Colicin Research Articles

Genetic and functional analysis of the bovine uterine microbiota. Part II: Purulent vaginal discharge versus healthy cows

The aim of this study was to characterize, using metagenomic shotgun DNA sequencing, the intrauterine microbial population and its predicted functional diversity within healthy cows and cows presenting purulent vaginal discharge (PVD). Twenty Holstein dairy cows from a single farm were enrolled in the study at 25 to 35 d postpartum. Purulent vaginal discharge was diagnosed by retrieving and scoring vaginal discharge using the Metricheck device (Simcro, Hamilton, New Zealand). Intrauterine samples for metagenomic analysis were collected by the cytobrush technique from 8 cows diagnosed with PVD and 12 healthy cows. Pair-end sequencing was performed using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). Metagenomic sequences were analyzed using the MG-RAST server (metagenomic rapid annotations using subsystems technology; http://metagenomics.anl.gov/), and the STAMP software (http://kiwi.cs.dal.ca/Software/STAMP) was used to study statistically significant differential abundance of taxonomic and functional features between the 2 metagenomes. Additionally, the total number of bacterial 16S rDNA copies was estimated by real-time PCR. Taxonomic analysis revealed that Bacteroidetes was the most abundant phylum in the uterine microbiota from cows with PVD, and Fusobacteria was almost completely absent in the healthy uterine microbiota. Moreover, species belonging to the genus Trueperella were present only in the uterine microbiota of PVD cows. The increased abundance of Fusobacteria and the unique presence of Trueperella in the PVD cows highlight the important role of these bacteria in the pathogenesis of PVD. Genes encoding cytolethal distending toxin were exclusive to the microbiota of PVD cows. Similarly, genes associated with lipid A modification were present only in samples from PVD cows; such modification is associated with greater resistance to cationic antimicrobial peptides. Conversely, genes encoding bacteriocins and ribosomally antibacterial peptide were exclusively found in the healthy uterine microbiota and dominated by tolerance to colicin E2. No difference was observed in total bacterial load between the 2 microbiotas. This study provides deep insight into the uterine microbial community in health and disease. The observations that the healthy microbiota is tolerant to colicin E2, whereas the uterine microbiota of PVD cows produces cytolethal distending toxins and modifies its lipopolysaccharides suggest that species-intrinsic factors may be more relevant than bacterial abundance to the development of disease or maintenance of health in the dairy cow postpartum uterus.

Hierarchical Post-transcriptional Regulation of Colicin E2 Expression in Escherichia coli.

Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system’s basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and reveals the importance of the specific regulatory components for the timing of toxin release.

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.

On the ability of molecular dynamics simulation and continuum electrostatics to treat interfacial water molecules in protein-protein complexes

Interfacial waters are increasingly appreciated as playing a key role in protein-protein interactions. We report on a study of the prediction of interfacial water positions by both Molecular Dynamics and explicit solvent-continuum electrostatics based on the Dipolar Poisson-Boltzmann Langevin (DPBL) model, for three test cases: (i) the barnase/barstar complex (ii) the complex between the DNase domain of colicin E2 and its cognate Im2 immunity protein and (iii) the highly unusual anti-freeze protein Maxi which contains a large number of waters in its interior. We characterize the waters at the interface and in the core of the Maxi protein by the statistics of correctly predicted positions with respect to crystallographic water positions in the PDB files as well as the dynamic measures of diffusion constants and position lifetimes. Our approach provides a methodology for the evaluation of predicted interfacial water positions through an investigation of water-mediated inter-chain contacts. While our results show satisfactory behaviour for molecular dynamics simulation, they also highlight the need for improvement of continuum methods.

