Biomedical subjects

R Brousseau

Publications and source records attributed to R Brousseau.

At least 37 records · Page 2Linked to original sources

PubMed1998

A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains.

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.

Animals↗

PubMed1998

Detection and confirmation of Mycoplasma pneumoniae in urogenital specimens by PCR.

Following the isolation of Mycoplasma pneumoniae from urogenital specimens (M. Goulet, R. Dular, J. G. Tully, G. Billows, and S. Kasatiya, J. Clin. Microbiol. 33:2823-2825, 1995), a study was undertaken to confirm the observations by PCR. Specific primers directed to the P1 adhesin gene of M. pneumoniae were used. A total of 300 genital specimens were tested for M. pneumoniae and Mycoplasma genitalium by culture and PCR. Of these, 15 were positive by culture and 17 were positive by PCR for M. pneumoniae. No M. genitalium was detected in any of the specimens by either method. The present study demonstrates that PCR is sensitive and rapid compared to cumbersome culture methods and can be used to detect M. pneumoniae in urogenital specimens in a routine diagnostic laboratory.

Adolescent↗

PubMed1997

Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors.

A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.

Animals↗

PubMed1997

Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering.

Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.

Bacillus thuringiensis↗

PubMed1997

A bacterial basic region leucine zipper histidine kinase regulating toluene degradation.

The two-component signal transduction pathways in bacteria use a histidine-aspartate phosphorelay circuit to mediate cellular changes in response to environmental stimuli. Here we describe a novel two-component todST system, which activates expression of the toluene degradation (tod) pathway in Pseudomonas putida F1. The todS gene is predicted to encode a sensory hybrid kinase with two unique properties--a basic region leucine zipper dimerization motif at the N terminus and a duplicated histidine kinase motif. Evidence from a synthetic peptide model suggests that TodS binds as a dimer to a pseudopalindromic sequence (5'-TGACTCA), which resembles the recognition sequence of the eukaryotic transcription factors Fos and Jun. These results provide additional evidence that bacteria and eukaryotes share common regulatory motifs. The todT gene product, a response regulator, was overproduced as a fusion protein in Escherichia coli, and the purified protein was found to bind specifically to a 6-bp palindromic DNA structure in the tod control region. The phosphorylated form of TodT appears to be the activator of tod structural genes. This is the first report of a two-component system that regulates aromatic metabolism in bacteria.

Amino Acid Sequence↗

PubMed1997

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

Amino Acid Sequence↗

PubMed1997

Effect of Bacillus thuringiensis toxins on the membrane potential of lepidopteran insect midgut cells.

To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae. In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min. It was sensitive to the K+ concentration and the oxygenation of the external medium. Addition of toxins to which L. dispar is highly [CryIA(a) and CryIA(b)] or only slightly [CryIA(c) and CryIC] sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane. CryIF, whose toxicity towards L. dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro. In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant effect. The basolateral-membrane potential was unaffected by CryIA(a) or CryIC when the toxin was applied to the basal side of the epithelium. In B. mori midguts, the apical-membrane potential was abolished by CryIA(a), to which silkworm larvae are susceptible, but CryIA(b) and CryIA(c); to which they are resistant, had no detectable effect. Although the technique discriminated between active and inactive toxins, the concentration required to produce a given effect varied much less extensively than the sensitivity of gypsy moth larvae, suggesting that additional factors influence the toxins' level of toxicity in vivo.

Animals↗

PubMed1995

Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation.

The activated 65 kDa lepidopteran-specific CryIA(a) toxin from the commercially most important strain Bacillus thuringiensis var. kurstaki HD-1 has been investigated by X-ray diffraction and for its ability to form channels in planar lipid bilayers. Its three-dimensional structure has been determined by a multiple isomorphous replacement method and refined at 2.25 A resolution to an R-factor of 0.168 for data with I > 2 delta (I). The toxin is made of three distinct domains. The N-terminal domain is a bundle of eight alpha-helices with the central, relatively hydrophobic helix surrounded by amphipathic helices. The middle and C-terminal domains contain mostly beta-sheets. Comparison with the structure of CryIIIA, a coleopteran-specific toxin, shows that although the fold of these two proteins is similar, there are significant structural differences within domain II. This finding supports the conclusions from genetic studies that domain II is involved in recognition and binding to cell surface receptors. The distribution of electrostatic potential on the surface of the molecule is non-uniform and identifies one side of the alpha-helical domain as negatively charged. The predominance of arginine residues as basic residues ensures that the observed positive charge distribution is also maintained in the highly alkaline environment found in the lepidopteran midgut. Structurally important salt bridges that are conserved across Cry sequences were identified and their possible role in toxin action was postulated. In planar lipid bilayers, CryIA(a) forms cation-selective channels, whose conductance is significantly smaller than that reported for CryIIIA but similar to those of other Cry toxins.

