Effect of Microwaves on Escherichia coli and Bacillus subtilis

Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.     

Thiouridine residues in tRNAs are responsible for a synergistic effect of UVA and UVB light in photoinactivation of Escherichia coli

Since different wavelengths of light impact different cellular targets, microorganisms exposed to natural sunlight experience a combination of multiple stressors. In order to better understand the effects of sunlight on microorganisms we, therefore, need to understand how different wavelength act alone and in combination. Here, we describe a synergistic effect between UVA and UVB irradiation on viability of Escherichia coli bacteria. To investigate the basis of this synergistic effect we analysed mutant strains that were obtained through selection for increased resistance to combined UVA and UVB. By identifying and reconstructing genetic changes in the resistant strains we provide evidence that UVA‐absorbing thiouridine residues in tRNAs are the key to the synergistic effect. Our study provides insights into how naturally occurring combinations of stressors can interact, and points to new ways for controlling microbial populations.     

过量表达Bacillus subtilis磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响

在大肠杆菌厌氧混合酸发酵途径中,磷酸烯醇式丙酮酸羧化酶(PPC)和磷酸烯醇式丙酮酸羧化激酶(PCK)皆可催化由磷酸烯醇式丙酮酸(PEP)到草酰乙酸(OAA)的反应。鉴于经由PCK催化的反应伴有ATP的生成,理论上更有利于菌体生长和产酸,本研究以大肠杆菌W3110(△pfl,△ldh)为出发菌株,利用λ-Red同源重组系统构建了其ppc缺陷菌株并在此基础上过量表达了Bacillus subtilispck基因。初步的厌氧发酵实验表明:过量表达pck可在一定程度上恢复初始菌株厌氧代谢葡萄糖的能力。其中又以ppc缺陷株更为明显,其耗糖能力和产酸能力分别为对照菌株的4.2和15.3倍。     

Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.     

High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases

Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.     

Bacteristatic activity of phenanthrolines against Escherichia coli and Bacillus subtilis

Using optical turbidimetry to measure the growth of Escherichia coli and Bacillus subtilis, we determined the mean lethal dose (LD50) values for various phenanthrolines. The dimethyl-substituted compounds are found to be more toxic to bacteria, with doses near 5 micrograms/mL reducing the number of viable cells by 50% over a 24-h period. 2,9-Dimethyl phenanthroline is the most potent compound against B. subtilis, being six times more effective than against E. coli. Bipyridine is the least toxic substance and is twice as effective against E. coli as it is against B. subtilis. Evidence is presented to show copper ions enhance the antibacterial action of phenanthrolines and may be required for activity.     

Purification of Intact Flagella from Escherichia coli and Bacillus subtilis

A procedure is described for the purification of bacterial flagella in the form of a filament-hook-basal body complex (intact flagella) free from detectable cell wall, membrane, or cytoplasmic material. Spheroplasts produced with lysozyme and ethylenediaminetetraacetic acid were lysed with Triton X-100, and the flagella were purified by (NH(4))(2)SO(4) precipitation, differential centrifugation, and CsCl gradient centrifugation. As much as 40% of the flagella were recovered, and they contained about one basal body per 4 to 6 mum of flagella. The same procedure developed for Escherichia coli was also successful for purifying intact flagella from Bacillus subtilis.     

Biosynthesis of Bacillus licheniformis penicillinase in Escherichia coli and in Bacillus subtilis.

Modified prepenicillinase was accumulated in both Escherichia coli and Bacillus subtilis treated with globomycin. Although the inhibitions of processings of prepenicillinase and prolipoprotein by globomycin in E. coli are qualitatively similar, they differ in the degree of inhibition at given concentrations of globomycin. The processing of prepenicillinase proceeds much more rapidly in E. coli than in B. subtilis.     

