Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters.

Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.     

Cyanotoxins in small artificial dams in Kenya utilised for cage fish farming – a threat to local people?

Nine small artificial dams located in different climatic regions of Kenya were studied. The local communities use the stored water for various purposes, such as irrigation, domestic use, watering of livestock and cage fish farming. Such intense use is commonly accompanied by eutrophication, including fast growth of cyanobacteria, which at times produce cyanotoxins threatening human and animal life. We studied the pelagic community, analysed abiotic variables and identified microcystins by means of high performance liquid chromatography and ELISA kits at monthly intervals over a period of one year. Mass spectrometry (MALDI-TOF MS) was used to identify structural variants of microcystins by their protonated masses (M + H). Three dams contained microcystins, with the highly toxic Microcystin-LR being identified as the most prominent substance. Cell content of the toxin varied from 7.2 to 686.7 fg cell^?1. Basic limnological variables that indicate the probability of toxin presence were also recorded. Non-parametric Mann–Whitney U-test revealed significant differences in soluble reactive phosphorous, nitrate-N, water depth, total hardness and post-Nauplii stages sampled between toxin-producing and non-toxin-producing dams. Although most of the samples did not contain high amounts of cyanobacteria, the cyanotoxin-problem was evident, suggesting the need for regular cyanotoxin monitoring programs.     

Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9(s-s)) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9(s-s), we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9(AF)) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.     

Aflatoxicosis in rabbits: Effectiveness of Egyptian raw bentonite in prevention or diminution the detrimental effects of naturally aflatoxin contaminated diets

Thirty five females and 15 males of New Zealand White mature rabbits about 6 months of age, were assigned to 1–5 dietary treatments (7 does+3 bucks for each): uncontaminated control diet, naturally aflatoxin contaminated diet without or with 1,2 and 3% bentonite. Rabbit fed with the aflatoxin-diet had a decreased (P<0.01 or 0.05) physical semen characteristics of bucks and a reproductive performance traits of does. The values of conception rate (%), gestation length (days), litter size (n) and litter weights (g) at birth and viability (%) of litters of doe rabbits, fed with the aflatoxin-diet, recorded, respectively: 64.5; 31.0; 4.4; 275.0 and 57.1 versus 85.6; 30.3; 7.9; 508.0; and 100 for those fed with the uncontaminated diet. Addition of bentonite to the aflatoxin contaminated diet improved in general the physical semen characteristics of buck and reproductive performance traits of doe rabbits. The results of the study demonstrate that adding 1% of Egyptian raw bentonite to the naturally aflatoxin contaminated rabbit diets can provide an effective, cheap and safe practical technique for preventing the aflatoxicosis in mature rabbits.     

Energy-dependent Immunity Protein Release during tol-dependent Nuclease Colicin Translocation