Bacillus thuringiensis↗

PubMed1995

The CryIA(c) receptor purified from Manduca sexta displays multiple specificities.

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

Acetylgalactosamine↗

PubMed1995

Codon-based combinatorial alanine scanning site-directed mutagenesis: design, implementation, and polymerase chain reaction screening.

Combinatorial alanine scanning mutagenesis is a powerful tool for the exploration of protein structure-function relationships. Unfortunately, combinatorial alanine replacement of multiple residues using standard site-directed mutagenesis is restricted to a subset of amino acids. To circumvent this limitation, an efficient procedure for combinatorial site-specific replacement by alanine of any residue in a given protein sequence has been established. The method, which involves simple procedures and commonly used materials, is based upon the use of codon-based mutagenesis. A defined ratio of alanine to wild-type codon was introduced at each predetermined triplet using the "column-splitting" technique during oligonucleotide synthesis. High-throughout genetic screening of mutant libraries was facilitated by the incorporation of diagnostic restriction sites at targeted codons followed by a PCR-based screening procedure. The method was tested on a set of 13 residues located at the interface between the variable domains of a Fab fragment of an antibody. The occurrence of alanine substitution was found to be comparable to the statistically predicted distribution.

Alanine↗

PubMed1995

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Amino Acid Sequence↗

PubMed1995

The SsoII and NlaX DNA methyltransferases: overproduction and functional analysis.

Overproduction of the NlaX DNA methyltransferase (M.NlaX) in an Escherichia coli host conferred resistance to SsoII restriction endonuclease (R.SsoII) digestion. This suggested an overlap of sequence specificity between M.NlaX and M.SsoII, the latter of which modifies the internal cytosine of the target sequence 5'-CCNGG-3'. A variant of M.NlaX (M.Sso/Nla), containing an N-terminal extension from M.SsoII, was also enzymatically active. Using deletion analysis, the N-terminal 71 amino-acid residues of M.SsoII were shown to be essential for modification activity.

Amino Acid Sequence↗

PubMed1995

Kinetics of Bacillus thuringiensis toxin binding with brush border membrane vesicles from susceptible and resistant larvae of Plutella xylostella.

An optical biosensor technology based on surface plasmon resonance was used to determine the kinetic rate constants for interactions between the CryIA(c) toxin from Bacillus thuringiensis and brush border membrane vesicles purified from susceptible and resistant larvae of diamondback moth (Plutella xylostella). CryIA(c) association and dissociation rate constants for vesicles from susceptible larvae were determined to be 4.5 x 10(3) M-1 s-1 and 3.2 x 10(-5) s-1, respectively, resulting in a calculated affinity constant of 7 nM. CryIE toxin did not kill susceptible or resistant larvae and did not bind to brush border vesicles. Contrary to expectations based on previous studies of binding in resistant P. xylostella, the binding kinetics for CryIA(c) did not differ significantly between susceptible larvae and those that were resistant to CryIA(c). Determination of the number of CryIA(c) receptors revealed an approximately 3-fold decrease in total CryIA(c) receptor numbers for resistant vesicles. These results suggest that factors other than binding may be altered in our resistant diamondback moth strain. They also support the view that binding is not sufficient for toxicity.

Animals↗

PubMed1995

Identification of a membrane protein and a truncated LysR-type regulator associated with the toluene degradation pathway in Pseudomonas putida F1.

A 3 kb DNA region upstream of the toluene degradation (tod) genes, todFC1C2BADEGIH, in Pseudomonas putida F1 (PpF1) was sequenced. Two divergently arranged open reading frames, todR and todX, were identified. A toluene-inducible promoter was localized in front of todX, and the transcription start point was mapped. This promoter is probably responsible for the expression of all tod structural genes. TodX was found to be a membrane protein. Its predicted amino acid sequence (453 residues; M(r) 48,265) exhibits considerable similarity with the FadL protein of Escherichia coli, an outer membrane protein required for binding and transport of long-chain fatty acids. An apparent function of TodX is likely to be involved in facilitating the delivery of exogenous toluene inside the PpF1 cells. The sequence of TodR (100 residues) exhibits extensive homology with the DNA-binding domain of transcriptional activators of the LysR family, but todR was found to have a negligible role in tod gene regulation.

