Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

Genes of human mitochondrial tRNA^Leu(UUR) (mtRNA^Leu(UUR)) and its mutant (mtRNA^Leu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively.E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNA^Leu(UUR) and mtRNA^Leu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters forE.coli LeuRS were measured. The results showed that the value of k_cal/ K_m of mtRNA^Leu(M) was about one fifth of that of mtRNA^Leu(UUR) and indicated the leucine acceptability of mtRNA^Leu(M) was much lower than that of mtRNA^Leu(UUR).     

Antibiotic-resistant Salmonella and Escherichia coli isolates with integrons and extended-spectrum beta-lactamases in surface water and sympatric black-headed gulls

Aim: To examine surface water from a pond in the northeastern part of the Czech Republic and young black‐headed gulls (Larus ridibundus) nesting on the same pond for the presence of antibiotic‐resistant Salmonella and Escherichia coli. Methods and Results: A total of 16% (n = 87) of water and 24% (n = 216) of gull samples yielded Salmonella. Salmonella Enteritidis PT8 and PT4 were the most prevalent. Antibiotic resistance was found in 12% (n = 14) of water and 28% (n = 51) of gull salmonellae. Escherichia coli were found in 83 (95%) and 213 (99%) of pond water and gull samples, respectively. Totals of 18% (n = 83) of water and 28% (n = 213) of gull E. coli isolates were resistant to antimicrobial agents tested. Class 1 integrons were found in 21% (n = 14) of water and 15% (n = 60) of gull antibiotic‐resistant E. coli isolates. Class 2 integrons and extended‐spectrum beta‐lactamase‐producing E. coli isolates (with bla_CTX‐M‐1, bla_CTX‐M‐15‐like, bla_SHV‐2 and bla_SHV‐12) were found in 13% (eight positive, n = 60 gull‐resistant E. coli isolates) and 3% (seven positive, n = 216 gull E. coli isolates) of gull isolates, respectively. Antibiotic‐resistant E. coli isolates with identical pulsed field gel electrophoresis (PFGE) patterns were found in either gulls or water, but not both. Salmonellae of the same serotype and PFGE profile were found in both gulls and water. Conclusion: A high prevalence of antibiotic‐resistant salmonellae and E. coli were found in both pond water and in sympatric black‐headed gulls. Significance and Impact of the Study: Intensive contamination of pond surface water by antibiotic‐resistant E. coli and salmonellae was documented. Black‐headed gulls were identified as important reservoirs of antibiotic‐resistant salmonellae and E. coli, including extended‐spectrum beta‐lactamase‐producing isolates.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.     

Expression of antimicrobial peptide LH multimers in <Emphasis Type="Italic">Escherichia coli</Emphasis> C43(DE3)

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni²⁺-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC₅₀ of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper.     

Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (<Emphasis Type="BoldItalic">adhE</Emphasis>) in <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5 and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg^-1 , respectively.     

Antimicrobial‐resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended‐spectrum beta‐lactamases in wild boars

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results: Rectal swabs or faeces collected during 2006–2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l^?1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended‐spectrum beta‐lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, n_E. coli = 242), 12% in wild ruminants and foxes (n_E. coli = 42), while no resistant isolates were detected in brown bears (n_E. coli = 16). In wild boars (Sus scrofa) (n_E. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, n_{rectal smears} = 293) multiresistant isolates producing ESBL were recovered: one isolate with bla_CTX‐M‐1 + bla_TEM‐1, three with bla_CTX‐M‐1 and one with bla_TEM‐52b. The bla_CTX‐M‐1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions: Antibiotic‐resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study: Wild mammals are reservoirs of antibiotic‐resistant E. coli including ESBL‐producing strains which were found in wild boars.     

Oxygen transfer correlation in high cell density culture of recombinant E. coli

Summary A recombinant E. coli BL21[pET3a-T2M2] was cultivated in fed-batch cultures and cell mass increased to more than 70g/L. The volumetric oxygen transfer coefficient was estimated in a range of various fermentation parameters (agitation speed, oxygen flow rate and cell mass concentration) and finally the oxygen transfer correlation in bioreactor containing the recombinant E. coli cultures was determined as: k_spla = 0.0195 (P_g/V)^0.55 (V_s)^0.64 (1+2.12X+0.20X²)^–0.25.     

Replacement of Methionine 208 in a Truncated <Emphasis Type="Italic">Bacillus</Emphasis> sp. TS-23 α-Amylase with Oxidation-Resistant Leucine Enhances Its Resistance to Hydrogen Peroxide

The methionine residues at positions 17, 104, 208, 214, 292, 315, 324, and 446 in the primary amino acid sequence of a truncated Bacillus sp. TS-23 α-amylase (His₆-tagged BLAΔNC) was changed to oxidative-resistant leucine by site-directed mutagenesis. The mutant enzymes with an apparent molecular mass of approximately 54 kDa were overexpressed in recombinant Escherichia coli. The specific activity for Met315Leu and Met446Leu was decreased by more than 76%, while Met17Leu, Met104Leu, Met208Leu, Met214Leu, Met292Leu, and Met324Leu showed 247, 128, 37, 260, 232, and 241%, respectively, higher activity than the wild-type enzyme. In comparison with wild-type enzyme, a lower K _m value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (k _cat/K _m) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in K _m values. Wild-type enzyme was sensitive to chemical oxidation, but Met208Leu was stable even in the presence of 500 mM H₂O₂. Except for Met214Leu, which was quite sensitive to H₂O₂, the other mutants showed a profile of oxidative inactivation similar to that of the wild-type enzyme. These observations indicate that the oxidative stability of His₆-tagged BLAΔNC can be improved by replacement of the critical methionine residue with leucine. Received: 12 April 2002 / Accepted: 8 June 2002     

An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L⁻¹) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis.     

