Functional domains of colicin M

Dreher, Ralf; Braun, Volkmar; Wittmann‐Liebold, Brigitte

doi:10.1007/bf00446975

Cited by 27 publications

(16 citation statements)

References 15 publications

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“…Previously, removal of the KR sequence at the C terminus of Cma with carboxypeptidase B inactivated Cma (9). Here, we constructed a genetic deletion, which resulted in Cma⌬(K270 R271) with an activity of 1% of that of wild-type Cma ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…This method was originally developed and widely used to release periplasmic proteins into the medium (20) and can be used to transfer colicins unspecifically into cells (2,5,9,22,33). The method is not quantitative but clearly distinguishes between active and inactive Cma and between defects in uptake and enzymatic activity.…”

Section: Resultsmentioning

confidence: 99%

“…Osmotic shock not only releases periplasmic proteins into the shock medium (20) but also transfers added proteins into the periplasm (2,5,9,33). The Cma sensitivity of cells after osmotic shock was tested with purified Cma samples.…”

Section: Methodsmentioning

confidence: 99%

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Mapping Functional Domains of Colicin M

Helbig

Braun

2011

J Bacteriol

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Colicin M (Cma) lyses Escherichia coli cells by inhibiting murein biosynthesis through hydrolysis of the phosphate ester between C 55 -polyisoprenol and N-acetylmuramyl (MurNAc)-pentapeptide-GlcNAc in the periplasm. To identify Cma functional domains, we isolated 54 point mutants and small deletion mutants and examined their cytotoxicity levels. Activity and uptake mutants were distinguished by osmotic shock, which transfers Cma into the periplasm independent of the specific FhuA receptor and the Ton system. Deletion of the hydrophobic helix ␣1, which extends from the compact Cma structure, abolished interference with the antibiotic albomycin, which is transported across the outer membrane by the same system as Cma, thereby identifying ␣1 as the Cma site that binds to FhuA. Deletion of the C-terminal Lys-Arg strongly reduced Cma translocation across the outer membrane after binding to FhuA. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. Asp226 is exposed at the Cma surface and is surrounded by Asp225, Asp229, His235, Tyr228, and Arg236; replacement of each with alanine inactivated Cma. We propose that Asp226 directly participates in phosphate ester hydrolysis and that the surrounding residues contribute to the active site. These residues are strongly conserved in Cma-like proteins of other species. Replacement of other conserved residues with alanine inactivated Cma; these mutations probably altered the Cma structure, as particularly apparent for mutants in the unique open ␤-barrel of Cma, which were isolated in lower yields. Our results identify regions in Cma responsible for uptake and activity and support the concept of a three-domain arrangement of Cma. Colicins are plasmid-encoded protein toxins released by Escherichia coli and taken up by sensitive E. coli cells (8, 35).Colicins are important traits of E. coli since they are produced by 40 to 50% of the natural isolates. Colicins are also the only proteins imported by E. coli. These enzymes or pore-forming proteins are equipped with receptor binding and translocation domains for their import into sensitive cells. They kill cells by degrading DNA or RNA in the cytoplasm, dissipating the membrane potential by forming pores in the cytoplasmic membrane, degrading murein, or inhibiting murein biosynthesis in the periplasm. Cma cleaves the phosphate bond between C 55 -polyisoprenol and N-acetylmuramyl (MurNAc)-pentapeptideGlcNAc (10) in the periplasm near the outside of the cytoplasmic membrane. The released C 55 -polyisoprenol no longer translocates MurNAc-pentapeptide-GlcNAc across the cytoplasmic membrane. Normally, C 55 -polyisoprenol leaves the biosynthetic reaction as a pyrophosphate ester in the periplasm and enters the reaction cycle as a monophosphate ester at the inner side of the cytoplasmic membrane. The same lipid reaction cycle incorporates O antigen into lipopolysaccharide, which is also inhibited by colicin M (14).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Mapping Functional Domains of Colicin M

Helbig

Braun

2011

J Bacteriol

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“…Colicin M lacking a 5-kDa N-terminal fragment binds to cells via the receptor FhuA but does not enter cells (23), and this fragment kills cells when it is translocated into the periplasm via osmotic shock, which bypasses the uptake system (24). Removal of the C-terminal Lys-Arg residues by carboxypeptidase B inactivates colicin M (23). Finally, randomly generated inactive point mutants are located in the C-domain (6).…”

