An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1- deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavagemediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts

Towards the development of a sustainable soya bean-based feedstock for aquaculture

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described. Includes supplementary materials

Towards the development of a sustainable soya bean-based feedstock for aquaculture

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described. Includes supplementary materials

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCE mecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance

Functional analysis of the cellular RNA-directed RNA polymerase (RdRP) in higher plants

The cellular RNA-directed RNA polymerase 2 (RdRP^2) full-length cDNA was successfully cloned and its authentic sequence has been deduced from the Lycopersicon esculentum cultivar Rentita (tomato). Insights into the possible biological function of the RdRP^2 were achieved by down-regulating expression of the endogenous tobacco RdRP^2 using RNA-mediated gene silencing technologies. Eight different RdRP sequence-containing constructs were produced and introduced into the Nicotiana tabacum cv. Petita Havana SR1. Results of the transgenic plants carrying RdRP^2-specific IR and DR transgene constructs indicated that the RdRP^2 could be involved in normal gene regulation. Appearance of phenotypic alterations in a plant line carrying a RdRP^2 DR transgene was indicative of partial down-regulation of the RdRP^2. Absence of any phenotypic alterations in plants carrying a RdRP^1-specific DR transgene construct supported the observations and conclusions that were drawn from plant lines carrying the RdRP^2 IR and DR transgenes. Expression of identical phenotypic alterations in plant lines containing RdRP^2 gene fragments, in either sense or antisense orientation, further supported the essential function of the RdRP^2. The same applies for the observation, that Nicotiana plants could not be infected with the recombinant PVX/RdRP^2. The presented data provided indirect experimental evidence that the RdRP^2 gene was indeed down-regulated by a gene silencing mechanism. These experiments included the generation of transgenic plants that carried RdRP gene constructs comprising highly conserved RdRP regions. Transgenic plants were genotypically characterised by Southern and PCR analysis and for most of them, T-DNA copy numbers as well as arrangements of the integrated DNA could be determined. These plants are now available for further experiments. In the frame of this work, homozygous T1 plant lines containing non-rearranged RdRP^2 constructs as a single copy insert were established. In addition to genotypical examinations, transgene expression levels were investigated by Northern analysis and by PCR amplification with cDNA

An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1- deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavagemediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts

Strains and plasmids used in these studies.

(DOCX)</p

ProT is the primary proline transporter in the presence of high salt.

Growth analysis of the following strains in CDM supplemented with 1 M NaCl: A) JE2, penta, Δ4-proT, Δ4-opuC, Δ4-opuD, Δ4-proP, and Δ4-putP B) JE2, penta, ΔproT, ΔopuC, ΔopuD, ΔproP, and ΔputP C) JE2, ΔproC, ΔproT, and ΔproT ΔproC D) JE2 empty vector, penta empty vector, penta Pcad::proT, penta Pcad::opuC, penta Pcad::opuD, penta Pcad::proP, and penta Pcad::putP reveal ProT is important for maximal growth under these conditions. Data are represented by the mean ± SD (n = 2–3).</p

Selection of Isolates Resistant to the Toxic Proline Analog 3,4 dehydro-DL-proline (DHP).

A) A lawn of JE2 ΔopuC ΔopuD ΔproP ΔputP was struck on modified CDM-P agar (see methods and materials). A sterile disk imbedded with 2 μg of DHP was placed in the center and incubated for 18 hours. Colonies in the zone of inhibition (left image) were isolated and subcultured on Tryptic Soy Agar (TSA). Isolates obtained from the zone of inhibition were fully resistant to DHP (right image; DHP-4) suggesting they were unable to transport DHP. Growth was supported on CDM-P (CDM lacking proline) agar through the biosynthesis of proline via ProC. B) JE2, JE2 ΔopuC ΔopuD ΔproP ΔputP, and DHP 1–4 were grown to stationary phase in 25 ml TSB in a 250 ml flask and amino acid consumption assays were performed on the spent medium. Note a lack of proline consumption in DHP 1–4 in comparison to WT JE2 and JE2 ΔopuC ΔopuD ΔproP ΔputP. We hypothesize that the accumulation of proline the DHP resistant strains is due to proteolysis of peptides in the TSB. C) Proline and arginine biosynthetic pathways in S. aureus. Arginine serves as a substrate for proline biosynthesis via RocF, RocD, and ProC. Proline serves as a substrate for arginine biosynthesis via PutA, RocD, ArcB1, ArgG and ArgH. The transcription of putA, rocD, arcB1, argG, argH, and rocF are repressed by CcpA. This is adapted from Reslane et al. and Halsey et al. (TIF)</p