Our newly discovered metalloprotease, designated as ALP NS12 was selected using gelatin agar plates with incubation at 100 °C. Subcloning of the fragments in to pUC118 to make E. coli HB101 (pPEMP01NS) with following two-step chromatography using diethylaminoethyl sepharose (DEAE-sepharose) and Sephadex G-100 columns to purify 97-kDa expressed enzyme was performed. Although activity of immobilized ALP NS12 on glass surface was established at temperatures between 70 and 120 °C and pH ranges 4.0-13.0, the optimum temperature and pH were achieved at 100 °C and 11.0, respectively. Enhancement of enzyme activity was obtained in the presence of 5 mM MnCl2 (91 %), CaCl2 (357 %), FeCl2 (175 %), MgCl2 (94 %), ZnCl2 (412 %), NiCl (86 %), NaCl (239 %), and Na-sulfate (81 %) while inhibition was observed with EDTA (5 mM), PMSF (3 mM), urea (8 M), and SDS (1 %) at 65, 37, 33, and 42 %, respectively. Consequently, the enzyme was well analyzed using crystallography and protein modeling. ALP NS12 can be applied in industrial processes at extreme temperatures and under highly basic conditions, chelators, and detergents.
Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Human infection with Plasmodium knowlesi is widely distributed in Southeast Asia. Merozoite surface protein-1₁₉ (MSP-1₁₉), which plays an important role in protective immunity against asexual blood stage malaria parasites, appears as a leading immunogenic antigen of Plasmodium sp. We evaluated the sensitivity and specificity of recombinant P. knowlesi MSP-1₁₉ (rMSP-1₁₉) for detection of malarial infection. rMSP-1₁₉ was expressed in Escherichia coli expression system and the purified rMSP-1₁₉ was evaluated with malaria, non-malaria and healthy human serum samples (n = 215) in immunoblots. The sensitivity of rMSP-1₁₉ for detection of P. knowlesi, Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale infection was 95.5%, 75.0%, 85.7% and 100%, respectively. rMSP-1₁₉ did not react with all the non-malaria and healthy donor sera, which represents 100% specificity. The rMSP-1₁₉ could be used as a potential antigen in serodiagnosis of malarial infection in humans.
The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.
The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
Universal access to clean water has been a global ambition over the years. Photocatalytic water disinfection through advanced oxidation processes has been regarded as one of the promising methods for breaking down microbials. The forefront of this research focuses on the application of metal-free photocatalysts for disinfection to prevent secondary pollution. Graphitic carbon nitride (g-C3 N4 ) has achieved instant attention as a metal-free and visible-light-responsive photocatalyst for various energy and environmental applications. However, the photocatalytic efficiency of g-C3 N4 is still affected by its rapid charge recombination and sluggish electron-transfer kinetics. In this contribution, two-dimensionally protonated g-C3 N4 was employed as metal-free photocatalyst for water treatment and demonstrated 100 % of Escherichia coli within 4 h under irradiation with a 23 W light bulb. The introduction of protonation can modulate the surface charge of g-C3 N4 ; this enhances its conductivity and provides a "highway" for the delocalization of electrons. This work highlights the potential of conjugated polymers in antibacterial application.
The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.
Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.
Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/growth & development
HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA.
Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmonella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size fractions were total or unfiltered, <250 μm, <20 μm, <2 μm, <0.7 μm, <0.2 μm and <0.02 μm. We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protist-bacteria coupling via the following correlations: Protistan bacterivory-Bacterial Production: r = 0.773, df = 11, p < 0.01; Protist-Bacteria: r = 0.586, df = 12, p < 0.05. However none of the bacterial decay rates were correlated with the biotic variables measured. E. coli and Salmonella decay rates were generally higher in the larger fraction (>0.7 μm) than in the smaller fraction (<0.7 μm) suggesting the more important role played by protists. E. coli and Salmonella also decreased in the <0.02 μm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella.
