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Articles by issue : Vol 1, No 3 (2007): December 2007
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Molecular Dynamics Analysis of Thermostable DNA Pol I ITB-1

HERTADI, RUKMAN ( Institut Teknologi Bandung ) , NURBAITI, SANTI ( Institut Teknologi Bandung ) , AKHMALOKA, . ( Institut Teknologi Bandung )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

One of the thermostable enzymes, which has been widely used in the biotechnological research, is DNA polymerase. The coding sequence of local DNA Pol I gene from a local thermophilic bacterium, namely DNA Pol I ITB-1, has been cloned, sequenced, and overexpressed. However, study on thermostability of this enzyme is very limited. In the present study, thermostability of the protein was evaluated by thermal unfolding simulation at 300, 400, and 500 K. Our simulation revealed that the secondary and tertiary structures of the protein was not significantly affected by thermal perturbation at 300 K, but they were affected and even gradually unfolded by that perturbation at 400 and 500 K. Evaluation of the root mean square fluctuation (RMSF) of individual residues from the simulation at 400 and 500 K revealed the distribution of the thermostability regions in the protein structure. From the RMSF analysis at 400 K, we found that thermostability of the 3’-5’ exonuclease domain was lower compared to that of the other domains. Where as from the RMSF analysis at 500 K, we found that in each domain of DNA pol I ITB-1 there was a single extraordinary thermostable a-helix which was likely to be the core of each corresponding domain. Thus our simulation provides a thermostability map of DNA Pol I ITB-1. Such information will be very valuable for the next genetic engineering work in determining a mutation target to modify thermostability of this enzyme.

Production of Recombinant Vaccine Cb Peritrophin-42 of Screwworm Fly in Escherichia coli and Saccharomyces cerevisiae

NATALIA, DESSY ( Institut Teknologi Bandung ) , MUHARSINI, SRI ( Research Institute for Veterinary Science, ) , MASDUKI, FIFI FITRIAH ( Institut Teknologi Bandung ) , WARDHANA, APRIL HARI ( Research Institute for Veterinary Science, ) , WARDANI, SAVIRA EKA ( Institut Teknologi Bandung ) , MARIA, ELIZABETH ( Sentra Biosains Dinamika ) , VAN DEN HEUVEL, JOOP ( German Research Centre for Biotechnology )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

The screwworm fly (Chrysomya bezziana) larva is an obligate parasite which causes myasis in mammals. Vaccination is thought to be a protective and an enviromentally friendly method for combating the pest. A gene encoding a peritrophic membrane protein Cb peritrophin-42 of C. bezziana was cloned and expressed in Escherichia coli and Saccharomyces cerevisiae.Cb peritrophin-42 fused with the outer membrane protein A signal sequence was produced as an aggregate in E. coli. Expression of an Cb peritrophin-42 gene fused with oligonucleotide of the invertase signal sequence in S. cerevisiae allowed the production of 14.4 mg l-1 soluble extracellular Cb peritrophin-42. Sheep vaccinated with recombinant Cb peritrophin-42 showed a strong immunological reaction. In vivo assay following vaccination with the recombinant Cb peritrophin-42 showed a 27% reduction in the weight of recovered larvae.

Thermostable Chicken Feather Degrading Enzymes from L-23 Isolate from Indonesia

LINTANG, ROSITA ( Universitas Sam Ratulangi ) , SUHARTONO, MAGGY THENAWIJAYA ( Bogor Agricultural University ) , HWANG, JAE KWAN ( Yonsei University Korea ) , PYUN, YU RYANG ( Yonsei University Korea )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

The thermostable chicken feather degrading protease enzymes used here was extracted and partially purified from thermophilic bacteria L-23 isolated from a coastal hot spring in North Sulawesi, Indonesia. The L-23 was grown in the selective medium containing 1% chicken feather powder at 70 °C and pH 7. The cell-free culture was precipitated with ammonium sulphate at 80% saturation, followed by heating at 65 °C for 1 h before applied onto Sephadex G-100. The molecular weight of the two enzymes identified were estimated as 47 and 64 kDa. The optimum pH of the mixed enzymes preparation was 7 while the optimum temperature was 65 °C. Zymogram analysis showed that one of the enzymes was still active after being heated at 100 °C for 20 min and was also resistant towards organic solvents and SDS. The activity was enhanced by addition of 1 mM FeCl3.

