In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB ؊ ) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme. IMPORTANCECyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the synthesis of active uptake systems. The Pst phosphate transport system is one such system, responsible for the internalization of phosphate when cells are in phosphate-limited environments. Our investigations reveal the presence of multiple Pst phosphate uptake systems that exist across three distinct operons in Nostoc punctiforme and functionally characterize the role of the gene product PstB1 as being crucial for the maintenance of phosphate accumulation. We demonstrate that the genes pstB2, pstB3, and pstB4 show alterations in expression to compensate for the deletion of pstB1. The overall outcomes of this work provide insights as to the complex transport mechanisms that exist in cyanobacteria like N. punctiforme, allowing them to thrive in low-phosphate environments. P hosphorus (P) is a crucial element for the survival and growth of all organisms, and it is commonly associated with oxygen in the form of inorganic phosphates (PO 4 Ϫ and P i ) in the environment. Phosphate has a central role in many of the metabolic and energy-producing pathways in ...

Over-expression of a GFP-PfRab1A fusion protein in Plasmodium falciparum schizonts produces a punctate pattern of fluorescence typical of rhoptries, secretory organelles involved in host cell invasion. The GFP-positive bodies were purified by a combination of differential and density gradient centrifugation and their protein content determined by MS/MS sequencing. Consistent with the GFP rhoptry-like pattern of transgenic parasites, four of the 19 proteins identified have been previously described to be rhoptry-associated and another four are ER or ER-associated proteins. Confirmation that GFP-PfRab1A decorates rhoptries was obtained by its co-localization with Rap1 and Ron4 in late phase schizonts. We conclude that PfRab1A potentially regulates vesicular traffic from the endoplasmic reticulum to the rhoptries in Apicomplexa parasites.

Malaria plagues one out of every 30 humans and contributes to almost a million deaths, and the problem could worsen. Our current therapeutic options are compromised by emerging resistance by the parasite to our front line drugs. It is thus imperative to better understand the basic biology of the parasite and develop novel drugs to stem this disease. The most facile approach to analyse a gene's function is to remove it from the genome or inhibit its activity. Although genetic manipulation of the human malaria parasite Plasmodium falciparum is a relatively standard procedure, there is no optimal method to perturb genes essential to the intraerythrocytic development cycle--the part of the life cycle that produces the clinical manifestation of malaria. This is a severe impediment to progress because the phenotype we wish to study is exactly the one that is so elusive. In the absence of any utilitarian way to conditionally delete essential genes, we are prevented from investigating the parasite's most vulnerable points. This review aims to focus on the development of tools identifying essential genes of P. falciparum and our ability to elicit phenotypic mutation.

No abstract