Supatra Areekit

Characterization of Thermophilic Halotolerant Aeribacillus pallidus TD1 from Tao Dam Hot Spring, Thailand

The bacterial strain TD1 was isolated from Tao Dam hot spring in Thailand. Strain TD1 was Gram positive, rod-shaped, aerobic, motile, and endospore forming. The cell was 2.0–40 μm in length and about 0.4 μm in diameter. The optimum growth occurred at 55–60 °C and at pH 7–8. Strain TD1 was able to grow on medium containing up to 10% NaCl. The DNA G+C content was 38.9 mol%. The cellular fatty acid content was mainly C16:0, which comprised 25.04% of the total amount of cellular fatty acid. 16S rDNA showed 99% identity to Aeribacillus pallidus DSM 3670T. Bayesian tree analysis strongly supported the idea that strain TD1 is affiliated with genus Aeribacillus, as Aeribacillus pallidus strain TD1. Although the 16S rDNA of A. pallidus strain TD1 is similar to that of A. pallidus DSM 3670T, some physiological properties and the cellular fatty acid profiles differ significantly. A. pallidus strain TD1 can produce extracellular pectate lyase, which has not been reported elsewhere for other bacterial strains in the genus Aeribacillus. A. pallidus strain TD1 may be a good candidate as a pectate lyase producer, which may have useful industrial applications.

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7–8.5, with an optimum pH of 7.5, and at temperatures in the range 30–45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites.

Isolation, cloning and molecular characterization of a thermotolerant xylanase from Streptomyces sp. THW31

A xylan-degrading Streptomyces sp. THW31 was isolated from rubbish compost in Thailand. Analysis of a genomic library of nucleotide sequences from Streptomyces sp. THW31 revealed that the complete open reading frame (ORF) of xylanase ( xlnW31 ) was 999 bp, and this gene encoded a member of the glycosyl hydrolase family 11. Sequence homology of the predicted amino acid sequence encoded by xlnW31 demonstrated that the enzyme consists of a signal peptide, catalytic and substrate-binding domains. The XlnW31 enzyme shared the highest identity (90%) to a xylanase family 11 member from Streptomyces lividans TK24. Cloning and expression of xlnW31 in Escherichia coli resulted in the production and purification of a 31.0 kDa enzyme. Purified XlnW31 show the highest activity at pH 6.0 and at a temperature 65 to 70°C. Enzyme stability tests indicated that XlnW31 retained its activity over a broad pH range (5.0 to 11.0) and at temperatures reaching 60°C for 2 h. Purified XlnW31 also exhibited endo-1,4-β -xylanase activity using xylan as a substrate and bound to insoluble xylan. Hydrolysis of xylan by the xylanase yielded xylobiose as the principal product. Keywords : Gene expression, hemicellulase, Streptomyces , xylan, xylanase African Journal of Biotechnology Vol. 12(4), pp. 427-437

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex

Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL−1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Development of loop-mediated isothermal amplification (LAMP) assay combined with malachite green as a rapid screening test for Candidatus Mycoplasma haemominutum infection in cats

Feline infectious anaemia is caused by a Gram-negative, uncultivable, cell wall-deficient, epierythrocytic parasitic bacteria known as feline haemoplasmas (FHM) in the genus Mycoplasma, namely, Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), and Ca. M. turicensis (CMtc). Here, a loop-mediated isothermal amplification (LAMP) targeting the 16S rRNA gene combined with malachite green (MG) based colorimetric assay was developed for the detection of feline CMhm infection. The limit of detection was determined using a ten-fold serial dilution of recombinant plasmid DNA (from 108 to 1 copies). The result indicated that the LAMP-MG assay could detect as low as 264 copies corresponding to 264 organisms/μl of CMhm feline blood. Comparison between the LAMP-MG and standard polymerase chain reaction (PCR) surveying 105 clinical samples suggested that 17 and 15 samples were positive for CMhm, respectively. Validity of the LAMP-MG assay was assessed and calculated within 95% confidence intervals (CIs). The sensitivity, specificity, prevalence and accuracy were 100.0%, 97.8%, 14.3%, and 98.1%, respectively. The degree of agreement between LAMP-MG and standard PCR assays was 92.6%, with a κ coefficient of 1 (CI: 82.5–100.0%). This LAMP-MG colorimetric assay may be applicable as a rapid screening point-ofcare testing for feline CMhm, as well as for blood donors prior to blood transfusion by using unsophisticated equipment, such as a heating block or water bath. This technique could provide robust results, which are easily distinguished within 60 min after amplification.