To overcome the biofouling challenge which faces membrane water treatment processed, the novel superhydrophobic carbon nanomaterials impregnated on/powder activated carbon (CNMs/PAC) was utilized to successfully design prepare an antimicrobial membrane. The research was conducted following a systematic statistical design of experiments technique considering various parameters of composite membrane fabrication. The impact of these parameters of composite membrane on Staphylococcus aureus growth was investigated. The bacteria growth was analyzed through spectrophotometer and SEM. The effect of CNMs' hydrophobicity on the bacterial colonies revealed a decrease in the abundance of bacterial colonies and an alteration in structure with increasing the hydrophobicity. The results revealed that the optimum preparative conditions for carbon loading CNMs/PAC was 363.04 mg with a polymer concentration of 22.64 g/100 g, and a casting knife thickness of 133.91 μm. These conditions have resulted in decreasing the number of bacteria colonies to about 7.56 CFU. Our results provided a strong evidence on the antibacterial effect and consequently on the antibiofouling potential of CNMs/PAC in membrane.
Surface acoustic wave mediated transductions have been widely used in the sensors and actuators applications. In this study, a shear horizontal surface acoustic wave (SHSAW) was used for the detection of food pathogenic Escherichia coli O157:H7 (E.coli O157:H7), a dangerous strain among 225 E. coli unique serotypes. A few cells of this bacterium are able to cause young children to be most vulnerable to serious complications. Presence of higher than 1cfu E.coli O157:H7 in 25g of food has been considered as a dangerous level. The SHSAW biosensor was fabricated on 64° YX LiNbO3 substrate. Its sensitivity was enhanced by depositing 130.5nm thin layer of SiO2 nanostructures with particle size lesser than 70nm. The nanostructures act both as a waveguide as well as a physical surface modification of the sensor prior to biomolecular immobilization. A specific DNA sequence from E. coli O157:H7 having 22 mers as an amine-terminated probe ssDNA was immobilized on the thin film sensing area through chemical functionalization [(CHO-(CH2)3-CHO) and APTES; NH2-(CH2)3-Si(OC2H5)3]. The high-performance of sensor was shown with the specific oligonucleotide target and attained the sensitivity of 0.6439nM/0.1kHz and detection limit was down to 1.8femto-molar (1.8×10(-15)M). Further evidence was provided by specificity analysis using single mismatched and complementary oligonucleotide sequences.
Ultraviolet (UV) radiation causes damage in all living organisms, including DNA damage that leads to cell death. Herein, we provide a new technique for UV radiation protection through intracellular short peptide expression. The late embryogenesis abundant (LEA) peptide, which functions as a shield that protects macromolecules from various abiotic stress, was obtained from the Polypedilum vanderplanki group 3 LEA protein. Recombinant Escherichia coli BL21 (DE3) expressing functional LEA short peptide in vivo were exposed to UVA and UVC radiation for 4, 6, and 8 h. E. coli transformants expressing the LEA peptide showed higher cell viability under both UVA and UVC treatment at all time points as compared with that of the control. Furthermore, the cells expressing LEA peptide showed a higher number of colony-forming units per dilution under UVA and UVC treatment. These results suggested that expression of the short peptide could be useful for the development of genetically modified organisms and in applications that require resilience of organisms to UV radiation.
A number of novel 5-substituted-2-((6-bromo-3,4-methylenedioxybenzyl)thio)-1,3,4-Oxadiazole derivatives (6a-l) have been synthesized to evaluate their antibacterial activity. Using aryl/aralkyl carboxylic acids (1a-l) as precursors, 5-substituted-1,3,4-Oxadiazol-2-thiols (4a-l) were yielded in good amounts. The derivatives, 4a-l, were subjected to electrophilic substitution reaction on stirring with 6-bromo-3,4-methylenedioxybenzyl chloride (5) in DMF to synthesize the required compounds. All the synthesized molecules were well characterized by IR, 1H-NMR, 13C-NMR and EIMS spectral data and evaluated for antibacterial activity against some bacterial strains of Gram-bacteria. The molecule, 6d, demonstrated the best activity among all the synthesized molecules exhibiting weak to moderate inhibition potential.