Nuclease colicins bind their target receptor in the outer membrane of sensitive cells in the form of a high affinity complex with their cognate immunity proteins. Upon cell entry the immunity protein is lost from the complex by means that are poorly understood. We have developed a sensitive fluorescence assay that has enabled us to study the molecular requirements for immunity protein release. Nuclease colicins use members of the tol operon for their translocation across the outer membrane. We have demonstrated that the amino-terminal 80 residues of the colicin E9 molecule, which is the region that interacts with TolB, are essential for immunity protein release. Using tol deletion strains we analyzed the cellular components necessary for immunity protein release and found that in addition to a requirement for tolB, the tolA deletion strain was most affected. Complementation studies showed that the mutation H22A, within the transmembrane segment of TolA, abolishes immunity protein release. Investigation of the energy requirements demonstrated that the proton motive force of the cytoplasmic membrane is critical. Taken together these results demonstrate for the first time a clear energy requirement for the uptake of a nuclease colicin complex and suggest that energy transduced from the cytoplasmic membrane to the outer membrane by TolA could be the driving force for immunity protein release and concomitant translocation of the nuclease domain.Membrane translocation is a formidable challenge for folded proteins. Eukaryotes have an array of dedicated translocation machineries to accomplish this feat, for example during mitochondrial import of cytosolic precursor proteins for which it has recently become clear that there is a surprising diversity in targeting signals, import routes, and translocation complexes (1, 2). It is now widely accepted that the mitochondrial genome originated from within the (eu)bacterial domain of life, so it should perhaps not come as a surprise that certain features of mitochondrial import have evolved from these ancestors.Gram-negative bacteria possess two membranes to protect them from the external world, separated by a layer of peptidoglycan and the periplasmic space. Their outer membrane, with its asymmetrical composition of lipopolysaccharide (LPS)² and phospholipids, forms an impressive barrier to most substances with the exception of small hydrophilic nutrients that can diffuse through the resident porins (3). Processes that require an energy input at the outer membrane, such as iron siderophore uptake, therefore often rely on energy generated by ion gradients at the cytoplasmic membrane (4). Energy-transducing systems such as the ton and tol systems in Escherichia coli harvest energy generated at the cytoplasmic membrane and transduce it to the outer membrane. These two systems have a number of features in common, and cross-complementation between the two systems has been observed (5).The energy transducing capacity of the ton system is somewhat better defined and is accomplished by three proteins: the cytoplasmic membrane proteins ExbB and ExbD, which form a heteromultimeric complex that interacts with TonB (4). As a result, TonB undergoes a conformational change in response to the PMF of the cytoplasmic membrane, which allows it to traverse the periplasm and make contact with nutrient-loaded outer membrane receptors, thereby facilitating active import (6). The homology between ExbB/D, TolQ/R, and the PMF-responsive flagellar motor proteins MotA and MotB is well established, and the cumulative evidence now suggests that they act as energy-harvesting complexes (7–9). Evidence of an evolutionary relationship between TolA and TonB comes from work demonstrating structural similarities between the Pseudomonas aeruginosa TolAIII globular domain and the carboxyl-terminal domain of E. coli TonB despite the very low sequence conservation (10). The activities of TonB and TolA are also critically dependent on a conserved SHLS motif in their transmembrane region, the mutation of which affects the interaction with their respective energy-harvesting complexes (11, 12). The cellular function of the tol system in E. coli is, however, less clear. It is thought that the Tol proteins play a role in maintaining cell envelope integrity through a network of interactions spanning the cytoplasmic membrane, periplasm, and outer membrane (13).Both energy-transducing systems have been parasitized by the colicins, plasmid-encoded antibacterial proteins produced by E. coli, and phages for their translocation into the cell, but the energy requirements for these processes are not unequivocal (14). Group A colicins use the tol system and group B colicins the ton system in a process whereby interactions of their amino-terminal translocation domains with the Tol or Ton proteins in the periplasm ultimately lead to the entry of their carboxyl-terminal cytotoxic domain into the cell (15, 16). In common with most colicins, the DNase-type colicin E9 consists of three functional domains: the killing activity is contained in its carboxyl-terminal DNase domain; the central section contains the receptor-binding domain, which binds the vitamin B₁₂ receptor, BtuB, in the outer membrane; and the amino-terminal translocation domain is needed for the entry of the cytotoxic domain into the target cell. The first 83 residues of this translocation domain, commonly referred to as the NDR, contain the OmpF and TolB binding sites (17, 18). Upon synthesis colicin E9 forms a high affinity interaction with its cognate immunity protein, Im9, also encoded by the colicin operon. This heterodimeric complex formation protects colicin-producing cells against DNA damage and potential suicide prior to release of the complex in the environment. The nature of the complex formation between colicin E9 and Im9 and other colicin-immunity complexes has been well characterized, and in the case of colicin E9-Im9 the interaction is strong, as reflected by its dissociation constant on the order of 10⁻¹⁴ m under physiological conditions (19). Despite the high avidity of this interaction, the DNase domain of colicin E9 appears to have only a marginally stabilizing effect on Im9 (20).Currently much progress is being made to unravel the early events that take place after receptor binding, where it has been shown that the colicin E9 NDR enters the periplasm through the OmpF lumen where it interacts with TolB, possibly displacing it from its interaction with Pal (18, 21–24). It was also recently demonstrated that the receptor binding and translocation domains remain in contact with their binding partners in the outer membrane and the periplasm, respectively, when the DNase domain gains access to the cytoplasm (25). In contrast, the molecular mechanisms that govern the loss of the immunity protein from the colicin complex and the cell entry of the DNase domain are less well documented. Because of the strength of the interaction between the colicin and its cognate immunity protein, it has been proposed that removal of the immunity protein from the complex would require a cellular energy source. One recent report investigating immunity protein loss from the colicin E2-Im2 complex qualitatively concluded that receptor binding alone does not lead to immunity protein release and that a functional tol translocation complex is required to establish immunity protein release (26).Here we have presented data that for the first time demonstrate a role for the individual Tol proteins and address the issue of energy requirements for immunity protein release. We observed, by using a previously described disulfide-“locked” colicin construct and domain deletion mutants thereof, that entry of the amino-terminal 80 residues of the colicin translocation domain and its interaction with TolB are essential factors for immunity protein release. We have also demonstrated a crucial role for TolA and its transmembrane region in this process, showing that immunity protein release from the colicin complex is an energy-dependent process governed by the cytoplasmic membrane PMF. Finally we have provided a rationale for how an energized Tol system might lead to immunity protein loss and concomitant colicin uptake in sensitive cells.