A MEMS based amperometric detector for E. Coli bacteria using self-assembled monolayers

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Similarities between a predicted secondary structure for the M1 RNA ribozyme and the tRNA binding center of 16 S rRNA from E. coli

We propose a new model for the secondary structure of the M1 RNA component of E. coli RNase P which is based on significant sequence homologies with parts of the E. coli 16 S rRNA. A large domain of the new model resembles closely the secondary structure of the tRNA binding center of 16 S rRNA. We suggest that this domain of M1 RNA when functioning as a ribozyme binds the mature part of the precursor tRNA.     

Cloning and Overexpression of a Tagged CMP-N-Acetylneuraminic Acid Synthetase from E. coli Using a Lambda Phage System and Application of the Enzyme to the Synthesis of CMP-N-Acetylneuraminic Acid

The gene coding from CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (Ec 2.7.7.43) was amplified from total DNA of E. coli strain K-235 through a primer-directed polymerase chain reaction. The gene was fused with a modified ribosome binding site of the original CMP-NeuAc synthetase gene and a decapeptide tag sequence which served as a marker for screening of expressed proteins. The gene was cloned into lambda ZAP vector at EcoRI and XbaI sites and overexpressed in E. coli Sure at a level approximately 1000 times that of the wild type. The decapeptide-containing enzyme retained almost the same specificity as indicated by the V_max and K_m values using CTP and NeuAc as substrates. A preparative synthesis of CMP-NeuAc based on the recombinant enzyme was demonstrated.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Cloning of a cDNA that encodes an invertebrate glutamate receptor subunit.

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terininal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Overexpression, purification, and characterization of selenomethionyl farnesyl diphosphate synthase of Bacillus stearothermophilus

To facilitate X-ray crystal structure solution of farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus, selenomethionyl recombinant enzyme was overproduced in a methionine (Met) auxotrophic strain of Escherichia coli, and purified to homogeneity by two chromatographic steps. About 50 mg of the pure selenomethionyl enzyme was obtained from 2 g of E. coli cells. Inductively coupled plasma (ICP) emission spectrometric analysis for selenium content showed that all of the Met residues in the FPP synthase were substituted by selenomethionine (SeMet). The selenomethionyl recombinant enzyme showed similar chromatographic behavior, heat stability, immunochemical property, product specificity, and kinetic parameters to those of the wild-type enzyme, indicating that SeMet substitution has little effect on the prenyltransferase with respect to substrate binding, enzymatic activity, and structure.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Catalytic domain of Salmonella typhimurium 2-oxoglutarate dehydrogenase is localized in N-terminal region

2-Oxoglutarate dehydrogenase (lipoamide) [OGDH or E1o: 2-oxoglutarate: lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinating); EC 1.2.4.2] is a component enzyme of the 2-oxoglutarate dehydrogenase complex. Salmonella typhimurium gene encoding OGDH (ogdh) has been cloned in Escherichia coli. The libraries were screened for the expression of OGDH by complementing the gene in E. coli E1o-deficient mutant. Three positive clones (named Odh-3, Odh-5 and Odh-7) contained the identical 2.9 kb Sau3AI fragment as determined by restriction mapping and Southern hybridization, and expressed OGDH efficiently and constitutively using its own promoter in the heterologous host. This gene spans 2878 bases and contains an open reading frame of 2802 nucleotides encoding a mature protein of 927 amino acid residues (M_r=110,000). The comparison of the deduced amino acid sequence of the cloned OGDH with E. coli OGDH shows 91% sequence identity. To localize the catalytic domain responsible for E. coli E1o-complementation, several deletion mutants lacking each portion of the ogdh gene were constructed using restriction enzymes. From the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a polypeptide which showed a complementation activity with an M_r of 30,000 was detected. The catalytic domain was localized in N-terminal region of the gene. Therefore, this is a first identification of the catalytic domain in bacterial ogdh gene.     

Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage λ causes transposition primarily into the vicinity of the λ operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Exo-inulinase of Aspergillus niger N402: A hydrolytic enzyme with significant transfructosylating activity

The purified exo-inulinase enzyme of Aspergillus niger N402 (AngInuE; heterologously expressed in Escherichia coli) displayed a sucrose:inulin (S/I) hydrolysis ratio of 2.3, characteristic for a typical exo-inulinase. The enzyme also had significant transfructosylating activity with increasing sucrose concentrations, producing various oligosaccharides. The AngInuE protein molecular mass was 57 kDa, close to the calculated value for the mature protein. AngInuE thus was active as a monomeric, non-glycosylated protein. Contradictory data on hydrolysis/transfructosylation activity ratios have been published for the (almost) identical (but monomeric or dimeric and glycosylated) exo-inulinases of other aspergilli. Our data clearly show that the AngInuE enzyme, produced in and purified from E. coli, is a broad specificity exo-inulinase that also has significant transfructosylating activity with sucrose. Analysis of site-directed mutants of AngInuE showed that the glycoside hydrolase family 32 conserved domain G is important for catalytic efficiency, with a clear role in hydrolysis of both sucrose and fructans.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.