Aris Tri Wahyudi

Design and application of a new cryptic-plasmid-based shuttle vector for Magnetospirillum magneticum.

ABSTRACT A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.

Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis.

Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 × 10−7 per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconju gants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1.

Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1

A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microaerobic respiration.

Penapisan Bakteri Filosfer Penghasil Senyawa Bioaktif Anti Xanthomonas oryzae pv. oryzae Penyebab Penyakit Hawar Daun Bakteri pada Padi

Ostrinia furnacalis is a corn stem-borer that develops complete metamorphosis and all stages in life cycle in corn. This research was aimed to examine several biology aspects of O. furnacalis such as life cycle, egg incubaton period, egg fertility, female fecundity, longevity of imago, and copulation time on artificial diet, based on the previous study. The results of the observations showed that the life cycle of artificial-diet-given O. furnacalis was between 27-34 days range. Female fecundity was 16-452 eggs with fertility rate of 61,97% and 3-5 days renge of egg incubation period. Longevity of imago was between 6-11 days range, and the longevity was longer in female compared to the male. The imago of O. furnacalis copulate on 0-3 days after emerge from pupae and the highest number in on the day 1. Copulation time was occurred at 3-8 hour after scotophase commenced and the highest was at third hour. The artificial diet used in this research can be used for O. furnacalis mass rearing purpose and performed shorter length of egg stage until pupal stage compared to mass rearing with natural diet.

Identification of nif D and nif H Genes of Methanotrophic Bacteria from Rice Field

Metanotrophic bacteria have ability to oxidize methane and fix atmospheric nitrogen, hence the bacteria has an important role as a nitrogen source provider on wetland area like rice fields. Nitrogen fixation process is catalyzed by the nitrogenase enzyme complex, encoded by nifD and nifH genes. However, characteristic of these genes from indigenous-methanotrophic bacteria still poorly understood. Hence, nifD and nifH genes of methanotrophic bacteria isolated from rice fields in Indonesia (BGM3, BGM9, SS1, SS3, SS10, ST18, SP3 and INP4) were identified and characterized. Detection of nifH and nifD genes was conducted by polymerase chain reaction (PCR) amplification. nifH and nifD gene sequences were analyzed using BLAST-X and phylogenetic trees were constructed using Neighbour Joining method. Based on nifH sequences analysis, SS1 closely related to Beijerinckia mobilis and SS3, SS10, ST 18 closely related to Beijerinckia indica subsp. indica ATCC 9039, while, BGM3, INP4, and BGM9 related to nifH of uncultured nitrogen-fixing bacterium. In other hand, sequence analysis of nifD gene showed that SS1, SS3, SS10, ST 18 closely related to B. indica subsp. indica ATCC 9039 and BGM3, BGM9, INP4 closely related to Xanthobacter autotrophicus Py2. Identification by 16 SrRNA indicated that SS1, SS3, SS10, and ST18 had closeness to Beijerinckia sp. P310-1, while INP4 closely related to Xanthobacter sp. M5C24.

Involvement of a Gene Encoding Putative Acetate Kinase in Magnetosome Synthesis in Magnetospirillum magneticum AMB-1

A nonmagnetic mutant of Magnetospirillum magneticum AMB-1, designated NMA40, was constructed by mini-Tn5 transposon mutagenesis to identify genes involved in magnetosome synthesis. Transposon delivery was carried out through conjugation between M. magneticum AMB-1 as a recipient and Escherichia coli S17-1 (λ pir) carrying pUTmini-Tn5Km1 as a donor strain. NAM40 did not respond to the magnetic fields and completely lacked of magnetosome in the cell. DNA sequence/gen interrupted by transposon (called flanking DNA) was isolated by inverse PCR and cloned into pGEM-T Easy. Alignment of the DNA sequence of the flanking DNA allowed the isolation of an open reading frame (ORF2) within an operon consisting of three genes. The amino acid sequence deduced from ORF2 showed homology with acetate kinase from Sinorhizobium meliloti (50% identity and 67% similarity), which function for acetate metabolism. Further analysis revealed that upstream of ORF2 is ORF1, had homology with phosphotransacetylase of S. meliloti (67% identity, 77% similarity), and ORF3 located downstream of ORF2, had homology with hypothetical protein of Thermotoga maritima (30% identity, 60% similarity). ORF2 was subsequently isolated, cloned, and overexpressed in Escherichia coli BL21 (DE3) pLysS as an ORF2-Histag fusion polypeptide. Key words: Magnetospirillum magneticum AMB-1, magnetosome synthesis, transposon mutagenesis, cloning, overexpression