Toxicity of the Colicin M Catalytic Domain Exported to the Periplasm Is FkpA Independent (original) (raw)
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Periplasmic chaperone FkpA is essential for imported colicin M toxicity
Molecular Microbiology, 2008
Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 104-fold dilutions killed fkpA+ cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.
Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone
Journal of Biological Chemistry, 2011
Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase (PPIase)/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1% and Cma(P107A) 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A) but not Cma(P176A) killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from P176. In a new PPIase assay, FkpA isomerized the Cma prolyl bond in peptide F-P(176) at a high rate but K-P(107) and L-P(260) at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, F-P(176) is most likely targeted by FkpA.
The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Pluckthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Pluckthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity.
Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?
Biochemical Society Transactions, 2012
Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis–trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA–TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolat...
We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cis-prolines. We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA.
Genome-wide screens: novel mechanisms in colicin import and cytotoxicity
Molecular Microbiology, 2009
Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last twenty-five years. Genome-wide screening using the "Keio collection" to test sensitivity to colicins A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the LPS inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of LPS and ECA. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Pal, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene-product has the necessary proteolysis function.
DpaA detaches Braun’s lipoprotein from peptidoglycan
bioRxiv (Cold Spring Harbor Laboratory), 2021
Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun's lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun's lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.
Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme
Journal of Biological Chemistry, 2010
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. . The abbreviations used are: ColM, colicin M; MurNAc, N-acetylmuramic acid;
Colicin M is only bactericidal when provided from outside the cell
Molecular & general genetics : MGG, 1990
The colicin M structural gene, cma, was subcloned in a vector which allowed temperature-inducible control of its expression. Induction of expression of cma in colicin M uptake proficient strains was lethal for the host cell when the colicin M immunity protein was not present. In liquid culture cells lysed, and no colonies were formed on solid media. These effects were not observed in mutants defective in the colicin receptor (FhuA) or uptake functions (TonB, TolM), nor in wild-type cells treated with trypsin prior to induction of cma expression. It was concluded that cytoplasmic colicin M is not toxic for the producing cell. To exert a lethal effect the colicin has to enter the cell from outside. Cells expressing cma released small amounts of colicin M.