A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species

Catálogo Taxonômico da Fauna do Brasil: setting the baseline knowledge on the animal diversity in Brazil

The limited temporal completeness and taxonomic accuracy of species lists, made available in a traditional manner in scientific publications, has always represented a problem. These lists are invariably limited to a few taxonomic groups and do not represent up-to-date knowledge of all species and classifications. In this context, the Brazilian megadiverse fauna is no exception, and the Catálogo Taxonômico da Fauna do Brasil (CTFB) (http://fauna.jbrj.gov.br/), made public in 2015, represents a database on biodiversity anchored on a list of valid and expertly recognized scientific names of animals in Brazil. The CTFB is updated in near real time by a team of more than 800 specialists. By January 1, 2024, the CTFB compiled 133,691 nominal species, with 125,138 that were considered valid. Most of the valid species were arthropods (82.3%, with more than 102,000 species) and chordates (7.69%, with over 11,000 species). These taxa were followed by a cluster composed of Mollusca (3,567 species), Platyhelminthes (2,292 species), Annelida (1,833 species), and Nematoda (1,447 species). All remaining groups had less than 1,000 species reported in Brazil, with Cnidaria (831 species), Porifera (628 species), Rotifera (606 species), and Bryozoa (520 species) representing those with more than 500 species. Analysis of the CTFB database can facilitate and direct efforts towards the discovery of new species in Brazil, but it is also fundamental in providing the best available list of valid nominal species to users, including those in science, health, conservation efforts, and any initiative involving animals. The importance of the CTFB is evidenced by the elevated number of citations in the scientific literature in diverse areas of biology, law, anthropology, education, forensic science, and veterinary science, among others

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase.

Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were "transcriptional regulation", "protein post-translational modifications" and "lipid metabolism". Further investigation of the "transcriptional regulation" cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators

Candidate Acc1p interactors with their respective soraphen A sensitivity scores.

<p>Candidate Acc1p interactors with their respective soraphen A sensitivity scores.</p

<i>SOR2</i> deletion reduced Acc1p activity and plasmid loss are not directly linked.

<p>(A) Strains transformed with pYC (empty vector) or pSNF1 (pYC::SNF1-3HA), both with <i>URA3</i> as a genetic marker, were grown for 48 h in liquid SD medium in the presence of selective pressure (without uracil supplementation). The percentage of plasmid-carrying cells was quantified by plating 200 cells of each culture on SC agar +ura or–ura, and colony counting after 3 da. For WT + pYC and WT + pYC:SNF1, n = 1; for <i>sor2Δ</i> + pYC and <i>sor2Δ</i> + pYC:SNF1, n = 3 (three different isolated clones were tested). (B) WT strain carrying pYC (empty vector) was grown for 48 h in liquid SD medium in the presence of selective pressure (without uracil), with the indicated concentrations of soraphen A. Plasmid stability was assessed as in (B) and the data are presented as the means from two experiments.</p

Acyl composition in total lipid extracts of WT and <i>SOR</i> null mutants after 24 h growth in rich YPD medium, as determined by GCMS.

<p>Acyl composition in total lipid extracts of WT and <i>SOR</i> null mutants after 24 h growth in rich YPD medium, as determined by GCMS.</p

<i>ACC1</i> transcriptional levels do not correlate with soraphen A sensitivity.

<p>Mutant strains with deletion of genes encoding transcriptional factors (TFs) that affect <i>ACC1</i> transcript levels were retested for soraphen A sensitivity as before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.g003" target="_blank">Fig 3</a>). Published <i>ACC1</i> transcript levels in these mutants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref032" target="_blank">32</a>] are shown. TFs mutant strains are marked in green or red, depending on whether <i>ACC1</i> transcription was down-regulated or up-regulated, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref031" target="_blank">31</a>]. TF mutant strains identified by our screen but with unaltered <i>ACC1</i> transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref030" target="_blank">30</a>] are marked in black. The <i>lys2Δ</i> strain was employed as a control. The results are representative of four independent experiments. ^{1, 2} Two <i>yjl206cΔ</i> strains were present in our collection and both were tested.</p