Fabien Gérard

Efficient Twin Arginine Translocation (Tat) Pathway Transport of a Precursor Protein Covalently Anchored to Its Initial cpTatC Binding Site

The thylakoid twin arginine protein translocation (Tat) system operates by a cyclical mechanism in which precursors bind to a cpTatC-Hcf106 receptor complex, which then recruits Tha4 to form the translocase. After translocation, the translocase disassembles. Here, we fine-mapped initial interactions between precursors and the components of the receptor complex. Precursors with (Tmd)Phe substitutions in the signal peptide and early mature domain were bound to thylakoids and photo-cross-linked to components. cpTatC and Hcf106 were found to interact with different regions of the signal peptide. cpTatC cross-linked strongly to residues in the immediate vicinity of the twin arginine motif. Hcf106 cross-linked less strongly to residues in the hydrophobic core and the early mature domain. To determine whether precursors must leave their initial sites of interaction during translocation, cross-linked precursors were subjected to protein transport conditions. tOE17 cross-linked to cpTatC was efficiently translocated, indicating that the mature domain of the precursor can be translocated while the signal peptide remains anchored to the receptor complex.

Human- and Plant-Pathogenic Pseudomonas Species Produce Bacteriocins Exhibiting Colicin M-Like Hydrolase Activity towards Peptidoglycan Precursors

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.

Non‐growing Escherichia coli cells starved for glucose or phosphate use different mechanisms to survive oxidative stress‡

Recent data suggest that superoxide dismutases are important in preventing lethal oxidative damage of proteins in Escherichia coli cells incubated under aerobic, carbon starvation conditions. Here, we show that the alkylhydroperoxide reductase AhpCF (AHP) is specifically required to protect cells incubated under aerobic, phosphate (Pi) starvation conditions. Additional loss of the HP‐I (KatG) hydroperoxidase activity dramatically accelerated the death rate of AHP‐deficient cells. Investigation of the composition of spent culture media indicates that ΔahpCF katG cells leak nutrients, which suggests that membrane lipids are the principal target of peroxides produced in Pi‐starved cells. In fact, the introduction of various mutations inactivating repair activities revealed no obvious role for protein or DNA lesions in the viability of ahp cells. Because the death of ahp cells was directly related to ongoing aerobic glucose metabolism, we wondered how glycolysis, which requires free Pi, could proceed. 31P nuclear magnetic resonance spectra showed that Pi‐starved cells consumed Pi but were apparently able to liberate Pi from phosphorylated products, notably through the synthesis of UDP‐glucose. Whereas expression of the ahpCF and katG genes is enhanced in an OxyR‐dependent manner in response to H2O2 challenge, we found that the inactivation of oxyR and both oxyR and rpoS genes had little effect on the viability of Pi‐starved cells. In stark contrast, the inactivation of both oxyR and rpoS genes dramatically decreased the viability of glucose‐starved cells.

Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.

It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.

In vivo assessment of the Tat signal peptide specificity in Escherichia coli

Abstract. Tat- and Sec-targeting signal peptides are specific for the cognate Tat or Sec pathways. Using two reporter proteins, the specificity and convertibility of a Tat signal peptide were assessed in vivo. The specific substitutions by RK, KR and KK for the RR motif of the TorA signal peptide had no effect on the exclusive Tat-dependent export of colicin V (ColV). By introducing multiple substitutions in a typical Tat signal peptide, altered signal peptides lacking the twin-arginine motif were obtained. Interestingly, some of these signal peptides preserved Tat-pathway targeting capacity, but resulted in a loss of exclusivity. In addition, further increasing the hydrophobicity of the n-region without modifying the h-region converted the Tat signal peptides to Sec signal peptides in the ColV transport. Replacement of positively charged residues in the c-region also abolished the Tat-exclusive targeting of ColV or green fluorescent protein (GFP), but the folded GFP could be transported only through the Tat pathway. These results strongly suggest that the overall hydrophobicity of the n-region is one of the determinants of Tat-targeting exclusivity.

Bactericidal Activity of Colicin V Is Mediated by an Inner Membrane Protein, SdaC, of Escherichia coli

Colicin V (ColV) is a peptide antibiotic that kills sensitive cells by disrupting their membrane potential once it gains access to the inner membrane from the periplasmic face. Recently, we constructed a translocation suicide probe, RR-ColV, that is translocated into the periplasm via the TAT pathway and thus kills the host cells. In this study, we obtained an RR-ColV-resistant mutant by using random Tn10 transposition mutagenesis. Sequencing analysis revealed that the mutant carried a Tn10 insertion in the sdaC (also called dcrA) gene, which is involved in serine uptake and is required for C1 phage adsorption. ColV activity was detected both in the cytoplasm and in the periplasm of this mutant, indicating that RR-ColV was translocated into the periplasm but failed to interact with the inner membrane. The sdaC::Tn10 mutant was resistant only to ColV and remained sensitive to colicins Ia, E3, and A. Most importantly, the sdaC::Tn10 mutant was killed when ColV was anchored to the periplasmic face of the inner membrane by fusion to EtpM, a type II integral membrane protein. Taken together, these results suggest that the SdaC/DcrA protein serves as a specific inner membrane receptor for ColV.

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

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 full-length 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 surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli.

Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system.

X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein

Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.

Characterization of Colicin M and its Orthologs Targeting Bacterial Cell Wall Peptidoglycan Biosynthesis

For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.