Matched MeSH terms: Escherichia coli/drug effects; Escherichia coli/growth & development
Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.
Microbial remediation has the potential to inexpensively yet effectively decontaminate and restore contaminated environments, but the virulence of pathogens and risk of resistance gene transmission by microorganisms during antibiotic removal often limit its implementation. Here, a cloned tetX gene with clear evolutionary history was expressed to explore doxycycline (DOX) degradation and resistance variation during the degradation process. Phylogenetic analysis of tetX genes showed high similarity with those of pathogenic bacteria, such as Riemerella sp. and Acinetobacter sp. Successful tetX expression was performed in Escherichia coli and confirmed by SDS-PAGE and Western blot. Our results showed that 95.0 ± 1.0% of the DOX (50 mg/L) was degraded by the recombinant strain (ETD-1 with tetX) within 48 h, which was significantly higher than that for the control (38.9 ± 8.7%) and the empty plasmid bacteria (8.8 ± 5.1%) (P 0.05). The efficient and safe DOX-degrading capacity of the recombinant strain ETD-1 makes it valuable and promising for antibiotic removal in the environment.
Escherichia coli (E. coli) from the B2 phylogenetic group is implicated in colorectal cancer (CRC) as it possesses a genomic island, termed polyketide synthetase (pks), which codes for the synthesis of colibactin, a genotoxin that induces DNA damage, cell cycle arrest, mutations and chromosomal instability in eukaryotic cells. The aim of this study was to detect and compare the prevalence of E. coli expressing pks (pks+ E. coli) in CRC patients and healthy controls followed by investigating the virulence triggered by pks+ E. coli using an in-vitro model. Mucosal colon tissues were collected and processed to determine the presence of pks+ E. coli. Thereafter, primary colon epithelial (PCE) and colorectal carcinoma (HCT116) cell lines were used to detect cytopathic response to the isolated pks+ E. coli strains. Our results showed 16.7% and 4.3% of CRC and healthy controls, respectively were pks+ E. coli. Further, PCE displayed syncytia and cell swelling and HCT116 cells, megalocytosis, in response to treatment with the isolated pks+ E. coli strains. In conclusion, pks+ E. coli was more often isolated from tissue of CRC patients compared to healthy individuals, and our in-vitro assays suggest these isolated strains may be involved in the initiation and development of CRC.
Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.
Biofilms are complex microbial communities that tend to attach to either biotic or abiotic surface. Enclosed in a self-produced extracellular polymeric substance (EPS) matrix, the biofilms often cause persistent infections. The objective of this study was to investigate the antibiofilm activity of dimethyl sulfoxide (DMSO) and afatinib against Gram-negative pathogens. Test microorganisms used in this study were Escherichia coli ATCC 1299, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhimurium ATCC 14028. Biofilms were developed in 96-well microplate at 37°C for 24 h. Following removal of non-adherent cells, analysis of biofilm viability, biofilm biomass, and extracellular polymeric substances (EPS) matrix were performed using resazurin assay, crystal violet assay, and attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy, respectively. Bradford protein assay was conducted to determine the total amount of EPS proteins. The results demonstrated that both 32% DMSO alone and its combination with 3.2 μg/mL afatinib were effective in killing biofilm cells and reducing biofilm biomass. IR spectral variations of EPS matrix of biofilms in the range between 1700 and 900 cm-1 were also observed. Reduction in EPS proteins verified the chemical modifications of EPS matrix. In conclusion, 32% DMSO alone and its combination with 3.2 μg/mL afatinib showed remarkable antibiofilm activities against Gram-negative pathogens. It was suggested that the biofilm inhibition was mediated by the chemical modification of EPS matrix.