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Articles by issue : Vol 3, No 1 (2009): April 2009
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Articles
Influenza A Virus: Phylogeny of Neuraminidase Primers and Amplification of Polymerase Basic Protein 2 and Neuraminidase Genes

OMEGA, MARIA, LAI, RICHARD, HEINE, HANS, BARNARD, ROSS

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Influenza A virus is a highly contagious agent that causes bird flu. To date, 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes are identified antigenically and can form any combinations or mutations with each other to confer non or low pathogenic to high pathogenic strains. Mutations in viral segments that are derived from avian isolates represent a novel subtypes to which human population is infected by influenza pandemics. In this work, polymerase basic protein 2 (PB2) gene segment of 8 different avian influenza subtypes were cloned to obtain more DNA samples for future work such as PB2 sequencing and to test HA primer annealing with PB2 gene. PCR amplification of NA gene segment of 3 different avian influenza subtypes was the second aim of this work to test primer universal for NA genes. Determination of the aligned sequences between 9 NA subtypes and NA primer PCR products was the second aim of this work, based on BLAST result homology 100% and phylogenetic trees of clustal

Genetic Diversity of Plant Growth Promoting Rhizobacteria of Bacillus sp. Based on 16S rRNA Sequence and Amplified rDNA Restriction Analysis

SYAMSUL BAHRI, SYAMSUL BAHRI, WAHYUDI, ARIS TRI, MUBARIK, NISA RACHMANIA

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Plant-growth promoting rhizobacteria (PGPR) are rhizosphere associated soil-borne bacteria that can enhance plant growth and inhibit the development of root pathogens. Many soil bacteria have been used as PGPR, and one of them is Bacillus sp. The implementation of PGPR is constrained by genotype fluctuation that makes it inactive on the rhizosphere. Our previous study had characterized and revealed that 11 Bacillus sp. isolated from the soybean plant rhizosphere were PGPR. To asses and compare the genetic diversity of these isolates, Amplified Ribosomal DNA Restriction Analysis (ARDRA) and DNA sequence analysis of 16S rRNA were conducted. The construction of Neighbor-joining trees and bootstrap analysis of 100 resamples of ARDRA and 16S rRNA gene sequences were performed using Treecon software for windows ver. 1.3b. ARDRA analysis was done by using four restriction enzymes (RsaI, HaeIII, CfrI and HinfI), resulting in four phylotypes, respectively phylotype I (Bacillus sp. Cr24, Cr33, Cr64 and Cr68), phylotype II (Bacillus sp. Cr 31 and Cr66), phylotype III (Bacillus sp. Cr44 and Cr71) and phylotype IV (Bacillus sp. Cr67, Cr28 and Cr69). Results of BLASTN from 16S rRNA gene sequences showed that these isolates are genetically diversed. The evolution relationship of Bacillus sp. could be shown by the 16S rRNA gene sequences analysis, while ARDRA based on the digestion sites showed their variability.

Effect of pH, Temperature and Medium Composition on Xylanase Production by Bacillus sp. AQ-1 and Partial Characterization of the Crude Enzyme

WAHYUNTARI, BUDIASIH, MUBARIK, NISA RACHMANIA, SETYAHADI, SISWA

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Bacillus sp. AQ-1 was isolated from household aquarium sediment. The isolate produced extracellular xylanolytic enzymes on xylan containing agar medium. Based on morphological, and physiological analysis, the isolate was identified as Bacillus sp. AQ1. The effect of temperature and pH on isolate growth and xylanase production were observed. The best condition observed for the enzyme production in Luria Broth supplemented with 0.5% oat spelt xylan medium was at 40 °C pH 7. The maximum enzyme production was 0.23 U mL-1 after 20 h of fermentation. Two different medium compositions (A and B) were examined for xylanase production. The maximum growth of the isolate and the xylanase production was better in A medium. Replacing oat spelt xylan in medium A with fruitless oil palm bunch in the medium caused the growth slightly slower than that of in the original formula. However, the xylanase production was 3 times higher in fruitless oil palm bunch medium. Optimum activity of the crude enzyme was observed at 60 °C and pH 7. Each ml of the crude enzyme contained 55.21 U xylanase, 8.12 U amylase and 0.50 U carboxymethylcellulase

Production and Purification of Xylanase From Indonesian Isolate Bacillus sp. AQ-1 Grown on Bunch Palm Oil

RAHAYU, PUJI, SETYAHADI, SISWA, HARMITA, .

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Xylanase (endoxylanase, EC 3.2.1.8) is a commercial enzyme that has been applied in the industrial production of fuel, food, textiles and paper. Xylanase was isolated from the culture supernatant of Bacillus sp. AQ-1 grown on Nakamura medium containing 0.5% powder bunch palm oil. The optimum pH and temperature of xylanase activity were pH 7.0 and 60 °C, respectively. The enzyme was purified by anion exchange chromatography using DEAE-Sepharose-Fast-Flow column and gel filtration chromatography using Sephacryl S-300 column. The results showed that purification of xylanase produced two forms of xylanase, which were identified as xylanase A and xylanase B. Xylanase A can be separated from xylanase B by ultrafiltration using a 30 kDa polyethersulfone membrane. The molecular weight of xylanase A and B were 15.7 and 57.7 kDa, respectively.

