Repression of RNA Polymerase II Transcription by a Drosophila Oligopeptide

Background: Germline progenitors resist signals that promote differentiation into somatic cells. This occurs through the transient repression in primordial germ cells of RNA polymerase II, specifically by disrupting Ser2 phosphorylation on its C-terminal domain. Methodology/Principal Findings: Here we show that contrary to expectation the Drosophila polar granule component (pgc) gene functions as a protein rather than a non-coding RNA. Surprisingly, pgc encodes a 71-residue, dimeric, alphahelical oligopeptide repressor. In vivo data show that Pgc ablates Ser2 phosphorylation of the RNA polymerase II C-terminal domain and completely suppresses early zygotic transcription in the soma. Conclusions/Significance: We thus identify pgc as a novel oligopeptide that readily inhibits gene expression. Germ cell repression of transcription in Drosophila is thus catalyzed by a small inhibitor protein

Metabolic adaptation to vitamin auxotrophy by leaf-associated bacteria

Auxotrophs are unable to synthesize all the metabolites essential for their metabolism and rely on others to provide them. They have been intensively studied in laboratory-generated and -evolved mutants, but emergent adaptation mechanisms to auxotrophy have not been systematically addressed. Here, we investigated auxotrophies in bacteria isolated from Arabidopsis thaliana leaves and found that up to half of the strains have auxotrophic requirements for biotin, niacin, pantothenate and/or thiamine. We then explored the genetic basis of auxotrophy as well as traits that co-occurred with vitamin auxotrophy. We found that auxotrophic strains generally stored coenzymes with the capacity to grow exponentially for 1-3 doublings without vitamin supplementation; however, the highest observed storage was for biotin, which allowed for 9 doublings in one strain. In co-culture experiments, we demonstrated vitamin supply to auxotrophs, and found that auxotrophic strains maintained higher species richness than prototrophs upon external supplementation with vitamins. Extension of a consumer-resource model predicted that auxotrophs can utilize carbon compounds provided by other organisms, suggesting that auxotrophic strains benefit from metabolic by-products beyond vitamins.ISSN:1751-7362ISSN:1751-737

The Ustilago maydis a2 Mating-Type Locus Genes lga2 and rga2 Compromise Pathogenicity in the Absence of the Mitochondrial p32 Family Protein Mrb1

The Ustilago maydis mrb1 gene specifies a mitochondrial matrix protein with significant similarity to mitochondrial p32 family proteins known from human and many other eukaryotic species. Compatible mrb1 mutant strains were able to mate and form dikaryotic hyphae; however, proliferation within infected tissue and the ability to induce tumor development of infected maize (Zea mays) plants were drastically impaired. Surprisingly, manifestation of the mrb1 mutant phenotype selectively depended on the a2 mating type locus. The a2 locus contains, in addition to pheromone signaling components, the genes lga2 and rga2 of unknown function. Deletion of lga2 in an a2Δmrb1 strain fully restored pathogenicity, whereas pathogenicity was partially regained in an a2Δmrb1Δrga2 strain, implicating a concerted action between Lga2 and Rga2 in compromising pathogenicity in Δmrb1 strains. Lga2 and Rga2 localized to mitochondria and Mrb1 interacted with Rga2 in the yeast two-hybrid system. Conditional expression of lga2 in haploid cells reduced vegetative growth, conferred mitochondrial fragmentation and mitochondrial DNA degradation, and interfered with respiratory activity. The consequences of lga2 overexpression depended on the expression strength and were greatly exacerbated in Δmrb1 mutants. We propose that Lga2 interferes with mitochondrial fusion and that Mrb1 controls this activity, emphasizing a critical link between mitochondrial morphology and pathogenicity

Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants.ISSN:0027-8424ISSN:1091-649

The 71-residue Pgc oligopeptide readily suppresses an early zygotic transcript in <i>Drosophila</i> somatic nuclei.

<p>(A) Fluorescent <i>in situ</i> hybridization with an antisense RNA probe for <i>CG3502</i>, a transcript in the anterior region of the embryo whose expression starts during the 11^th cleavage cycle. <i>CG3502</i> mRNA accumulates in the nucleus before the 13^th cleavage division (A, B) and redistributes to the cytoplasm during cellularization (C). (D) No detectable <i>CG3502</i> mRNA one hour after purified Pgc is injected into the anterior region of the embryo. The three representative embryos are developmentally beyond the 11^th cleavage division and should thus be expressing <i>CG3502</i>.</p

The <i>pgc</i> locus encodes an alpha-helical monomeric and dimeric oligopeptide protein.

<p>(A) <i>pgc</i> contains two open reading frames, encoding potential 71 and 75 residue oligopeptide proteins (Pgc ORF1 and ORF2, respectively). (B) The two candidate oligopeptides are predicted to contain alpha-helical secondary structure. (C) <i>E. coli</i> expressed and purified Pgc ORF1 is a soluble protein, in contrast to ORF2, which fails to refold <i>in vitro</i>. (D) Size-exclusion chromatography of PGC ORF1 reveals two chromatographic species, one migrating at 18 kDa, the other at 36 kDa, consistent with monomer and dimer fractions of (His)₆-tagged Pgc ORF1. Absorbance signal at 280 nm (blue) and 260 nm (red). (E) Far-UV circular dichroism spectroscopy spectrum of purified, recombinant PGC ORF1 reveals that recombinant Pgc ORF1 protein contains intrinsic alpha-helical structure. Peak *1 (red), corresponding to the dimeric Pgc ORF1 complex, contains a higher alpha-helical content than monomeric Pgc1 (peak *2, blue).</p

Microinjection of recombinant Pgc protein strongly reduces CTD Ser2 phosphorylation in <i>Drosophila</i> embryonic nuclei.

<p>Pgc was injected (arrows) into the anterior region of stage 3–4 <i>Drosophila</i> embryos. Representative images of embryos fixed 30 minutes after the injection of Pgc protein (A–H) or buffer only (I–L). DNA detected by Hoechst stain (purple, A, C, D, G, I, K) and CTD phospho-Ser2 (green, B, E, F, H, J, L). Ser2 phosphorylation strongly decreases at the site of injection (arrow, B). In comparison, somatic cell nuclei in the posterior area of the same embryo show no changes in Ser2 phosphorylation (H). Ser2 phosphorylation is gradually lost when moving away from the site of injection (E, F, arrow, left, denotes anterior region). Ser2 phosphorylation does not decrease with control injections (J) when compared to the uninjected area (L).</p

Phosphorelay through the bifunctional phosphotransferase PhyT controls the general stress response in an alphaproteobacterium.

Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography–mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.ISSN:0021-9258ISSN:1083-351