PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA

The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Clostridium acetobutylicum is a promising biocatalyst for the production of n-butanol at high yield from renewable resources. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it was previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remained unknown until this study, were necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purified, identified and characterized the proteins responsible for both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase activities and demonstrated the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrated the yield of butanol formation was limited by the level of expression of CAC_0764, the ferredoxin-NADP+ reductase encoding gene. The integration of these enzymes into metabolic engineering strategies introduces new opportunities for developing a homobutanologenic C. acetobutylicum strain.

First come, first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.

First come, first served: Superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition.

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.

Hexose fluxes, mediated by vacuolar SWEET transporters, are important for xylem development in the inflorescence stem of Arabidopsis thaliana

ABSTRACTIn higher plants, the development of the vascular system is controlled by a complex network of transcription factors. However, how nutrient availability in the vascular cells affects their development remains to be addressed. At the cellular level, cytosolic sugar availability is regulated mainly by sugar exchanges at the tonoplast through active and/or facilitated transport. In Arabidopsis thaliana, among the tonoplastic transporters, SWEET16 and SWEET17 have been previously localized in the vascular system. Here, using a reverse genetic approach, we propose that sugar exchanges at the tonoplast, mediated by SWEET16, are important for xylem cell division as revealed in particular by the decreased number of xylem cells in the swt16 mutant and the expression of SWEET16 at the procambium-xylem boundary. In addition, we demonstrate that transport of hexoses mediated by SWEET16 and/or SWEET17 is required to sustain the formation of the xylem secondary cell wall. This result is in line with a defect in the xylem cell wall composition as measured by FTIR in the swt16swt17 double mutant and by upregulation of several genes involved in secondary cell wall synthesis. Our work therefore supports a model in which xylem development is partially dependent on the exchange of hexoses at the tonoplast of xylem-forming cells.

Biogenesis and functions of aminocarboxypropyluridine in tRNA

Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.

Structural and phenotypic analysis of Chikungunya virus RNA replication elements

Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3′ to 5′ exoribonuclease Atrimmer 2 in Arabidopsis

The 3′ end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3′ to 5′ exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3′ to 5′ trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1. Unexpectedly, we observed a marked increase of 3′ to 5′ trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.