Resolving the 3D spatial orientation of helix I in the closed state of the colicin E1 channel domain by FRET. Insights into the integration mechanism

Current evidence suggests that the closed-state membrane model for the channel-forming domain of colicin E1 involves eight amphipathic α-helices (helices I–VII and X) that adopt a two-dimensional arrangement on the membrane surface. Two central hydrophobic α-helices in colicin E1 (VIII and IX) adopt a transmembrane location–the umbrella model. Helices I and II have been shown to participate in the channel by forming a transmembrane segment (TM1) in the voltage-induced open channel state. Consequently, it is paramount to determine the relative location and orientation of helix I in the two-dimensional arrangement of the membrane. A new, low-resolution, three-dimensional model of the closed state of the colicin E1 channel was constructed based on FRET measurements between three naturally occurring Trp residues and three sites in helix I, in addition to previously reported FRET distances for the channel domain. Furthermore, a new mechanism for the channel integration process involving the transition of the soluble to membrane-bound form is presented based on a plethora of kinetic data for this process.

The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import?

The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.

Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in Escherichia coli

With the increasingly serious global environment problem caused by slather use of fossil fuels, numerous efforts have been made in developing renewable alternatives. Converting lignocellulose to bioenergy has accomplished by cellulases but the production is limited, therefore the recombinant expression platforms in E. coli have been established. Since E. coli is partly restricted by its inability to secrete enzymes to extracellular membrane, we constructed a synthetic circuit to coexpress cellulases and colicin E7 lysis to solve this problem. The pBAD-RBS-lysis-TT (BBa_K117010) sequence from iGEM was added to form a chimeric plasmid based on pET system, which harboring cellulases from Bacillus subtilis or Thermobifida fusca. The presence of arabinose for the promoter pBAD, leading to induce lysis and further breakdown of the host membrane to release cellulase to extracellular membrane. The extracellular activities increase to 48.1 and 55.5% under lysis gene functioned on Cel5 and CD1, respectively. This is the first attempt to show that cellulases have successfully expressed and released to extracellular membrane without cumbersome steps. Finally, this synthetic circuit simplifies and achieves the simultaneous release and heterologous expression of the cellulases as well as obtain a long-term stable enzyme in E. coli system.

TRNA-targeting ribonucleases: molecular mechanisms and insights into their physiological roles.

Most bacteria produce antibacterial proteins known as bacteriocins, which aid bacterial defence systems to provide a physiological advantage. To date, many kinds of bacteriocins have been characterized. Colicin has long been known as a plasmidborne bacteriocin that kills other Escherichia coli cells lacking the same plasmid. To defeat other cells, colicins exert specific activities such as ion-channel, DNase, and RNase activity. Colicin E5 and colicin D impair protein synthesis in sensitive E. coli cells; however, their physiological targets have not long been identified. This review describes our finding that colicins E5 and D are novel RNases targeting specific E. coli tRNAs and elucidates their enzymatic properties based on biochemical analyses and X-ray crystal structures. Moreover, tRNA cleavage mediates bacteriostasis, which depends on trans-translation. Based on these results and others, cell growth regulation depending on tRNA cleavage is also discussed.

Microcin determinants are associated with B2 phylogroup of human fecal Escherichia coli isolates.

Escherichia coli strains are classified into four main phylogenetic groups (A, B1, B2, and D) and strains of these phylogroups differ in a number of characteristics. This study tested whether human fecal E. coli isolates belonging to different phylogroups differ in prevalence of bacteriocinogenic isolates and prevalence of individual bacteriocinogenic determinants. A set of 1283 fecal E. coli isolates from patients with different diseases was tested for the presence of DNA regions allowing classification into E. coli phylogroups and for the ability to produce bacteriocins (23 colicins and 7 microcins). Of the isolates tested, the most common was phylogroup B2 (38.3%) followed by phylogroups A (28.3%), D (26.3%) and B1 (7.2%). Altogether, 695 bacteriocin producers were identified representing 54.2% of all tested isolates. The highest prevalence of bacteriocin producers was found in group B2 (60.3%) and the lowest in group B1 (44.6%). Determinants encoding colicins E1, Ia, and microcin mV were most common in phylogroup A, determinants encoding microcins mM and mH47 were most common in phylogroup B2, and determinant encoding mB17 was most common in phylogroup D. The highest prevalence of bacteriocinogeny was found in phylogroup B2, suggesting that bacteriocinogeny and especially the synthesis of microcins was associated with virulent and resident E. coli strains.

Identification and functional analysis of a bacteriocin, pyocin S6, with ribonuclease activity from a Pseudomonas aeruginosa cystic fibrosis clinical isolate.

