Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.     

The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes

A naturally occurring small RNA molecule ( micF RNA), complementary to the region encompassing the Shine-Dalgarno sequence and initiation codon of the ompF mRNA, is known to block the expression of that mRNA in E. coli. We have constructed a plasmid that produces a complementary RNA to the E. coli lpp mRNA (mic[Ipp] RNA). Induction of the mic(Ipp) gene efficiently blocked lipoprotein production and reduced the amount of lpp mRNA. Two mic(ompC) genes were similarly engineered and their expression was found to inhibit drastically production of OmpC. Analysis of several types of mic(ompA) genes suggests that micRNAs complementary to regions of the mRNA likely to come in contact with ribosomes were most effective. The novel capabilities of this artificial mic system provide great potential for application in both procaryotic and eucaryotic cells.     

Overexpression of L-isoaspartate O-methyltransferase in Escherichia coli increases heat shock survival by a mechanism independent of methyltransferase activity

Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein. An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.     

Identification and network of outer membrane proteins regulating streptomysin resistance in Escherichia coli

Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, Delta tolC, Delta ompT, Delta dps, Delta tolC-R, Delta ompT-R, Delta dps-R and Delta fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli.     

Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: A proteome-level study

Vitreoscilla haemoglobin (VHb) expression in heterologous host was shown to enhance growth and oxygen utilization capabilities under oxygen-limited conditions. The exact mechanism by which VHb enhances the oxygen utilization under oxygen-limiting conditions is still unknown. In order to understand the role of VHb in promoting oxygen utilization, changes in the total protein profile of E. coli expressing the vgb gene under its native promoter was analysed. Two-dimensional difference gel electrophoresis (2D DIGE) was employed to quantify the differentially expressed proteins under oxygen-limiting conditions. Overexpression of proteins involved in aerobic metabolic pathways and suppression of proteins involved in non-oxidative metabolic pathways shown in this study indicates that the cells expressing VHb prefer aerobic metabolic pathways even under oxygen limitation. Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, the significance of which is not clear as of now.     

Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers Raman spectroscopy.

BACKGROUND: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. METHODS: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. RESULTS: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm(-1), are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. CONCLUSION: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.     

Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli

Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.     

耐辐射球菌pprI在大肠杆菌中表达增强细胞抗氧化能力的研究

将耐辐射球菌(Deinococcus radiodurans)与DNA修复有关的开关基因—pprI通过穿梭质粒pRADZ3导入大肠杆菌TG1中,使其在正常培养条件下(不需诱导剂)表达PprI蛋白,并通过Western blot证实该基因在TG1中可稳定表达。与转化了空白质粒pRADZ3 TG1对照,观察了改造后的两种大肠杆菌在有H2O2氧化压力下的存活率和大肠杆菌中两种过氧化氢酶（KatE, KatG）的活性表达差异。结果表明,无论在指数生长期还是稳定生长期,能表达PprI蛋白的大肠杆菌比对照的存活率要高出10％左右;非变性电泳结果表明,耐辐射球菌pprI 在大肠杆菌中的表达使得KatE活性在指数生长期与稳定生长期分别增加1.5～2倍和2.5～3倍。证明耐辐射球菌pprI 在大肠杆菌中的表达能够增强细胞抗氧化能力。     

Nitric oxide stimulates macrophage inflammatory protein-2 expression in sepsis

NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-kappaB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IkappaB and activation of NF-kappaB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-L-arginine. Despite having relatively modest effects on NF-kappaB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-L-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.     

鸡氨肽酶N的高效可溶性表达及生物学功能分析

本研究旨为克隆鸡氨肽酶N(chAPN)基因,高效表达可溶性目的蛋白,并测定其生物学功能。应用RT-PCR方法从鸡胚肾细胞中克隆chAPN的基因片段,经测序鉴定后再克隆至原核表达载体pCOLD-TF,构建重组原核表达质粒pCOLD-TF-chAPN,在大肠杆菌BL21(DE3)中经不同条件诱导表达目的蛋白;利用镍柱亲和层析法纯化可溶性蛋白,并进行SDS-PAGE、Western blotting鉴定;Leu-PNA酶促反应和ELISA等方法检测目的蛋白生物学功能。结果显示,重组质粒pCOLD-TF-chAPN在大肠杆菌中以可溶形式高效表达;酶促反应及ELISA结果显示该蛋白具有酶活性,可结合传染性支气管炎病毒(IBV),并表现为剂量依赖性。这为今后研究chAPN的酶活性、作为IBV受体及抗病毒功能奠定了实验基础。     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the gene's second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.