Multiplex PCR for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia

NOVIANA, HERA ( Institut Teknologi Bandung ) , NURACHMAN, ZEILY, RAMDANI, MAELITA, NOER, AS

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

Mycobacterium tuberculosis resistant to rifampin and isoniazid, known as multidrug-resistant M. tuberculosis (MDR-TB) strains, is an emerging problem of great importance to public health, with higher mortality rates than for drug-sensitive strains. Rifampin resistance is due to mutations on the hot spot region of the rpoB gene, especially at positions 526 and 531, and isoniazid resistance is due to mutation on katG at position 315. Mechanisms of resistance are an appropriate target for molecular genotyping diagnostic methods. Here we examined the multiplex PCR assays for the rapid detection targeting rpoB526, rpoB531, and katG mutations. Sixty-one M. tuberculosis strains were studied based on rpoB526, rpoB531, and katG315 assays employing multiplex PCR. Of the 61 strains, the susceptibility tests determined 42 isolates were MDR-TB strains, 10, 4, and 5 isolates were resistant to rifampin, isoniazid, and at least to six drugs which prescibed for TB, respectively. The mutation profiles of the 42 MDR strains assayed by multiplex PCR were 81 and 38.1% on rpoB and katG, respectively. Six rifampin-resistant isolates (60%) had a mutation on rpoB, 25% isoniazid-resistant isolates had mutation on katG, and 20% of the isolates that were sensitive to all drugs tested had a mutation on rpoB. Sequencing analysis revealed sensitivity of the multiplex PCR assay for rpoB was 98.4% and was 100% for katG. There was a 19% difference between phenotype and genotype properties of all isolates detected. In conclusion, the sensitivity of multiplex PCR method was sufficient for preliminary detection of rpoB and katG mutations, but resistance M. tuberculosis to rifampin and isoniazid were not always conferred by mutated alleles on rpoB and, especially, on katG.

Immunological Detection of Avian Influenza Virus in Infected Ducks by Monoclonal Antibodies Against AIV-H5N1

ASTAWA, NYOMAN MANTIK ( Universitas Udayana ) , WINAYA, IDA BAGUS OKA ( Universitas Udayana ) , AGUSTINI, LUH PUTU ( Disease Investigation Centre Regional VI Denpasar ) , HARTANINGSIH, NINING ( Disease Investigation Centre Regional VI Denpasar )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

In order to establish a detection method for avian influenza virus (AIV) infection in ducks, monoclonal antibodies (MAbs) against the virus were produced. The virus used for the production of the monoclonal antibodies was AIV-H5N1 of Indonesian origin. Immortal mouse myeloma were fused with the lymphocytes derived from the spleen of mice immunized with the virus. The MAbs were tested for their specificity by enzyme linked immunosorbent assay (ELISA) and western blotting using formaldehyde inactivated virus and normal allantoic fluid as a negative control. Twelve MAbs which were specific against AIV were isolated and 8 of them were used for detecting of AIV antigen in duck’s tissues. AIV antigen was detected in paraffin embedded tissues of AIV-infected ducks by immunohistochemistry using MAbs. AIV antigen was not detected in ducks, which were confirmed to be AIV negative. In the infected ducks, high intensity of AIV infection was detected in proventricle gland and small intestine. The AIV antigen with a lesser intensity was also detected in lungs, spleen, and bursa of Fabricius, but hardly detected in muscle, brain, and several other issues. This study shows a clear evidence that MAbs produced in this study are applicable for use in immunological detection of AIV in infected duck tissues.

Selection and Identification of Cellulase-Producing Bacteria Isolated from the Litter of Mountain and Swampy Forest

WIZNA, . ( Universitas Andalas ) , ABBAS, HAFIL ( Universitas Andalas ) , RIZAL, YOSE ( Universitas Andalas ) , DHARMA, ABDI, KOMPIANG, I PUTU ( Research Institute for Animal Production )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

The isolation and selection of cellulase-producing bacteria was conducted to identify the species of cellulolytic Bacillus. The bacteria were isolated from the litter of swampy forest in Pesisir Selatan and mountain forest in Lembah Anai Tanah Datar.  These bacteria were cultivated in selective media to obtain bacteria from the genus Bacillus. Six Bacillus isolates were obtainedfrom swampy forest and three Bacillus isolates from mountain forest. These isolates were cultivated in agar medium with carboxymethylcellulose as the carbon source. Colonies which produced clear zones were assumed to be cellulolytic Bacillus.  Based on biochemical and morphological examinations the result indicated that these two isolates were Bacillus coagulans and B. amyloliquefaciens. The cellulase activity of B. coagulans and B. amyloliquefaciens were 0.812 and 1 200 unit ml-1 to C1(b-exoglucanase) respectively, 0.368 and 0.488 unit ml-1 to Cx(b-endoglucanase) respectively.