Bioaugmentation of the decolorization rate of acid red GR by genetically engineered microorganism <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109 (pGEX-AZR)

The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r _dye,max, K _s and K _i , were found to be 42.45 mg g⁻¹ h⁻¹, 584.93 mg L⁻¹ and 556.89 mg L⁻¹, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

A sensitive fluorescence method for detection of E. Coli using rhodamine 6G dyeing

Negatively charged bacteria combined with positively charged alkaline dye rhodamine 6G (Rh6G) in NaH₂PO₄–Na₂HPO₄ buffer solution pH 7.4, by electrostatic interaction. The dyed bacteria exhibited a strong fluorescence peak at 552 nm and fluorescence intensity was directly linear to Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) concentrations in the range of 7.06 × 10⁴ to 3.53 × 10⁷, 4.95 × 10⁵ to 2.475 × 10⁸ and 32.5 to 16250 colony forming unit/mL (cfu/mL) respectively, with detection limits of 3.2 × 10⁴ cfu/mL E. coli, 2.3 × 10⁵ cfu/mL B. subtilis and 16 cfu/mL S. aureus, respectively. Samples were cultured for 12 h, after which the linear detection range for E. coli was 2 to 88 cfu/mL. This simple, rapid and sensitive method was used for the analysis of water and drinking samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs^b, Cs^c) and the wild-type (Cs^a) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs^a > Cs^c > Cs^b > UM255, and their susceptibility to hydrogen peroxide (H₂O₂) showed UM255 > Cs^b > Cs^c > Cs^a. We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs^b, Cs^c and Cs^a. Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H₂O₂ is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

E. coli (P450pyrTM‐GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g^?1 cdw) for the biohydroxylation of N‐benzylpyrrolidine 1 and a GDH activity of 106 U g^?1 protein. The E. coli cells were used as efficient biocatalysts for highly regio‐ and stereoselective hydroxylation of alicyclic substrates at non‐activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N‐benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio‐ and stereoselectivity, giving (S)‐N‐benzyl‐3‐hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM‐GDH) was found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpyrrolidin‐2‐one 3 , improving the regioselectivity from 90% of the wild‐type P450pyr to 100% and giving (S)‐N‐benzyl‐4‐hydroxylpyrrolidin‐2‐one 4 in 99% ee as the sole product. A high activity of 15.5 U g^?1 cdw was achieved and (S)‐ 4 was obtained in 19.4 mM. E. coli (P450pyrTM‐GDH) was also found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpiperidin‐2‐one 5 , increasing the ee of the product (S)‐N‐benzyl‐4‐hydroxy‐piperidin‐2‐one 6 to 94% from 33% of the wild‐type P450pyr. A high activity of 15.8 U g^?1 cdw was obtained and (S)‐ 6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM‐GDH) represents the most productive system known thus far for P450‐catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM‐GDH) provide with simple and useful syntheses of (S)‐ 2 , (S)‐ 4 , and (S)‐ 6 that are valuable pharmaceutical intermediates and difficult to prepare. Biotechnol. Bioeng. 2013; 110: 363–373. © 2012 Wiley Periodicals, Inc.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

Gene cloning, expression and purification of human mitochondrial tRNA~(Leu(UUR)) and its mutant

Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repu-rified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal/Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR).     

Inactivation of bacteriophage k, Escherichia coli, and Candida albicans by ozone

The effects of ozone (O₃) on three types of microbes were studied. Test suspensions were exposed to 600 ppm O₃ at room temperature. Control experiments were performed under identical conditions using oxygen gas. Bacteriophage λ was completely inactivated at 10 min while Escherichia coli and Candida albicans were only inactivated by factors of 10⁵ and 10⁴ respectively at 40 min. Exposure of a mixed microbial suspension to O₃ for 5 min resulted in 100% killing of bacteriophages while the viability of E. coli remained unchanged. Various body fluids containing phages were exposed to O₃. Compared to buffered solution, the decrease in phage titers was significantly slower in whole blood, plasma, and albumin. Both E. coli and  C. albicans had increased production of thiobarbituric-acid-reactive substances with increased O₃ exposure. ³H-labelled amino acids were incorporated into E. coli. O₃ treatment resulted in a loss of radioactivity, indicating leakage of cytoplasmic contents. The data indicate that microbes are inactivated by O₃ at different rates, possibly related to differential membrane permeability. The milieu in which microbes are present determines the effectiveness and outcome of O₃ treatment. Received: 15 October 1997 / Accepted: 24 February 1998