Engineered ZnO and TiO(2) nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli

Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environmental impact and possible ecotoxicity. This study explores the toxicity mechanism of ZnO and TiO(2) ENPs in a gram-negative bacterium, Escherichia coli. Internalization and uniform distribution of characterized bare ENPs in the nano range without agglomeration was observed in E. coli by electron microscopy and flow cytometry. Our data showed a statistically significant concentration-dependent decrease in E. coli cell viability by both conventional plate count method and flow cytometric live-dead discrimination assay. Significant (p<0.05) DNA damage in E. coli cells was also observed after ENP treatment. Glutathione depletion with a concomitant increase in hydroperoxide ions, malondialdehyde levels, reactive oxygen species, and lactate dehydrogenase activity demonstrates that ZnO and TiO(2) ENPs induce oxidative stress leading to genotoxicity and cytotoxicity in E. coli. Our study substantiates the need for reassessment of the safety/toxicity of metal oxide ENPs.     

Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.     

Transformation of Escherichia coli and Bacillus subtilis with a hybrid plasmid molecule.

A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leu-E. coli, converting them to leucine prototrophy, but fails to survive in strains of B. subtilis 168.     

Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis

Summary A hybrid plasmid, pOXN29 (10.4 Mdal), coding the xylanase (xynA) and -xylosidase (xynB) genes of Bacillus pumilus IPO was constructed by the ligation of pBR322 and a 7.7 Mdal PstI-fragment of chromosomal DNA as reported in our previous paper (Panbangred et al. 1983). A deletion plasmid of pOXN29, pOXN293 (9.2 Mdal), which contains xynA and xynB, was ligated with pUB110 at an EcoRI site, and used to transform B. subtilis MI111. Two selected clones of B. subtilis as xylanase hyper-producers contained plasmids pOXW11 (4.2 Mdal) and pOXW12 (4.0 Mdal), both consisting of only pUB110, xynA, and its flanking regions, as the result of spontaneous deletion. These B. subtilis clones produced 2.7–3.0 times as much xylanase as B. pumilus. Escherichia coli and B. subtilis clones harbouring the hybrid plasmids synthesized xylanase and -xylosidase constitutively, whereas both enzymes were induced by xylose in B. pumilus.Xylanase synthesized by B. subtilis harbouring pOXW11 or pOXW12 was excreted into the medium like that of B. pumilus IPO, but xylanase synthesized in E. coli harbouring pOXN29, 293 or pOXW1 coding xynA was intracellular. In a previous investigation (Panbangred et al. 1983), xylanase was found to be located in the cytoplasm, not the periplasm nor the membrane fraction in E. coli cells harbouring pOXN29 derivatives. In spite of the abnormal location of xylanase synthesized in E. coli, the signal peptide was processed in the same way as in B. pumilus, with the same molecular weight and the same amino terminal sequences of xylanase prepared from E. coli cells and B. pumilus culture fluid.     

枯草杆菌及大肠杆菌异柠檬酸脱氢酶的酶学性质研究

对枯草杆菌异柠檬酸脱氢酶（BsIDH）、大肠杆菌异柠檬酸脱氢酶（EcIDH）和大肠杆菌异柠檬酸脱氢酶的突变体酶（EmIDH）进行了纯化和酶学性质鉴定。BsIDH和EcIDH对辅酶NADP^＋的特异性与NAD^＋相比,分别是NAD^＋的1330倍和3890倍。而EmIDH对NAD^＋的特异性与NADP^＋相比,是NADP^＋的122倍。因此BsIDH和EcIDH是NADP^＋依赖性异柠檬酸脱氢酶,而EmIDH的辅酶特异性已转换为NAD^＋依赖性。EcIDH、BsIDH和EmIDH对底物异柠檬酸的Km值分别为67.4 μmol/L、60.6 μmol/L和105.6 μmol/L。BsIDH和EcIDH的最适反应pH分别为8.2和8.0,EmIDH的最适pH为7.0。BsIDH和EmIDH的最适反应温度是45℃,EcIDH的最适温度为43℃。三种IDH的活性依赖于不同的二价金属离子的存在,Mn^2＋ 、Mg^2＋存在时酶活性最强,Cu^2＋ 、Ca^2＋ 、Zn^2＋和Ni2＋强烈抑制酶的活性。系统的酶学性质研究为深入认识IDH的催化与调节机制提供了更多依据。