The b’x Region of Yeast Protein Disulfide Isomerase is Not Essential for Saccharomyces cerevisiae Viability at 30 °C

PURKAN, ., SAVALAS, LALU RUDYAT TELLY, SINDUMARTA, MULIAWATI, NATALIA, DESSY

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Protein disulfide isomerase (PDI) catalyzes thiol oxidation, reduction and isomerization of disulphide bond of cell surface and secreted proteins. Yeast PDI1 consists of two catalytic domains (a and a’) which are separated by two non-catalytic domains (b and b’), and a x region linked the b’ and a domains. The b’ domain is important for the non-covalent binding of partially folded protein. To understand the contribution of b’ domain and x-linker of yeast PDI1 we have deleted the b’x and investigated its functional role in vitro and in vivo. Yeast PDI1 without b’x region retained only 50% activity and became more sensitive toward Proteinase K. Interestingly, yeasts containing full length PDI1 and pdi1Db’x showed approximately the same growth rate. However, the yeast pdi1Db’x mutant growth impaired severely at 37 °C compared to that of the full length PDI1. Our results suggested that the a-b-a’-c domains of PDI seems to be sufficient to support the growth of yeast cells in normal condition, but the b’x region might be essential in assisting refolding of highly accumulated unfolded protein at high temperature (37 °C).

Detection of Listeria monocytogenes in Pasteurized Milk Sold in Bogor and Its Relationship with Human Health

SANJAYA, AGATHA WINNY, SUDARWANTO, MIRNAWATI, ROBERT, KIBUUKA

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Many countries have established a zero tolerance policy, under which ready-to-eat foods are contaminated with Listeria monocytogenes at a detectable level. The research was done in two parts. The first part was to qualitatively identify the presence of L. monocytogenes in pasteurized milk (n=32 samples) sold in different supermarkets in Bogor. The method was adopted from the Bacteriological Analytical Manual/Food and Drug Administration. All samples tested resulted negative to L. monocytogenes. The second part of the research was to evaluate the growth of L. monocytogenes in sterilized milk stored in an incubator set at 4oC and monitored for 7 days. The original L. monocytogenes culture at a concentration of 1x109 cfu mL-1 was diluted with buffered phosphate water 0.1% to reach a cell concentration of approximately 1.0 x 102 cfu mL-1. Growth was observed on the first, second, third, fourth and fifth day. On the sixth and seventh day, the numbers of colony forming units observed were almost similar (2.5-2.8 x 105 cfu mL-1). A population of 10 cells is sufficient to cause serious listeriosis infection in human.

Membrane Topology of Subunit 8 Variant of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase

ARTIKA, I MADE

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

The yeast mitochondrial F1F0-ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. There is no homologue of subunit 8 found in bacteria. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. Subunit 8 has been shown to adopt a transmembrane topology with the central hydrophobic domain spans the inner mitochondrial membrane once. In order to elucidate the need of subunit 8 to maintain transmembrane topology for its functioning, a severely functionally defective subunit 8 variant that has been introduced with double-charged residues within the central hydrophobic domain was analysed. A gene encoding this variant was expressed in a yeast strain lacking endogenous subunit 8. The subunit 8 variant was then targeted into mitochondria. Following its assembly into mitochondrial ATP synthase complex, its membrane topology was determined. The results obtained showed that subunit 8 was obligatory to maintain a transmembrane topology for providing proper functioning. The transmembrane topology may be critical for subunit 8’s proposed tructural roles as part of the stator stalk of the mitochondrial ATP synthase complex.

Neighboring Plants Alleviate Aluminum Toxicity on The External Hyphae of Gigaspora margarita

ROHYADI, AGUS

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

Excessive soluble aluminum (Al3+) in acidic soils is toxic to the external hyphae of arbuscular mycorrhizal fungi but it can be alleviated by other soil factors. A glasshouse experiment was conducted to study the effect of increased Al3+ concentration on the growth of the external hyphae of Gigaspora margarita in the presence of other plants near the host plants. The experiment used compartmentalized pots to facilitate the growth of mycorrhizal-inoculated host plants, external hyphae of the fungus and not mycorhizal-inoculated neighboring plants in different compartments; and measuring the effects of Al3+ and of the neighboring plants on the growth of the fungal hyphae independently. Increased concentration of Al3+ in soil affected the growth of external hyphae of G. margarita negatively. However, the hyphal length density of the fungus was much higher in the pots with neighboring plants than that in the other ones, despite the Al toxicity. This indicates that the hyphae could be taken away from the toxic effect of Al3+ by the stimulating growth from roots of the neighboring plants.

T-cell Epitopes of Mycobacterium tuberculosis Antigen 85 Complex Potential for Generating Antibody: an Immunoinformatics Study

SIMANJUNTAK, YOGY

Microbiology Indonesia Vol 3, No 1 (2009): April 2009
Publisher : Indonesian Society for microbiology

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Abstract

The present study was conducted to predict T-cell epitopes of the antigen 85 complex capable of stimulating antibody generation by using immunoinformatics approaches. By applying computational biology software, the available data of the antigen 85 complex and related-epitopes would be turned into more constructive and useful scientific informations for the development of multiepitope-based anti-TB vaccine. The identification of T-cell epitopes capable of generating antibody was done by the 3DEX program, discotope analysis and PyMol program. Selected peptides having individual amino acids localized on the predicted antibody-binding sites were subjected to antigenic property analysis, including their hydrophilicity, flexibility and antigenic propensity. The 3DEX program identified 17 peptides having at least four individual amino acids located on the antigen surface. However, after homology analysis with preselected distance of 7 Å and taking into account the spatial neighborship, only seven peptides of antigen 85A, 85B and 85C (3, 3 and 1 peptide(s) respectively) had individual amino acids overlapping the predicted antibody-binding site. Peptides 17838, 21780, 21275 and 36131 had an average score of antigenic propensity above 1.0. In conclusion, there are seven peptides representing T-cell epitope of antigen 85 complex that could potentially be capable of generating an antibody response. The seven peptides, P17838, P21093, P36131, P21275, P21780, P21796 and P10839, are suitable candidates for further study in order to develop a subunit-based multiepitope anti-TB vaccine.