重金属对大肠杆菌生长繁殖的影响研究

汞、镉、铅、砷、铬等是工业废物中常见的重金属。本论文采用琼脂扩散法,寻求对大肠杆生长繁殖无影响的重金属界点浓度,以此为大肠杆菌的重金属的抗性、浓缩研究及大肠杆菌的驯化及其生物治理应用等方面的深入探讨提供实验依据。     

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.     

应用微量量热法研究大肠杆菌在甘草液中的代谢作用

应用微量量热法测定了大肠杆菌在不同浓度的甘草液中生长的热功率 -时间曲线 ,应用非限制性条件下微生物生长的模型进行数学处理 ,得出生长速率常数 ,并用计算机拟合出非线性方程 ,确定了大肠杆菌在甘草液中的最佳生长浓度和临界生长浓度。     

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.     

含铜铁素体抗菌不锈钢的杀菌机理的初步研究

通过杀菌性能、蛋白质溶出、添加半胱氨酸等实验，并利用生物透射电镜技术，对含铜铁素体抗菌不锈钢对大肠杆菌的杀灭作用机理进行了初步研究。结果表明抗菌不锈钢对大肠杆菌的杀菌率达到99．99％。进一步的研究发现，大肠杆菌与抗菌不锈钢作用后，其细胞壁和细胞膜破裂，细胞膜脂质和含-SH等还原基团酶发生氧化，细胞内出现了明显的内含物溢出现象。随着与抗菌不锈钢作用时间的延长，细菌细胞内的蛋白质漏出量不断增加，最终致使细菌死亡。     

The yin-yang driving urinary tract infection and how proteomics can enhance research,diagnostics, and treatment

Bacterial urinary tract infections (UTIs) afflict millions of people worldwide both in the community and the hospital setting. The onset, duration, and severity of infection depend on the characteristics of the invading pathogen (yin), as well as the immune response elicited by the infected individual (yang). Uropathogenic Escherichia coli (UPEC) account for the majority of UTIs, and extensive investigations by many scientific groups have elucidated an elaborate pathogenic UPEC life cycle, involving the occupation of extracellular and intracellular niches and the expression of an arsenal of virulence factors that facilitate niche occupation. This review will summarize the current knowledge on UPEC pathogenesis; the host immune responses elicited to combat infection; and it will describe proteomics approaches used to understand UPEC pathogenesis, as well as drive diagnostics and treatment options. Finally, new strategies are highlighted that could be applied toward furthering our knowledge regarding host-bacterial interactions during UTI.     

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Escherichia coli can produce H₂ from glucose via formate hydrogen lyase (FHL). In order to improve the H₂ production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H₂ production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H₂ production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H₂ yield; 1.80 mol/mol glucose under high H₂ pressure or 2.11 mol/mol glucose under reduced H₂ pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdABdirected most of the carbons from glucose to the glycolytic pathway leading to H₂ production by FHL, not to the pentose phosphate pathway.     

The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases

Human mucus proteinase inhibitor (MPI) consists of 107 aminoacids arranged in two domains showing high homology to eachother. This protein is an inhibitor of different serine proteinasesincluding trypsin, chymotrypsin, leukocyte elastase and cathepsinG. On the basis of sequence comparisons it has been suggestedthat the first domain inhibits trypsin, whereas the second onewas thought to be active against chymotrypsin and elastase.To prove the location of the different inhibitory activitiesgene fragments for both domains have been cloned separatelyand expressed in Escherichia coli. Inhibition assays with theisolated recombinant domains showed that the second domain isactive against chymotrypsin, neutrophil elastase and trypsin,whereas for the first domain only a weak activity against trypsincould be detected. These results suggest that the inhibitoryactivities of the native molecule towards these three proteinasesare all located in the second domain.     

A model laboratory system to study the synergistic interaction and growth of environmental Escherichia coli with macrophytic green algae

Escherichia coli, an important fecal indicator bacterium, is known to persist and reproduce in association with Cladophora and other macrophytic algae and environmental substrates. Recent increases in the growth of Cladophora and other macrophytic algae in many of the Laurentian Great Lakes result in the accumulation of large amounts of algal biomass along the shoreline and on beaches. While the Cladophora–E. coli association may pose substantial public health risks, detailed laboratory-based studies have not been done to investigate the bases of the interaction. This is due, in large part, to past inabilities to culture many macrophytic algae under near axenic conditions. Here we describe the development and experimental use of laboratory microcosms to study the synergistic interaction and growth of the green alga Pithophora, a close relative of Cladophora, with environmental E. coli. In the absence of exogenous organic carbon supply, the E. coli population attached to algal filaments increased approximately three orders of magnitude within 72 h of inoculation. Growth of E. coli on Pithophora appeared to be limited by dissolved nitrogen with a concentration of ≥ 66 μg/mL N allowing maximal bacterial growth. In contrast, an environmental strain of Salmonella did not grow under identical conditions in the microcosms, suggesting that this bacterium requires additional growth factors not provided by Cladophora. Since the alga can be maintained in the laboratory for long periods of time, this system allows for further experimentation and understanding of macroalga–microbe interactions.