Antibiotic Colicin Research Articles

Depicting the Translocation Process of the Protein Antibiotic Colicin E9 through OmpF

Depicting the Translocation Process of the Protein Antibiotic Colicin E9 through OmpF

The Effect of Lipopolysaccharide Core Oligosaccharide Size on the Electrostatic Binding of Antimicrobial Proteins to Models of the Gram Negative Bacterial Outer Membrane.

Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate-rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different-sized core oligosaccharide regions we demonstrate the role of uncharged sugars in blocking short-range electrostatic interactions between the cationic antibiotics and the vulnerable anionic phosphate groups.

Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin

The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity.

Self-recognition by an intrinsically disordered protein

The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.

When an ATPase is not an ATPase: at low temperatures the C-terminal domain of the ABC transporter CvaB is a GTPase.

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg2+ and Mn2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37 degrees C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10 degrees C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.

Processing of Colicin V-1, a Secretable Marker Protein of a Bacterial ATP Binding Cassette Export System, Requires Membrane Integrity, Energy, and Cytosolic Factors

Extracellular secretion of the peptide antibiotic colicin V (ColV) in Escherichia coli is mediated by a dedicated exporter system consisting of host TolC protein and the products of two specific genes, cvaA and cvaB, the latter being a member of the ATP binding cassette (ABC) superfamily. An amino-terminal export signal of ColV is specific for the CvaA-CvaB-TolC exporter and is processed concomitant with secretion. In this study, we attempt to characterize this processing with a secretable marker protein, ColV-1, using a newly developed in vitro assay. Processing is found to be dependent on both CvaA-CvaB transporters and the TolC protein and to require membrane integrity. An additional cytoplasmic soluble factor(s) is also necessary for the processing. Although the sequence of the cleavage site suggests it could be a substrate, ColV-1 cannot be processed in vitro by the purified leader peptidase I. Moreover, ColV-1 processing is inhibited by antipain and N-ethylmaleimide. Furthermore, the processing requires energy in the form of nucleotide hydrolysis. These results indicate that the processing of ColV-1 is specific and more complex than expected, requiring the CvaA-CvaB-TolC transporter intact in the membrane, energy, and cytosolic factors for rapid cleavage.

Use of colicin e3 for biological containment of microorganisms

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms.

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

The protein antibiotic colicin N forms ion-permeable channels through planar lipid bilayers. Channels are induced when positive voltages higher than +60 mV are applied. Incorporated channels activate and inactivate in a voltage-dependent fashion. It is shown that colicin N undergoes a transition between an "acidic" and a "basic" channel form which are distinguishable by different voltage dependences. The single-channel conductance is non-ohmic and strongly dependent on pH, indicating that titratable groups control the passage of ions through the channel. The ion selectivity of colicin N channels is influenced by the pH and the lipid composition of the bilayer membrane. In neutral membranes the channel undergoes a transition from slightly cation-selective to slightly anion-selective when the pH is changed from 7 to 5. In lipid membranes bearing a negative surface charge the channel shows a more pronounced cation selectivity which decreases but does not reverse upon lowering the pH from 7 to 5. The high degree of similarity between the channel characteristics of colicin A and N suggests that the channels share common features in their molecular structure.

Crystallization and preliminary X-ray diffraction studies of colicin E 3 immunity protein

Immunity protein, an inhibitor of the ribonuclease activity of the protein antibiotic colicin E 3, crystallizes in the orthorhombic space group C222 with cell dimensions a = 78·7 A ̊ , b = 54·1 A ̊ , c = 36·1 A ̊ and one molecule of M r 9800 per asymmetric unit. The crystals are suitable for high resolution X-ray analysis.

Synthesis of colicin E1 in a cell-free coupled transcription-translation system

Protein was synthesized in a cell-free coupled transcription-translation (S30) system on plasmid Col E1 and phage T2 and Sd DNA templates. Phage T2 DNA and closed plasmid DNA proved to be the most active templates. The latter controlled the synthesis of eight individual proteinsin vitro, including the biologically active protein antibiotic colicin. The titer of colicin synthesized in this cell-free system reached 1024 units/ml, two orders of magnitude higher than the titer of the antibiotic in populations of colicinogenic bacteria.

Synthesis of E colicins in Escherichia coli.

Mitomycin C treatment of Escherichia coli cells containing one of the ColE plasmids results in specific inhibition of chromosomal protein synthesis and a high rate of protein synthesis from about 35% of the plasmid genome, whereas similar treatment of plasmid-free cells has no measureable effect on protein synthesis. In the case of ColE2- and ColE3-containing cells, the antibiotic colicin protein (molecular weight about 78000) and two others (molecular weight about 11000 and 6000) are coordinately synthesized in the approximate molar ratio 1:4:1, while in ColE1-containing cells only the colicin protein is synthesized in large amounts. Partially purified colicin E2 isolated from the outer cell surface is associated with the two small proteins in the approximate molar ratio 1:1:1, indicating that not are they only synthesized coordinately but are released as a ternary complex.

The killing of sensitive cells by colicin D

1. At low multiplicities, the large molecular weight protein antibiotic colicin D inhibits protein synthesis in sensitive cells without markedly affecting the synthesis of DNA or RNA. In this respect it is very much like colicin E3 but unlike those colicins which appear to inhibit oxidative phosphorylation. It was previously suggested that the lethal action of colicin D was the inhibition of oxidative phosphorylation. 2. Chloramphenicol causes an immediate decrease in the sensitivity of cells to the action of colicin D, but does not similarly alter the sensitivity of cells to colicin E3. This may indicate that these two colicins are not completely identical in their modes of action. 3. Although the lethal adsorption of colicin D to sensitive cells follows single-hit kinetics, on average 100 molecules are required to kill a cell. Thus, only 1 % of adsorbed colicin molecules actually effects the lethal action.