Gene-based identification of bacterial colonies with an electric chip

A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.     

Anaerobiosis and low specific growth rates enhance hemolysin BL production by <Emphasis Type="Italic">Bacillus cereus</Emphasis> F4430/73

Bacillus cereus F4430/73 produced the highest levels of hemolysin BL (HBL) when grown under anaerobiosis in MOD medium. Anaerobic cells grown in a chemostat at low specific growth rate (0.1–0.2 h^–1) expressed up to sevenfold more HBL than did cells held at a faster growth rate. At 0.2 h^–1, the presence of 90 mM glucose resulted in inhibition of HBL production. Glucose was found to repress HBL induction at the mRNA level, indicating the potential involvement of catabolite repression in the regulation of HBL. Based on these data, it is suggested that growth rate could be an effector of catabolite regulation of HBL.     

酶联免疫吸附试验检测A型产气荚膜梭菌肠毒素

本实验建立了双抗体夹心ＥＬＩＳＡ检测产气荚膜梭菌肠毒素的实验体系。以家兔单特异ＩｇＧ包被酶标板,辣根过氧化物酶标记的绵羊ＩｇＧ作为指示物,可检测出１．２５ｎｇ／ｍｌ（０．１２５ｎｇ）的产气荚膜梭菌肠毒素,线性范围大,重复性及稳定性好,对培养上清及粪便滤液检查无非特异反应。是产气荚膜梭菌食物中毒实验室诊断的一种快速可靠的检测方法。     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

大肠杆菌肠毒素的基因融合及其免疫原性的研究

应用重组基因工程技术,将热敏肠毒素Ｂ亚基（ＬＴ－Ｂ）基因与含有部分前体的耐热肠毒素（ｐｒｏ－ＳＴ）基因融合在一起,构建了表达ＬＴ－Ｂ／ｐｒｏ－ＳＴ融合蛋白的重组质粒．该融合蛋白不仅保持结合神经节苷脂（ＧＭ１）的能力,而且具有热敏肠毒素和耐热肠毒素的抗原性．乳鼠实验证明,融合蛋白虽然含有野生耐热肠毒素,但不具耐热肠毒素的生物毒性．腹腔免疫和鼻饲免疫均能激发产生抗ＥＴＥＣ两种肠毒素的抗体．     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)₂, similar to the transposition intermediates described for a number of IS elements.     

Autoradiographic demonstration of specific binding sites for E. coli enterotoxin in various epithelia of the North American opossum

Summary In the North American opossum, heat-stable specific binding sites for E. coli enterotoxin are observed (i) in epithelial cells lining the small intestine, colon, gall bladder, cystic duct, common bile duct and trachea, and (ii) in epithelial cells forming the duodenal (Brunner's) glands, liver, kidneys (metanephros, mesonephros) and testis, as demonstrated by autoradiography. Enterotoxin-specific binding sites in the intestinal tract are only found in intestinal epithelial cells with the highest concentration in the microvillus border. Enterotoxin-specific binding sites also occur in epithelial cells comprising the secretory tubules and ducts of the duodenal glands. In the kidneys (metanephros and mesonephros), enterotoxin-specific binding sites are confirmed primarily to the proximal tubules, whereas in the testis they are localized in seminiferous tubules. In the liver, enterotoxin-specific binding sites are confined primarily to hepatocytes. E. coli enterotoxin caused a 7-fold increase of cGMP in the liver and a 30-fold increase in the duodenal glands. The liver responded in about half of the animals studied, whereas the duodenal glands gave a consistent response in each case. Likewise, the duodenal glands consistently showed strong labelling for ¹²⁵I-enterotoxin, whereas receptor labelling of hepatocytes was inconsistent in nearly half the incubations and corresponds to the observed cGMP measurements.Supported by a Weldon Springs Grant, University of Missouri and by funds from the Medical Research Service, Department of Veteran's Affairs     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli

We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.     

Mode of action of heat-stable Escherichia coli enterotoxin Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have exmined the tissue specificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP oncentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase in liver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.