S‐type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor‐binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore‐forming activity. Pyocins S2, S3, S4, and S5 recognize TonB‐dependent ferri‐siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin–immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor‐binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3‐equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron‐limiting conditions. In the S1‐pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6‐type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6‐type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one‐fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non‐CF clinical isolates.

Unraveling the Outer Membrane Translocation Mechanism of a Protein Antibiotic using Single-Molecule Microbiology and Computational Biophysics

Colicins are natural protein antibiotics deployed by Escherichia coli to kill closely related bacterial competitors - they act by delivering a toxic protein domain intracellularly. A mechanistic understanding of how colicins manipulate endogenous protein-protein interactions to gain cell entry may lead to the development of novel therapeutics against antibiotic-resistant bacteria. By combining in vivo, in vitro and in silico biophysical techniques, we recently discovered that promiscuous interactions occur between the outer membrane proteins (OMPs) BtuB and OmpF, which are the receptor and translocator proteins for colicin E9 (ColE9), respectively. These promiscuous protein-protein interactions may contribute to the observed self-association of OMPs to form outer membrane (OM) islands. Super-resolution imaging, i.e. stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM), has been used to reveal the size and surface distribution of colicin-receptor complexes in these OM islands. Using confocal fluorescence recovery after photo-bleaching (FRAP) microscopy, we have for the first time detected in vivo assembly of the translocon complex observed as tethering of the inner membrane protein TolA by the N-terminus of ColE9 in a BtuB and TolB dependent manner. These in vivo studies have demonstrated that delivery of the intrinsically unstructured N-terminus of ColE9 to the periplasm is sufficient to assemble a periplasm-spanning translocon complex and that this occurs while the colicin molecule remains receptor-bound at the extracellular surface. Work is underway to elucidate the role of trans-periplasmic energy transduction in the formation and maintenance of this translocon complex.

Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI.

Escherichia coli strain MRE600 was originally identified for its low RNase I activity and has therefore been widely adopted by the biomedical research community as a preferred source for the expression and purification of transfer RNAs and ribosomes. Despite its widespread use, surprisingly little information about its genome or genetic content exists. Here, we present the first de novo assembly and description of the MRE600 genome and epigenome. To provide context to these studies of MRE600, we include comparative analyses with E. coli K-12 MG1655 (K12). Pacific Biosciences Single Molecule, Real-Time sequencing reads were assembled into one large chromosome (4.83 Mb) and three smaller plasmids (89.1, 56.9, and 7.1 kb). Interestingly, the 7.1-kb plasmid possesses genes encoding a colicin E1 protein and its associated immunity protein. The MRE600 genome has a G + C content of 50.8% and contains a total of 5,181 genes, including 4,913 protein-encoding genes and 268 RNA genes. We identified 41,469 modified DNA bases (0.83% of total) and found that MRE600 lacks the gene for type I methyltransferase, EcoKI. Phylogenetic, taxonomic, and genetic analyses demonstrate that MRE600 is a divergent E. coli strain that displays features of the closely related genus, Shigella. Nevertheless, comparative analyses between MRE600 and E. coli K12 show that these two strains exhibit nearly identical ribosomal proteins, ribosomal RNAs, and highly homologous tRNA species. Substantiating prior suggestions that MRE600 lacks RNase I activity, the RNase I-encoding gene, rna, contains a single premature stop codon early in its open-reading frame.

Determinants encoding fimbriae type 1 in fecal Escherichia coli are associated with increased frequency of bacteriocinogeny.

