A functional protein pore with a "retro" transmembrane domain.
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Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif. (+info)
Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish.
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We produced monoclonal antibodies (MAbs) to the extracellular proteins of Listeria monocytogenes EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from Listeria ivanovii. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of L. monocytogenes. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from L. ivanovii and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between L. monocytogenes PC-PLC and L. ivanovii sphingomyelinase. The reactions of the MAbs with catfish isolates of L. monocytogenes suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of L. monocytogenes in food and clinical samples and for detecting L. ivanovii in veterinary clinical samples. (+info)
Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.
(35/3113)
Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C. (+info)
Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.
(36/3113)
Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297. (+info)
A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxII antigen for diagnosis of pleuropneumonia in pig herds.
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For the surveillance of pig herds infected with porcine pleuropneumonia, an enzyme-linked immunosorbent assay (ELISA) using the recombinant Actinobacillus pleuropneumoniae ApxII protein as species- but not serotype-specific antigen was developed. Using this ELISA, 243 of 400 animals from 22 A. pleuropneumoniae-infected herds were classified as seropositive. (+info)
Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.
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Genes cry26Aal and cry28Aal were cloned from Bacillus thuringiensis ssp. finitimus strain B-1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aal gene product included seven residues determined to be an N-terminal part of a chymotrypsin-treated delta-endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore-associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aal in both types of crystal bodies is synthesized under the control of one and the same genomic locus. (+info)
Further evolution of a strain of Staphylococcus aureus in vivo: evidence for significant inactivation of flucloxacillin by penicillinase.
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A strain of Staphylococcus aureus (no. FAR4) has been isolated at intervals, for 32 months, from the sputum of a patient with cystic fibrosis of the lung. Changes in the properties of isolates of this strain over the first 18 months have been reported previously (Lacey et al., 1973 and 1974). During the last 14 months (May 1973 to July 1974), further evolution has occurred to produce a total of 31 distinct phenotypes. Recent changes are as follows. 1. The ability of isolates to produce penicillinase in vitro was closely correlated with flucloxacillin therapy. Inactivation of flucloxacillin by penicillinase was demonstrated by diffusion testing (but not MIC determination) in vitro and may have occurred to a significant extent in vivo. 2. Lincomycin-resistant mutants slowly disappeared from the sputum after the termination of clindamycin therapy. 3. All of the recent isolates were resistant to erythromycin, possibly because of the linkage of the genes coding for erythromycin resistance with those coding for the production of delta-haemolysin; delta-haemolysin may be an important "virulence factor". (+info)
A novel protein of Erysipelothrix rhusiopathiae that confers haemolytic activity on Escherichia coli.
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Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, produces a haemolysin. A recombinant plasmid, pHLY, conferring haemolytic activity on Escherichia coli was isolated from a genomic library of Ery. rhusiopathiae strain Tama-96. This plasmid was stable in RecA- E. coli, but unstable in a RecA+ strain. A spontaneous deletion plasmid, pMini-HLY, also conferring haemolytic activity was derived from pHLY. Two ORFs were detected in pHLY. Analysis of pMini-HLY and other deletion clones established that ORF2 was associated with haemolytic activity. The sequence of ORF1 was highly homologous to those of transposases in the IS30 family. The deletion which generated pMini-HLY was between two short direct repeat (DR) sequences flanking the ORF1 sequence, and there were inverted repeat sequences inside the two DR sequences, suggesting an insertion element. No sequence homology to the deduced amino acid sequence of ORF2 was detected in the databases, but its sequence was characteristic of a surface lipoprotein. Western blot analysis, using antiserum against the 16 kDa protein produced from ORF2, found the protein to be commonly distributed in all Erysipelothrix species. (+info)