Lipoic acid metabolism in Leishmania major

Protozoan parasites of the genus Leishmania are the causative agents of a complex of diseases referred to as leishmaniasis. Leishmania have a digenetic life cycle that involves a sand fly vector (promastigote stage) and a mammalian host (amastigote stage). The parasites reside within very different environmental niches in the two different hosts, and therefore must be able to adapt their energy metabolism to the available carbon and nitrogen sources. Lipoic acid (LA) is a multifaceted molecule, and plays an important role as a water- and fat-soluble antioxidant. LA is also an essential cofactor of the alpha-ketoacid dehydrogenase complexes (alpha-KADHs) and of the glycine cleavage complex (GCC). The alpha-KADHs include the pyruvate dehydrogenase (PDH), branched-chain alpha-ketoacid dehydrogenase (BCKDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH), each of which is integral to cellular energy metabolism. In some organisms, LA can be acquired through salvage and biosynthesis pathways, and yet others only encode enzymes that permit one of the two pathways. Lipoylation of the PDH has been demonstrated in a parasite related to Leishmania called Trypanosoma brucei; however there have not been any investigations into the enzymes involved in LA metabolism in either Leishmania or Trypanosoma brucei. In silico analyses identified genes encoding for proteins involved in both LA biosynthesis and salvage (lipoic acid synthase (LIPA), octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LIPB) and lipoate protein ligase (LPLA), respectively), and it was predicted that all three proteins possess mitochondrial targeting peptides. Targeting of these proteins to the mitochondrion was verified by a green fluorescence protein (GFP) reporter system, and by subcellular pre-fractionation using digitonin followed by western blotting. Functionality of L. major putative LIPA, LIPB and LPLA genes was determined by showing that the genes complemented the no-growth phenotype of bacteria deficient in either lipA or lipB genes on minimal medium. Bioinformatics analyses also showed that L. major possesses genes encoding all of the subunits comprising the different alpha-KADHs and the GCC, and the subunits were predicted to possess mitochondrial targeting peptides. Western blotting of promastigote protein with an antibody recognising protein-bound LA (alpha-LA antibody) identified four proteins, which based upon predicted molecular sizes, correspond to the lipoylated transacylase subunits of the three alpha-KADHs and the H-protein of the GCC. Interestingly, the lipoylation pattern changes throughout promastigote growth in vitro, with alpha-KGDH being lipoylated throughout promastigote life while PDH and BCKDH are not lipoylated and presumably not active in metacyclic promastigotes. These findings indicate that modification of alpha-KADHs and the GCC by lipoylation is a dynamic process, possibly reflecting adaptations in the parasite’s energy metabolism during their developmental cycle. Three approaches were taken to study the relative importance of the LA biosynthesis and salvage pathways in L. major promastigotes. First, LA analogues 8’ bromooctanoic acid (8-BOA) and octanoic acid (OA) were tested for their effects on growth in L. major maintained in lipid-depleted medium. The IC50 for 8-BOA was relatively high when compared to that determined in other organisms, suggesting that LA biosynthesis can compensate for a decrease in LA salvage in medium deficient in LA. Second, attempts to replace either LIPA or LPLA genes with selectable markers were unsuccessful. LPLA could however, be knocked-out when an extra copy of the gene was introduced into the parasite’s genome. These data suggest that both LA acquisition pathways might be essential for promastigote growth and development. Third, overexpression of C-terminal His-tagged versions of LIPB (LIPB-His), LPLA (LPLA-His) and a LPLA active site mutant, LPLAH118A (LPLAH118A-His), resulted in slow-growth phenotypes. Overexpression of LIPB-His and LPLAH118A-His resulted in lipoylation of the PDH and BCKDH in metacyclic promastigotes, which is not observed in wild-type metacyclic promastigotes. It is hypothesised that LA biosynthesis and salvage enzymes could have differential substrate-specificities in L. major. A number of avenues require further investigation, including the mechanism that permits a relatively rapid turnover of lipoylated protein, and whether lipoylation patterns differ depending upon the carbon sources that are provided in the growth medium. Also, it will be interesting to determine whether LIPB and LPLA have intrinsic substrate-specificities, and whether this is sufficient to explain the fact that both LIPA and LPLA are essential in the promastigote stage in vitro

Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species

An RFX-binding site is shown to be conserved in the promoters of a subset of ciliary genes and a subsequent screen for this site in two Drosophila species identified novel RFX target genes that are involved in sensory ciliogenesis

Systematic, continental scale temporal monitoring of marine pelagic microbiota by the Australian Marine Microbial Biodiversity Initiative

Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and processed fashion. Currently 952 samples are publically available for bacteria and archaea which include 88,951,761 bacterial (72,435 unique) and 70,463,079 archaeal (24,205 unique) 16 S rRNA v1-3 gene sequences, and 388 samples are available for eukaryotes which include 39,801,050 (78,463 unique) 18 S rRNA v4 gene sequences

"Reporting The Media":The Occupational Subculture Of The Sports Journalist

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (> 90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.Publisher PDFPeer reviewe

Soil DNA chronosequence analysis shows bacterial community re‐assembly following post‐mining forest rehabilitation

Mining activities modify both aboveground and belowground ecological communities, presenting substantial challenges for restoration. The soil microbiome is one of these impacted communities and performs important ecosystem functions but receives limited focus in restoration. Sequencing soil DNA enables accurate and cost-effective assessment of soil microbiota, allowing for comparisons across land use, environmental, and temporal gradients. We used amplicon sequencing of the bacterial 16s rRNA gene extracted from soil samples across a 28-year post-mining rehabilitation chronosequence to assess soil bacterial composition and diversity following rehabilitation at a bauxite mine in Western Australia's jarrah forest. We show that while bacterial alpha diversity did not differ between reference and rehabilitated sites, bacterial community composition changed dramatically across the chronosequence, suggesting strong impacts by mining and rehabilitation activities. Bacterial communities generally became increasingly similar to unmined reference sites with time since rehabilitation. Soil from sites rehabilitated as recently as 14 years ago did not have significantly different communities to reference sites. Overall, our study provides evidence indicating the recovery of soil bacterial communities toward reference states following rehabilitation. Including several ecological reference sites revealed substantial natural variability in bacterial communities from within a single mine site. We urge future restoration chronosequence studies to sample reference sites that geographically span the restored sites and/or are spatially paired with restored sites to ensure this variability is captured and to improve any inferences on recovery

Next generation restoration metrics: using soil eDNA bacterial community data to measure trajectories towards rehabilitation targets

In post-mining rehabilitation, successful mine closure planning requires specific, measurable, achievable, relevant and time-bound (SMART) completion criteria, such as returning ecological communities to match a target level of similarity to reference sites. Soil microbiota are fundamentally linked to the restoration of degraded ecosystems, helping to underpin ecological functions and plant communities. High-throughput sequencing of soil eDNA to characterise these communities offers promise to help monitor and predict ecological progress towards reference states. Here we demonstrate a novel methodology for monitoring and evaluating ecological restoration using three long-term (>25 year) case study post-mining rehabilitation soil eDNA-based bacterial community datasets. Specifically, we developed rehabilitation trajectory assessments based on similarity to reference data from restoration chronosequence datasets. Recognising that numerous alternative options for microbiota data processing have potential to influence these assessments, we comprehensively examined the influence of standard versus compositional data analyses, different ecological distance measures, sequence grouping approaches, eliminating rare taxa, and the potential for excessive spatial autocorrelation to impact on results. Our approach reduces the complexity of information that often overwhelms ecologically-relevant patterns in microbiota studies, and enables prediction of recovery time, with explicit inclusion of uncertainty in assessments. We offer a step change in the development of quantitative microbiota-based SMART metrics for measuring rehabilitation success. Our approach may also have wider applications where restorative processes facilitate the shift of microbiota towards reference states

Comparison of the DCBB set of genes with the Ciliary proteome and Ciliome databases

Venn diagram presenting the overlaps between the three datasets: the cilia proteome [46,48]; the ciliome [47,49], and the DCBB (Additional data file 2). Asterisks indicate this study. Note that only 412 common genes are found in the three datasets. The number of genes also found in the 1,462, 412 or 83 X-box gene lists (Table 4), respectively, are noted in parentheses. The numbers of genes selected in the different studies to construct each dataset are given in Additional data file 3.<p><b>Copyright information:</b></p><p>Taken from "Identification of novel regulatory factor X (RFX) target genes by comparative genomics in species"</p><p>http://genomebiology.com/2007/8/9/R195</p><p>Genome Biology 2007;8(9):R195-R195.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2375033.</p><p></p