Microarray analysis of Escherichia coli strains from interstitial beach waters of Lake Huron (Canada).

DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes.

Development of a new integrated diagnostic test for identification and characterization of pathogens.

Animal diseases directly cause multi-million dollar losses world-wide. Therefore a rapid, highly specific, cost-effective diagnostic test for detecting a large set of bacterial virulence and antimicrobial resistance genes simultaneously is necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group, proposes developing a powerful molecular tool (DNA microarray) to detect a broad range of infectious agents, their endogenous main virulence factors and antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide microarray capable of detecting the presence or absence of 169 Escherichia coli virulence genes or virulence marker genes as well as their variants, in addition to 30 principal antimicrobial resistance genes previously characterized in E. coli strains was developed by our group. This microarray was validated with a large collection of well characterized pathogenic and reference E. coli strains. Moreover, we are developing a new powerful clinical diagnostic microarray tool, to identify pathogenic bacteria of veterinary interest. The commercialization of this assay would allow same day diagnosis of infectious agents and their antibiotic resistance resulting in early treatment. In addition, this technology is also applicable to microbial quality control of food and water.

Implication of tyrosine kinase receptor and steel factor in cell density-dependent growth in cervical cancers and leukemias.

Cell-cell interaction is important in the expansion of leukemic cells and of solid tumors. Steel factor (SF) or Kit ligand is produced as a membrane-bound form (mSF) and a soluble form. Because both primary gynecological tumors and primary leukemic cells from patients with acute myeloblastic leukemia (AML) have been shown to coexpress c-Kit and SF, we addressed the question of whether mSF could contribute to cell interaction in these cancers. Investigations on primary cervical carcinomas have been hindered by the fact that the cells do not grow in culture. We report herein the establishment of two cervical carcinoma cell lines, CALO and INBL, that reproduce the pattern of SF/c-Kit expression observed in primary tumor samples. In addition, these cells exhibit marked density-dependent growth much in the same way as AML blasts. Using an antisense strategy with phosphorothioate-modified oligonucleotides that specifically target SF without affecting other surface markers, we provide direct evidence for a role of mSF and c-Kit in cell interaction and cell survival in these gynecological tumor cell lines as well as in primary AML blasts. Finally, our study defines the importance of juxtacrine stimulation, which may be as important, if not more, than autocrine stimulation in cancers.

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.

Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells.

The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

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.

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Construction of a family of universal expression plasmid vectors.

A family of universal expression vectors based on the pUC8 and pUC9 plasmids has been constructed. These vectors cover all three possible reading frames in both directions, allowing any synthetic DNA, genomic DNA or cDNA to be expressed under control of the lac promoter. The four new vectors retain the useful features of the pUC plasmids, including the blue to white color change on X-gal plates indicating the presence of an insert. This family of expression vectors is expected to be quite useful in allowing direct immunological screening of cDNA or genomic DNA banks.

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli.

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.

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.

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.

Synthesis of a human insulin gene. VI. Expression of the synthetic proinsulin gene in yeast.

The construction of plasmid vectors for the controlled expression of a synthetic human proinsulin gene in the yeast Saccharomyces cerevisiae is described. Attempts to express the proinsulin gene using the yeast ADH1 promoter alone did not yield detectable levels of proinsulin. Successful expression was achieved when the proinsulin gene was fused with the promoter and protein leader sequence of the GAL1 gene (coding for yeast galactokinase) in the yeast-Escherichia coli plasmid vector pYT7810. Two different-length leader sequences were employed; the longer leader (about 280 amino acids, fusion plasmid pPS13) gave about five times greater expression than the shorter leader fusion (30 amino acids, plasmid pPS5). Both fusions gave soluble protein products, and the proinsulin could be cleaved by cyanogen bromide treatment from the leader polypeptide. Proinsulin was detected by radioimmunoassay for human C-peptide only in cells induced with galactose, and was not detected in the gene fusions that were out of phase with the GAL1 leader sequence. Methods of improving the level of expression of the proinsulin gene in yeast using this system are discussed.

Synthesis of a human insulin gene. V. Enzymatic assembly, cloning and characterization of the human proinsulin DNA.

To form a 258-bp sequence coding for human proinsulin, 41 synthetic deoxyribo-oligonucleotide fragments of 11 to 15 nucleotides in length were assembled by enzymatic methods. The coding sequence is preceded by ATG and following by TGA for translation start and stop signals, and terminated in an EcoRI and a BamHI recognition sequence. The complete synthetic sequence was ligated to a plasmid and cloned in Escherichia coli. The cloned DNA was shown to have the correct human proinsulin coding sequence.

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.

We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli. The plasmids include the E. coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx. 590 or 450 amino acids. The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein. Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames. We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E. coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin. The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.

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.

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.

Bacillus thuringiensis insecticidal crystal toxins: gene structure and mode of action.

Thanks to the techniques of recombinant DNA, there is now abundant sequence information on several endotoxin genes of Bacillus thuringiensis. The task of correlating this sequence information with the economically important aspects of the toxins such as insect specificity, LD(50) and speed of kill is now under worldwide investigation. Progress has also been made on understanding the mechanism of action of the toxins and on identifying the parts of the protoxin which are important in toxicity. Taken together, the mechanistic data and the sequence information allow the first attempts at rational design of mutant endotoxin genes and greatly facilitate the transfer of those genes to other organisms such as plants. More information is still needed, however, as to the nature of the binding site of the toxin and on the three-dimensional structure of the activated toxins.

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera.

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD(50)]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD(50) of HD-1 with the FFD(50) expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.

The heliothis virescens 170 kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis Cry1A delta-endotoxin binding and pore formation.

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.