玫瑰茄醇提物的抑菌活性及其作用机制

玫瑰茄具有多种药理活性,包括抗肿瘤、抗氧化和抑菌作用等,但目前关于玫瑰茄的抑菌作用研究较少,有关抑菌机制方面的研究尚未见报道. 本文通过测定玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌细胞膜、蛋白质和核酸的影响,及其与DNA的作用方式等,系统阐述玫瑰茄的抑菌作用机制. 电导率和大分子物质的测定结果显示,玫瑰茄醇提物只对菌体的细胞膜造成微小损伤,其抑菌作用的靶点不在细胞膜. SDS-PAGE和DAPI结果显示,玫瑰茄醇提物可抑制大肠杆菌和金黄色葡萄球菌蛋白质和核酸的合成. 琼脂糖凝胶电泳和紫外吸收光谱结果显示,玫瑰茄醇提物可与DNA结合,当DNA与药物的浓度比较低时,玫瑰茄醇提物与DNA以嵌入结合为主,当二者的浓度较高时,两者间发生的是氢键结合. 上述结果证明,玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌抑菌机制,主要是药物通过与DNA发生嵌入结合和氢键结合,使DNA不能进行正常的复制和转录,降低核酸的含量,进而影响蛋白质的合成,最终导致菌体生物学功能的丧失.     

The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.     

The adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli on cotton, polyester and their blends

The adherent behaviour of the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and the Gram-negative Escherichia coli on cotton, polyester and their blends through contact in aqueous suspensions was studied. Staphylococcus epidermidis was found to adhere to fabrics much more so than Staph. aureus. The adherence of both Staph. epidermidis and Staph. aureus to fabrics increased as the content of polyester fibres in the fabrics increased. The attachment of E. coli to all fabrics was very low and was not affected by the fibre contents. Total numbers of adherent bacteria on cotton and polyester fabrics were related directly to the concentrations of the bacterial suspensions. The extents of adherence, expressed by the percentage of adherent bacteria from the suspension, however, were independent of the concentration. The length of contact with bacteria was also found to affect the adherence of bacteria on fabrics studied.     

The adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli on cotton, polyester and their blends

The adherent behaviour of the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and the Gram-negative Escherichia coli on cotton, polyester and their blends through contact in aqueous suspensions was studied. Staphylococcus epidermidis was found to adhere to fabrics much more so than Staph. aureus. The adherence of both Staph. epidermidis and Staph. aureus to fabrics increased as the content of polyester fibres in the fabrics increased. The attachment of E. coli to all fabrics was very low and was not affected by the fibre contents. Total numbers of adherent bacteria on cotton and polyester fabrics were related directly to the concentrations of the bacterial suspensions. The extents of adherence, expressed by the percentage of adherent bacteria from the suspension, however, were independent of the concentration. The length of contact with bacteria was also found to affect the adherence of bacteria on fabrics studied.     

Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase.     

An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae.A yeast/Escherichia colishuttle Cm-resistance (Cm^R)marker was constructed by inserting the CAT coding sequence from Tn9,and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator. When present on a 2 μm-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2. When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3. Industrial yeast strains were transformed with both the replicating and integrating plasmids. The Cm^R marker could also efficiently transform E. coli. This versatile and efficient performance is currently unique for a yeast dominant marker.     

Sensitivity and resistance of Escherichia coli and Staphylococcus aureus to chlorhexidine

Escherichia coli PC1349 and Staphylococcus aureus 6571 were sensitive to low concentrations of chlorhexidine diacetate, as determined by minimal inhibitory concentration tests. Lack of bactericidal response to 30 μ g/ml was due to the fact that adsorption of biocide to the cells was very slight in suspensions of high cell density and was not due to emerging resistance. Attempts by various methods to induce stable resistance in these organisms failed, despite reports that resistant strains have been isolated.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

Genetic activity of carbamate herbicides in Escherichia coli and Saccharomyces cerevisiae

DNA-damaging activity and herbicide-induction of gene point mutations, and intragenic mitotic recombination were studied in bacteria and the yeast tester strains. Herbicide (eptam, triallate, tillam, surpass) were not effective in DNA-damaging and mitotic recombination tests. Of the 4 chemicals, only eptam was strongly mutagenic. Dose response curves for eptam differed in bacteria and yeast; maximum mutagenic activity was registered in bacteria at 5 mg/l. Maximum yield of prototrophs was observed after 2 h incubation time. Triallate was moderate, tillam and surpass being weak mutagens for the strains used.     

Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli

The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.     

Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae

Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B (blue or red) and chromatofocusing on polybuffer PBE 94^tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps.The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it desintegrated into assumingly identical smaller subunits (E. coli rom M_r 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). Both forms, dimer and monomer were found catalytically active.Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.Abbreviations APS adenylylsulphate - APS kinase - ATP adenylylsulphate 3-phosphotransferase - DCPIP 2,6-dichlorophenol indophenol - DTT dithiothreitol - GSH reduced glutathione - GSSG oxidized glutathione - HPLC high performance liquid chromatography - -MSH -mercaptoethanol - PAPS 3-phosphoadenylylsulphate - TNBS 2,4,6 tri-nitrobenzenesulphonic acid     

Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

Study of an artificial endoassociation between Saccharomyces cerevisiae and Escherichia coli

The establishment of an artificial endoassociation between Escherichia coli (JC 5466, trp, his, recA 56, lac delta X 74, SpcR, harbouring the plasmid pRD1 which confers on it the capacity to produce penicillinase), and Saccharomyces cerevisiae (3.2, a, ade, ura, lys) was carried out in order to study its behaviour and stability. The pattern of protoplast reversion to whole cells, the penicillinase production capacity, the stability without selective pressure and the bacterial localization in the yeast cells, is described and discussed.     

大肠杆菌和酵母PCR模板制备的优化

对快速制备大肠杆菌和酵母基因组DNA PCR模板的方法进行了优化。实验证明加入0.1%Triton X-100和0.1%SDS的煮沸法能够明显提高大肠杆菌基因组模板的扩增效率,加入0.1%SDS的煮沸法在提高酿酒酵母基因组模板的扩增效率方面也有类似效果,但加入0．1％Triton X—100的煮沸法对酿酒酵母基因组模板的扩增仅有较小的作用。     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Regulation of glucosamine utilization in Staphylococcus aureus and Escherichia coli.

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.     

Thermal stability of phosphoenolpyruvate carboxykinases from Escherichia coli,Trypanosoma brucei,and Saccharomyces cerevisiae

The quaternary structure of ATP-dependent phosphoenolpyruvate (PEP) carboxykinases is variable. Thus, the carboxykinases from Escherichia coli, Trypanosoma brucei, and Saccharomyces cerevisiae are monomer, homodimer, and homotetramer, respectively. In this work, we studied the effect of temperature on the stability of the enzyme activity of these three carboxykinases, and have found that it follows the order monomer > dimer > tetramer. The inactivation processes are first order with respect to active enzyme. The presence of substrates leads to an increase in the thermal stability of all three PEP carboxykinases. The protection effect of the substrates on the thermal inactivation of these enzymes suggests similarities in the substrate-bound form of these proteins. We propose that the higher structural complexity of some PEP carboxykinases could be related to the acquisition of properties of relevance in vivo.