Construction of a chimeric gene cluster for the biosynthesis of apoemulsan with altered molecular weight

Acinetobacter venetianus RAG-1 produces an extracellular protein/high-molecular-weight (HMW) polysaccharide complex termed emulsan. As an emulsion stabilizer, emulsan has potential industrial applications. To control the molecular weight of the polymer, a stable chromosomal mutant was generated where RAG-1 wza, wzb, wzc genes were replaced by Escherichia coli homologs. The heterologous Wza, Wzb, Wzc proteins restored production of HMW polysaccharide. The polymer produced was of higher molecular weight than from the parent strain and with the cells exhibiting modified hydrophobicity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression ofMycobacterium tuberculosis genes inEscherichia coli

TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.     

毒力岛基因ibeT有助于大肠杆菌抵抗人脑微血管内皮细胞溶酶体的降解

大肠杆菌是导致新生儿细菌性脑膜炎最常见的革兰氏阴性致病菌．为探讨毒力岛基因ibeT在大肠杆菌K1株致病过程中的作用,构建了ibeT基因缺失的大肠杆菌K1株,细菌在细胞内存活试验结果显示,ibeT基因缺失抑制了大肠杆菌K1株在人脑微血管内皮细胞中的生长．利用激光共聚焦扫描显微镜观察到,在细菌侵袭进入人脑微血管内皮细胞后,与野生型相比,ibeT基因缺失突变株较多地滞留在溶酶体内;透射电镜结果进一步显示,ibeT基因缺失使大肠杆菌K1株逃逸ECV(含有大肠杆菌的囊泡)的能力发生了下降,继而使其在细胞浆内的复制减少．利用体外模拟的弱酸性环境,检测大肠杆菌菌体胞内的缓冲容量,发现ibeT基因缺失突变株菌体胞内的缓冲能力较野生型低．这些结果提示,在大肠杆菌K1株侵袭进入人脑微血管内皮细胞后,ibeT基因有利于大肠杆菌降解ECV膜,避免与溶酶体融合,进而促使大肠杆菌逃逸进入细胞浆并进行复制．     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation

Summary Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to -rays but not to ultraviolet light. One new mutant of this type, named rorB, was isolated. This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN. Unlike previously reported mutants of E. coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks. The rorB gene maps close to ilvGEDAC at 84.5 min of the E. coli chromosome.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

Identification of CED-3 substrates by a yeast-based screening method

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.     

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.     

Effect of 6059-S,a novel oxacephem,on cross-linking reaction of peptidoglycan biosynthesis in Escherichia coli,Pseudomonas aeruginosa and Serratia marcescens

The effect of 6059-S, a novel 1-oxacephem, on peptidoglycan synthesis was investigated using ether-treated cells of Escherichia coli K 12, Pseudomonas aeruginosa KM 338 and Serratia marcescens IFO 12648. The cross-linking reaction of peptidoglycan synthesis in these organisms was inhibited by markedly low concentration of 6059-S.Non-standard abbreviations PBP penicillin binding protein - MIC minimum inhibitory concentration - ETB ether treated bacterial cells - SDS sodium dodecylsulfate     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli

Summary To measure the degree of phr gene induction by DNA-damaging agents, the promoter region was fused to the coding region of the lacZ gene in plasmid pMC1403. The new plasmids were introduced into Escherichia coli cells having different repair capabilities. More efficient induction of phr gene expression was detected in a uvrA ^– strain as compared with the wild-type strain. In addition, obvious induction was detected in uvrA ^– cells treated by 4-nitroquinoline 1-oxide and mitomycin C. Nalidixic acid, an inhibitor of DNA gyrase, also induced phr gene expression. In contrast, little induced gene expression was noted in UV-irradiated lexA ^– and recA ^– strains. It is suggested from these results that induction of the phr gene is one of the SOS responses. Possible nucleotide sequences which could be considered to constitute an SOS box were found at the regulator region of the phr gene.Abbreviations phr photoreactivation - UV ultraviolet light - 4NQO 4-nitroquinoline 1-oxide - MMC mitomycin C - PRE photoreactivating enzyme - E. coli Escherichia coli     

反刍动物产志贺毒素大肠杆菌的分离鉴定和致病潜力分析

产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）是重要的食源性病原,而STEC往往以正常菌群的形式存在于牛羊等反刍动物肠道。[目的] 本研究对牛羊粪便样品中的STEC分离和鉴定并对分离株进行致病潜力分析。从江苏、云南和河北等地共分离到羊源STEC菌株11株,牛源STEC菌株1株,另新疆农业大学佟盼盼组馈赠牛源菌株10株。[方法] 通过细菌选择培养及特异性基因stx1和stx2的检测进行分离鉴定;并通过Vero细胞毒性试验、溶血活性试验和毒力因子的检测分析STEC分离株的致病潜力。[结果] 分离到羊源分离株11株,分离率17.5%（11/63）;分离得到牛源分离株1株,分离率0.7%（1/134）;11株羊源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性;11株牛源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性。11株羊源STEC分离株eae基因携带率为63.6%（7/11）,而11株牛源STEC分离株eae基因携带率仅为9.0%（1/11）。[结论] 结果表明羊源STEC菌株的分离率和致病潜力高于牛源菌株,所以,除牛外,羊作为STEC菌株宿主也应该得到更多的重视。     

Molecular imaging of c-Met tyrosine kinase activity

Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Construction,characterization, and screening of a transformation-competent artificial chromosome library of peach

As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination. TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for gene cloning and genetic engineering in peach.