BackgroundTo screen whether E. coli strains encoding type 1 fimbriae, isolated from fecal microflora, produce bacteriocins more often relative to fimA-negative E. coli strains of similar origin.MethodsPCR assays were used to detect presence of genes encoding 30 bacteriocin determinants (23 colicin- and 7 microcin-encoding genes) and 18 virulence determinants in 579 E. coli strains of human and animal origin isolated from hospitals and animal facilities in the Czech and Slovak Republic. E. coli strains were also classified into phylogroups (A, B1, B2 and D).ResultsfimA-negative E. coli strains (defined as those possessing none of the 18 tested virulence determinants) were compared to fimA-positive E. coli strains (possessing fimA as the only detected virulence determinant). Strains with identified bacteriocin genes were more commonly found among fimA-positive E. coli strains (35.6 %) compared to fimA-negative E. coli strains (21.9 %, p < 0.01) and this was true for both colicin and microcin determinants (p = 0.02 and p < 0.01, respectively). In addition, an increased number of strains encoding colicin E1 were found among fimA-positive E. coli strains (p < 0.01).ConclusionsfimA-positive E. coli strains produced bacteriocins (colicins and microcins) more often compared to fimA-negative strains of similar origin. Since type 1 fimbriae of E. coli have been shown to mediate adhesion to epithelial host cells and help colonize the intestines, bacteriocin synthesis appears to be an additional feature of colonizing E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0530-5) contains supplementary material, which is available to authorized users.

An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system.

Biological containment is a genetic technique that programs dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotrophy for a substance not found in the natural environment is an ideal biological containment. Here, we constructed an Escherichia coli strain that cannot survive in the absence of the unnatural amino acid 3-iodo-L-tyrosine. This synthetic auxotrophy was achieved by conditional production of the antidote protein against the highly toxic enzyme colicin E3. An amber stop codon was inserted in the antidote gene. The translation of the antidote mRNA was controlled by a translational switch using amber-specific 3-iodo-L-tyrosine incorporation. The antidote is synthesized only when 3-iodo-L-tyrosine is present in the culture medium. The viability of this strain rapidly decreased with less than a 1 h half-life after removal of 3-iodo-L-tyrosine, suggesting that the decay of the antidote causes the host killing by activated colicin E3 in the absence of this unnatural amino acid. The contained strain grew 1.5 times more slowly than the parent strains. The escaper frequency was estimated to be 1.4 mutations (95% highest posterior density 1.1–1.8) per 105 cell divisions. This containment system can be constructed by only plasmid introduction without genome editing, suggesting that this system may be applicable to other microbes carrying toxin-antidote systems similar to that of colicin E3.

Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins

Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.

Transfer-messenger RNA and SmpB mediate bacteriostasis in Escherichia coli cells against tRNA cleavage.

RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells.

Activity of Species-specific Antibiotics Against Crohn's Disease-Associated Adherent-invasive Escherichia coli.

Crohn's disease (CD) is associated with bacterial dysbiosis that frequently includes colonization by adherent-invasive Escherichia coli (AIEC). AIEC are adept at forming biofilms and are able to invade host cells and stimulate the production of proinflammatory cytokines. The use of traditional antibiotics for the treatment of CD shows limited efficacy. In this study, we investigate the use of species-specific antibiotics termed colicins for treatment of CD-associated AIEC. Colicin activity was tested against a range of AIEC isolates growing in the planktonic and biofilm mode of growth. Colicins were also tested against AIEC bacteria associated with T84 intestinal epithelial cells and surviving inside RAW264.7 macrophages using adhesion assays and gentamicin protection assay, respectively. Uptake of colicins into eukaryotic cells was visualized using confocal microscopy. The effect of colicin treatment on the production of proinflammatory cytokine tumor necrosis factor alpha by macrophages was assessed by an enzyme-linked immunosorbent assay. Colicins show potent activity against AIEC bacteria growing as biofilms when delivered either as a purified protein or through a colicin-producing bacterial strain. In addition, colicins E1 and E9 are able to kill cell-associated and intracellular AIEC, but do not show toxicity toward macrophage cells or stimulate the production of proinflammatory cytokines. Colicin killing of intracellular bacteria occurs after entry of colicin protein into AIEC-infected macrophage compartments by actin-mediated endocytosis. Our results demonstrate the potential of colicins as highly selective probiotic therapeutics for the eradication of E. coli from the gastrointestinal tract of patients with CD.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has “chosen” highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.

Preorganization of the catalytic Zn2 +-binding site in the HNH nuclease motif—A solution study

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn2+-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn2+-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn2+-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site – existing in the wild-type enzyme but not in the W464 mutants – was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.