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.     

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.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0°, 22°, 90°) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14° were measured for the E. coli detection, as compared to 2° due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.     

Synthesis and microbial inhibition study of novel 5-imidazolyl substituted isoxazolidines

Cycloaddition of C-imidazolyl-N-phenylnitrones with monosubstituted alkenes afforded 5-imidazolyl substituted isoxazolidines with high regioselectivity. Novel isoxazolidines were screened for their antibacterial activities against S. aureus, E. coli and B. subtilis by using streptomycin as a positive control. They were also tested for their antifungal activities against F. moniliforme, A. niger and C. acremonium by using nystatin as a positive control. Isoxazolidines, 4a and 4f exhibited more potent inhibition towards antifungal activity than the other isoxazolidines prepared.     

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.     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Acquired resistance to echinostomes in four Biomphalaria glabrata strains

Four strains of Biomphalaria glabrata showed a distinctive pattern of acquired resistance to each of 3 echinostome species. Juvenile albino B. glabrata from our laboratory NIH stock developed a strong resistance to Echinostoma lindoense but only a weak one to E. paraensei and a moderate one to E. liei. Juvenile B. glabrata 10-R2 strain developed a strong acquired resistance to E. lindoense but a weak one to E. paraensei and E. liei. Juvenile B. glabrata M-RLc strain developed a strong acquired resistance to E. lindoense and a moderate one to E. paraensei and E. liei. Juvenile B. glabrata 641 strain developed a moderate acquired resistance to E. lindoense, a weak one to E. liei and no measurable resistance to E. paraensei.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

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.     

Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with K_i = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Capacity of irradiated echinostome sporocysts to protect Schistosoma mansoni in resistant Biomphalaria glabrata

Three closely related species of Echinostoma flukes each has a distinctive pattern of protection of Schistosoma mansoni in schistosome-resistant Biomphalaria glabrata host snails. Protection of developing S. mansoni by irradiated E. paraensei sporocysts in the schistosome-resistant snail host was strong; protection induced by irradiated E. lindoense and E. liei sporocysts was weak or not measurable. The capacity of irradiated E. paraensei sporocysts to interfere with the host's innate anti-schistosome response also differed between strains of B. glabrata. Protection of S. mansoni strain Lc-1 was greater in B. glabrata strain 10-R2 than it was in strain M-RLc snails. Irradiated E. paraensei sporocysts also induced a different response to the two schistosome strains in a single host strain. Irradiated E. paraensei sporocysts induced in B. glabrata 10-R2 snails a stronger protection of S. mansoni strain PR-1 than of strain Lc-1. Exposure of each snail to the irradiated E. paraensei miracidia usually protected the following challenge schistosome infection better when 30 rather than 10 irradiated echinostome miracidia were used.     

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HIyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA     

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.     

Enumeration of heterotrophs, fecal coliforms and Escherichia coli in water: comparison of 3M Petrifilm plates with standard plating procedures

A total of 177 naturally contaminated water samples were analyzed by membrane filtration according to the Standard Methods for the Examination of Water and Wastewater published by the American Public Health Association. Filters were incubated in parallel on mHPC-agar and 3M™ Petrifilm™ Aerobic Count Plates (Petrifilm™ AC plates) for heterotrophic counts. Fecal coliforms and Escherichia coli were enumerated on mFC-agar and 3M™ Petrifilm™ E. coli/Coliform Count Plates (Petrifilm™ EC plates). Typical colonies on each media type were confirmed following standard procedures. Heterotrophic counts were between 10³ and 10⁴ CFU/mL and the average log₁₀ counts obtained on Petrifilm™ AC plates were about two-fold lower than on mHPC-agar. Counts for fecal coliforms and E. coli were between 10² and 10³ CFU/mL. Average log₁₀ counts for confirmed fecal coliforms obtained on Petrifilm™ EC plates were slightly lower than on mFC agar with a correlation coefficient of 0.949. The average log₁₀ counts for confirmed E. coli on Petrifilm™ EC plates and on mFC agar were statistically not different (P=0.126) with a correlation coefficient of 0.879. Specificity of Petrifilm™ EC plates and mFC agar was evaluated by comparing typical colony counts with confirmed counts. On mFC agar, counts for typical colonies were by 2 log₁₀ CFU higher than the actual confirmed counts. In contrast, on Petrifilm™ EC plates typical colony counts were almost identical to confirmed colony counts for both fecal coliforms and E. coli. This comparison illustrates the high specificity of Petrifilm™ EC plates for enumeration of both fecal coliforms and E. coli in water.