at physiological pH (6). Such compounds, termed compatible solutes (7), may be endogenously synthesized or imported from the surrounding medium (3,8). Imported compatible solutes generally confer a high degree of osmotic tolerance to injured cells. Among these so-called osmoprotectants, glycine betaine (GB) 1 is by far the most effective and the most commonly assayed for hyperosmotic purposes.In addition to the obvious predominant role they play in cellular osmotic adjustment, internalized and accumulated osmoprotectants should directly participate in other intracellular processes. Protective as well as stabilizing effects of betaine and other solutes on proteins denaturation because of increased salinity or temperature have been reported (9 -12). It is tempting to extrapolate these results in vivo; however, bacteria submitte...

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124 -271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the fulllength colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surfaceexposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.