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The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1) that exhibits ATP- and Zn2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.

SummaryThe apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a D-loop/bi-directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA-protein interactions at a plDNA replication ori region and report the identification of a nuclearencoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJA interacted with the minor groove of the DNA double-helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti-PfDnaJA antibodies confirmed identity of the protein in DNA-binding experiments with organellar protein fractions. The DNA-binding domain of the~69 kDa PfDnaJA lay within the N-terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJA in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non-specific manner. Our results suggest a role for PfDnaJA in replication/repair of the apicoplast genome.

The isolation of good quality metagenomic DNA from diverse soil, in appreciable amount, is a prerequisite for metagenomics. The availability of commercial kits for isolation of genomic DNAs from soil has drastically expedited the application of metagenomics approach for identifying novel sources of industrially important enzymes. The quantitative and qualitative assessment of metagenomic DNA isolated using either the manual method or the kit-based method should be performed prior to its use in downstream applications. The metagenomic DNA isolated from six different soil samples, using three methods, were analyzed in terms of yield, quality and downstream application as template for PCR amplification. The yield of DNA was approximately 3.52, 7.35, and 232.42 μg of DNA per gram of soil sample for the kit-based method, kit-modified method, and manual method, respectively. The manual method seems to be promising based on better yield and lesser humic acid content than the other two methods. The maximum yield was obtained in the soil collected from teak forest with all the three methods, indicating maximum microbial content and diversity. Furthermore, in terms of its suitability as template DNA for PCR amplification using 16S RNA primer, all methods are equally well. Thus, comparative assessment of three methods revealed suitability of manual method based on DNA yield and humic acid content, which could be important for many downstream applications like library preparations during metageomics approach.

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