Amino Acid Sequence↗

PubMed1994

Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1.

The todFC1C2BADE gene cluster in Pseudomonas putida F1 encodes enzymes for the first four steps of toluene degradation, leading to the formation of 2-hydroxypenta-2,4-dienoate (HPD). Here, we report the nucleotide (nt) sequence and expression of the remaining three genes of the tod pathway, downstream from todE and arranged in the order, todGIH. The deduced amino acid (aa) sequences of TodG [HPD hydratase (268 aa)], TodH [4-hydroxy-2-oxovalerate (HO) aldolase (352 aa)] and TodI [acylating aldehyde (AA) dehydrogenase (316 aa)] are compared with the isofunctional proteins present in the meta-cleavage pathways of other bacteria. New sequence motifs are identified. The highly conserved TodH and TodI sequences are potentially useful DNA probes for biomonitoring purposes.

Aldehyde Oxidoreductases↗

PubMed1994

Stable immobilization of lipid vesicles for kinetic studies using surface plasmon resonance.

In order to study the kinetics of binding between membrane vesicle surface receptors to the lepidopteran insecticidal toxins from Bacillus thuringiensis using surface plasmon resonance, we have developed a technique to immobilize membrane vesicles purified from the brush border of dissected guts from the lepidopteran insect pest Choristoneura fumiferana. Two methods using immobilized immunoglobulins against either avidin or biotin were successful in achieving stable immobilization of the vesicles (> 1.5 h). Specificity of the immobilized receptors exposed on the vesicle surface was demonstrated, in part, by the inability of bovine serum albumin to bind to the immobilized brush border membrane vesicles. Homologous and heterologous competition experiments further demonstrated specific binding of trypsin-activated CryIA(c) toxin to the cell-surface receptors on the vesicles. Kinetic rate constants for activated cryIA(b) toxin binding to brush border vesicles were determined, revealing the presence of a high-affinity receptor on the surface of the immobilized brush border membrane vesicles.

Animals↗

PubMed1994

The NlaIV restriction and modification genes of Neisseria lactamica are flanked by leucine biosynthesis genes.

The genes encoding the Neisseria lactamica restriction endonuclease IV (R.NlaIV) and its cognate DNA methyltransferase (M.NlaIV), both of which recognize the sequence GGNNCC, have been cloned in Escherichia coli and overexpressed using the T7 polymerase/promoter system. Analysis of a sequenced 3.58 kb fragment established the gene order, leuD-M.NlaIV-R.NlaIV-leuB. The predicted primary sequence of M.NlaIV (423 amino acids) shows the highest degree of identity to a pair of cytosine-specific methyltransferases, M.BanI (44.9%) and M.HgiCI (44.3%), which recognize the sequence GGYRCC (Y, pyrimidines; R, purines). In contrast, the R.NlaIV protein sequence (243 amino acids) is unique in the existing data-base, a situation that holds for most endonucleases. Flanking the NlaIV modification and restriction genes are homologues of the leuD and leuB genes of enteric bacteria, which code for enzymes in the leucine biosynthesis pathway. This gene context implies a possible new mode of gene regulation for the RM.NlaIV system, which would involve a mechanism similar to the recently discovered leucine/Lrp regulon in E. coli.

Amino Acid Sequence↗

PubMed1994

Specific targeting of antisense oligonucleotides to neutrophils.

The ability of three different hydrophobic ligands (cholic acid, cholesterol, and the tetrapeptide fMLFY) to increase the uptake of an antisense (anti-actin) oligomer into neutrophils was analyzed. In agreement with the literature (Boutorin et al., 1989; Letsinger et al., 1989), we found that cholic acid and cholesterol conjugates greatly enhance the uptake of anti-actin oligomer. When fMLFY is the ligand, the cellular uptake is much less than that of anti-actin oligomer alone, but the biological consequences are much more significant. Our results are consistent with the hypothesis that the fMLFY conjugate of the anti-actin oligomer is internalized via a different route, and reaches its target site most efficiently.

Actins↗