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confidence: 99%

Crystal Structure of Colicin M, a Novel Phosphatase Specifically Imported by Escherichia coli>

Zeth

Römer

Patzer

et al. 2008

Journal of Biological Chemistry

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Colicins are cytotoxic proteins secreted by certain strains of Escherichia coli. Colicin M is unique among these toxins in that it acts in the periplasm and specifically inhibits murein biosynthesis by hydrolyzing the pyrophosphate linkage between bactoprenol and the murein precursor. We crystallized colicin M and determined the structure at 1.7 Å resolution using x-ray crystallography. The protein has a novel structure composed of three domains with distinct functions. The N-domain is a short random coil and contains the exposed TonB box. The central domain includes a hydrophobic ␣-helix and binds presumably to the FhuA receptor. The C-domain is composed of a mixed ␣/␤-fold and forms the phosphatase. The architectures of the individual modules show no similarity to known structures. Amino acid replacements in previously isolated inactive colicin M mutants are located in the phosphatase domain, which contains a number of surface-exposed residues conserved in predicted bacteriocins of other bacteria. The novel phosphatase domain displays no sequence similarity to known phosphatases. The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins. The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.Colicins are plasmid-encoded toxic proteins produced by Escherichia coli to kill E. coli strains that lack the respective colicin-encoding plasmid (1). Approximately half of the natural E. coli isolates produce one or more of these toxins. Colicins either specifically cleave DNA, rRNA, tRNA or form pores in the cytoplasmic membrane, thereby dissipating the electrochemical potential.Colicins are not translocated uni-directionally like most other translocated bacterial proteins, i.e. from the cytoplasm to the cytoplasmic membrane, the periplasm, the outer membrane, and the environment of the cell, but are rather translocated in both directions, i.e. released by the producer cells and imported by colicin-sensitive cells. The mechanism of colicin import is highly specific and involves cognate outer membrane receptor proteins and an energy-coupled transport across the outer membrane. Energy driving the import across the outer membrane is provided by the proton motive force across the cytoplasmic membrane. Energy coupling between the outer and cytoplasmic membranes is mediated by either the Ton system or the Tol system. The Ton system consists of the three membrane proteins TonB, ExbB, and ExbD.Colicin M belongs to the group of colicins whose uptake requires the Ton system (2, 3). These colicins contain a consensus sequence of seven residues at the N terminus, designated as the TonB box, which is also present in all outer membrane transport proteins that require the Ton system and the proton motive force for the import of substrates (4, 5). Both colicin M and its receptor FhuA contain the TonB box, and both are required for the import of colicin M (6...

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“…This result was not unexpected, because it has been shown for all colicins studied to date that the central domain is important for binding to the outer membrane receptor proteins and the amino-terminal portion is involved in the uptake of colicins (7,(11)(12)(13)29). These processes are different for colicins A and B. Colicin A requires a complex receptor structure, consisting of the vitamin B12 receptor BtuB, the OmpF porin, and lipopolysaccharide (8,9).…”

Section: Discussionmentioning

confidence: 88%

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors

Schramm¹,

Mende²,

Braun³

et al. 1987

J Bacteriol

163

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Colicins are plasmid-encoded proteins which are synthesized by Escherichia coli and kill sensitive strains of E. coli and closely related species. Colicin B belongs to the group of channel-forming colicins (33), which include colicins A, E1, and I (11, 22, 31, 34, 35, 39). These transmembrane toxins depolarize the cytoplasmic membrane, leading to dissipation of cellular energy. Colicin B is usually encoded on large self-transmissible plasmids (15, 38), frequently in proximity to colicin M (30), It consists of a single polypeptide chain (6) with a molecular weight of about 60,000 (33).Comparison of the channels formed by colicins A and B in lipid bilayers under identical conditions showed a similar conductance characteristic. However, the ion flux through the colicin B channels was larger than through the A channels. In addition, the colicin B cha,nnels were more stable and less dependent on added CaCl2 than the colicin A channels (33). Crystals of the channel-forming domain of colicin'A have been obtained (40)

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Functional domains of colicin M

Cited by 27 publications

References 15 publications

Mapping Functional Domains of Colicin M

Mapping Functional Domains of Colicin M

Crystal Structure of Colicin M, a Novel Phosphatase Specifically Imported by Escherichia coli>

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors

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