Isolation and Screening of Endophytic Microbes from Morinda citrifolia and their Ability to Produce Anti-Microbial Substances

KUMALA, SHIRLY ( Universitas Pancasila ) , SISWANTO, ENDRO BUDI ( Universitas Pancasila )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

Assaying for, and isolation and screening of endophytic microbes from the Morinda citrifolia plant and their ability to produce anti-microbial substances was carried out. Endophytic microbes are microorganisms that live asymptomatically within the living tissue of host plants. Microorganisms such as bacteria, fungi, and yeast can be associated with the host and produce secondary metabolites. These secondary metabolites may be enzymes and other bioactive substances with medicinal activity such as anti-arthritic, anti-cancer, and anti-microbial compounds. The aims of these experiments was to investigate the ability of endophytic microbes isolated from M. citrifolia to produce secondary metabolites which can act as anti-microbial agents. A direct seed inoculating technique was used by planting the plant sample onto the surface of Nutrient Agar and Potato-Dextrose Agar. Assessment of their ability to produce anti-microbial substances was conducted by growing the endophyte isolates in Muller Hinton Broth for bacterial isolates, and in Potato-Dextrose Yeast Broth for fungal isolates. The agar diffusion method using paper disk was applied to assay the anti-microbial activity of each substance. The results of the endophyte isolation in these experiments gave five bacterial isolates and eleven fungal isolates. All of the bacterial isolates showed a broad antimicrobial spectrum while ten of the fungal isolate demonstrated broad anti-microbial activity and four out of the ten fungal isolates had activity towards Candida albicans.

Host Plant Mediated the Effect of Phosphorus on the Growth of External Hyphae of Gigaspora margarita

ROHYADI, AGUS ( Universitas Mataram )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

The effect of soil phosphorus (P) administered to the host plant on the growth of external hyphae of Gigaspora margarita was investigated in a glasshouse experiment using pots divided into three compartments i.e. one for donor plants (DPC), one for external fungal hyphae (EHC) and one for receiver plants (RPC) respectively. The DPC was filled with a sterilized soil/sand mix previously set up to have 5, 16 or 26 mg Bray-1 P kg-1 soil (P1, P2,or P3; categorized as low, intermediate or high level of P-availability) at pH 5.3 and inoculated with and without the fungal inoculums, while the RPC was filled with P3 without inoculation. Two pre-germinated seeds of cowpea were then grown there for 2 weeks before filling the EHC with the original sterilized soil/sand mix having pH 4.6 and 12 mg Al3+ kg-1 soil. These plants were harvested after further grown for 4-8 weeks. P fertilizer induced different growth conditions of host plants, which could control the production of external hyphae by the fungal partner. In supporting G. margarita to develop an optimum extent of external hyphae in acidic soils with a toxic level of Al3+, cowpea plants required soil P availabilities at about the intermediate level.

SHORT COMMUNICATION: On Scientific Publications

PADLAN, EDUARDO AGUSTIN ( University of the Philippines )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

Maybe we can make our local journals internationally “visible” and thus be included in international indices (the sign of international recognition). That may not be too difficult to achieve. If we could convince our more productive local scientists and our compatriots abroad, who are able toproduce internationally competitive results, to publish seminal papers or review papers in local journals and afterwards cite those (local) papers in their other (international) publications, then the internationalcommunity will become aware of our local science and our local journals. 

In Vitro Recombination of Poliovirus with Coxsackie A Virus Serotype 18 at Downstream Nonstructural Protein-Coding Regions

UTAMA, ANDI ( Lembaga Ilmu Pengetahuan Indonesia ) , SHIMIZU, HIROYUKI ( National Institute of Infectious Diseases )

Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

Many genetic recombinations of poliovirus (PV) are to be found in excreted viruses, including viruses from vaccineassociated paralytic poliomyelitis (VAPP) as well as healthy vaccine recipients. Most recombinations were among different serotypes of PVs. However, recombination can also occur between PV and other enteroviruses. It was predicted that the hot spot of the recombination is in the nonstructural protein-coding regions, but the exact site is may be different in each recombination. We have demonstrated that the construct recombinant virus between PV and coxsackie A virus serotype 11 (CAV-11), or with CAV-17 with recombination site in the N-term of 2C-coding region, were viable. However, the recombination of PV with CAV-18 at this site was not viable. To determine if the recombination between PV and CAV-18 can occur at other sites, eight chimeric cDNAs (between PV [isolate PJ156] and CAV-18 [PJ156/CAV-18]), all having different recombination sites (2C-8, 2C-133, 2C-235, 2C-268, 2C-287, 2C-327, 3A-67, 3C-60) were constructed using the long-PCR method. The cDNA was then transcribed in vitro and then transfected into the HEp-2 cell-line. As expected, the recombinant virus PJ156/CAV-18, with recombination sites 2C-327, 3A-67, and 3C-60 were viable, while all the others were not. The recombinant viruses displayed a slightly smaller plaque size, but  emonstrated quite similar growth as compared to the parental control PJ156. Since analysis for similarity has shown that the homology between PV and CAV-18 was high around these regions, these results supported the copy-choice mechanism of enterovirus recombination.