何谓肠出血性大肠杆菌O104：H4及防治

所谓肠出血性大肠杆菌0104：H4（enterohem．orrhagicE．coli,0104：H4）,是由德国分离的一种新菌珠0104：H4型大肠杆菌,含有志贺毒素2（vtx2a）的基因,没有（enterohemorrhagicEscherichiacoli,EHEC）溶血素、志贺毒素1、肠致病性大肠埃希菌（enteropathogenicEscherichiacoli）基因毒力岛基因;但含有肠集聚性粘附大肠杆菌（enteroaggragtiveEscherichiacoli）（第5类致泻性大肠杆菌）毒力质粒上的3个基因：aatA、aggR、aap。显示其为EHEC家族的一个新成员。     

The rnh gene is essential for growth of Escherichia coli.

We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli. A strain was made with two copies of the rnh gene by lysogenizing an E. coli strain with a lambda phage bearing a copy of the rnh gene. Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene. In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene. Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death. An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained. The technique described is probably generally applicable for assessing the requirement for other E. coli genes.     

Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells.

Enteropathogenic Escherichia coli (EPEC), a major cause of pediatric diarrhea, adheres to epithelial cells and activates host cell signal transduction pathways. We have identified five proteins that are secreted by EPEC and show that this secretion process is critical for triggering signal transduction events in epithelial cells. Protein secretion occurs via two pathways: one secretes a 110-kDa protein and the other mediates export of the four remaining proteins. Secretion of all five proteins was regulated by temperature and the perA locus, two factors which regulate expression of other known EPEC virulence factors. Amino-terminal sequence analysis of the secreted polypeptides identified one protein (37 kDa) as the product of the eaeB gene, a genetic locus previously shown to be necessary for signal transduction. A second protein (39 kDa) showed significant homology with glyceraldehyde-3-phosphate dehydrogenase, while the other three proteins (110, 40, and 25 kDa) were unique. The secreted proteins associated with epithelial cells, and EaeB became resistant to protease digestion upon association, suggesting that intimate interactions are required for transducing signals.     

ATP is essential for protein translocation into Escherichia coli membrane vesicles.

The energy requirement for translocation of alkaline phosphatase and the outer membrane protein OmpA into Escherichia coli membrane vesicles was studied under conditions that permit posttranslational translocation and, hence, prior removal of various components necessary for protein synthesis. Translocation could be supported by an ATP-generating system or, less well, by the protonmotive force generated by D-lactate oxidation; the latter might act by generating ATP from residual bound nucleotides. However, when protonmotive force inhibitors were used or when ATP was further depleted by E. coli glycerol kinase, D-lactate no longer supported the translocation. Furthermore, ATP could still support protein translocation in the presence of proton uncouplers or with membranes defective in the F1 fraction of the H+-ATPase. We conclude that ATP is required for protein translocation in this posttranslational system (and probably also in cotranslational translocation); the protonmotive force may contribute but does not appear to be essential.     

Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures

Trehalose accumulates dramatically in microorganisms during heat shock and osmotic stress and helps protect cells against thermal injury and oxygen radicals. Here we demonstrate an important role of this sugar in cold-adaptation of bacteria. A mutant Escherichia coli strain unable to produce trehalose died much faster than the wild type at 4 degrees C. Transformation of the mutant with the otsA/otsB genes, responsible for trehalose synthesis, restored trehalose content and cell viability at 4 degrees C. After temperature downshift from 37 degrees C to 16 degrees C ("cold shock"), trehalose levels in wild-type cells increased up to 8-fold. Although this accumulation of trehalose did not influence growth at 16 degrees C, it enhanced cell viability when the temperature fell further to 4 degrees C. Before the trehalose build-up, levels of mRNA encoding OtsA/OtsB increased markedly. This induction required the sigma factor, RpoS, but was independent of the major cold-shock protein, CspA. otsA/B mRNA was much more stable at 16 degrees C than at 37 degrees C and contained a "downstream box," characteristic of cold-inducible mRNAs. Thus, otsA/otsB induction and trehalose synthesis are activated during cold shock (as well as during heat shock) and play an important role in resistance of E. coli (and probably other organisms) to low temperatures.     

Multiple joined genes prevent product degradation in Escherichia coli.

A method is described that allows the expression of a stable human proinsulin product in Escherichia coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of beta-galactosidase. In the unfused system, the proinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacterial Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, when multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by cyanogen bromide treatment.