Tribolium castaneum–which is a widespread generalist pest of stored cereals. The beetles complete their entire life cycle in flour, which thus serves multiple functions: habitat, food, and a source of microbes. We determined key factors that shape the T. castaneum microbiome, established protocols to manipulate it, and tested its consequences for host fitness. We show that the T. castaneum microbiome is derived from flour-acquired microbes, and varies as a function of (flour) resource and beetle density. Beetles gain multiple fitness benefits from their microbiome, such as higher fecundity, egg survival, and lifespan; and reduced cannibalism. In contrast, the microbiome has a limited effect on development rate, and does not enhance pathogen resistance. Importantly, the benefits are derived only from microbes in the ancestral resource (wheat flour), and not from novel resources such as finger millet, sorghum, and corn. Notably, the microbiome is not essential for beetle survival and development under any of the tested conditions. Thus, the red flour beetle is a tractable model system to understand the ecology, evolution and mechanisms of host-microbiome interactions, while closely mimicking the host species’ natural niche.A large body of ongoing research focuses on understanding the mechanisms and processes underlying host-microbiome interactions, and predicting their ecological and evolutionary outcomes. To draw general conclusions about such interactions and understand how they are established, we must synthesize information from a diverse set of species. We analysed the microbiome of an important insect model–the red flour beetle  Most animals harbor specific microbes that alter various aspects of their biology, ranging from development to behavior and reproduction –3. HowevTribolium castaneum. Globally, this species infests a large diversity of cereal flours. However, it is best adapted to wheat flour, on which the beetles can complete egg-to-adult development within ~4–6 weeks. As such, they are easy to maintain in the laboratory, and have been used for over a century as model systems to address problems in ecology and evolutionary biology [T. confusum (which is closely related to T. castaneum); this effect was rescued by re-introducing bacteria isolated from larvae [T. castaneum populations [T. castaneum microbiome is important for the host to mount an immune priming response (a form of immune memory) against a bacterial pathogen [T. castaneum may derive diverse fitness benefits from its microbiome. However, the flour beetle microbiome and its consequences for host fitness have not been systematically analysed thus far.Here, we present such a system: the red flour beetle  biology . Importam larvae . On the ulations –althoughpathogen . TogetheWe addressed this using a large outbred laboratory population of flour beetles. We analysed the flour beetle microbiome and tested the impact of sex, life stage, and flour resource. We also established protocols to disrupt the flour microbiome, and analysed its impact on multiple traits relevant for fitness. Our results demonstrate that although flour beetle fitness is enhanced by flour-acquired microbes, it is not an obligate relationship, and it breaks down in novel resources. Therefore, we suggest that this is an excellent model system to understand the processes and mechanisms driving host-microbiome interactions in changing habitats.We reared beetles at 33°C (±1°C) in laboratory incubators, in round airtight plastic boxes  containing commercially available cereal flour. In most cases, we reared beetles on whole wheat flour (henceforth “wheat” or “wheat flour”), on which beetles have highest reproductive fitness and rapid development. As novel resources, we used corn, finger millet, or sorghum flour; all of these are cultivated in India, but are typically not heavily infested by flour beetles. To remove any prior insect infestation, we froze flour packets at -80°C for at least 4 hours and thawed the flour at room temperature before using it for experiments. To test the efficacy of this method, we added 500 eggs in 50 g wheat flour, followed the freeze-thaw protocol, and observed the flour sample for 30 days. We did not see any live beetles, confirming that freezing removes prior infestation.For all experiments, we used beetles from a large, genetically diverse laboratory stock population, generated using flour beetles collected from 12 different locations across India. To propagate stocks, we allowed 2500–3000 individuals to oviposit in fresh wheat flour for 1 week. We then removed the adults, let the progeny develop for 4–5 weeks, and transferred newly eclosed adults to fresh flour to initiate the next generation. We maintained the stock population for ~10–12 generations in the laboratory before starting experiments.To minimize contamination while handling flour and insects, we sterilized all equipment and tools  by autoclaving, wiping or flaming with 70% ethanol, or exposing to UV radiation in a laminar flow hood as appropriate. We conducted all microbiome manipulation experiments in a laminar flow hood, and placed all containers with flour or insects inside a larger sealed airtight container (with no holes in the lid), before placing them in incubators.To deplete the flour microbial community, we spread thin layers of flour on autoclaved plastic petri dishes (90 mm diameter), using sterilized plastic spoons. We exposed the flour to UV radiation (UV-C at ~254 nm) in a laminar airflow hood for 2 h. We weighed the flour before and after UV treatment to confirm that the treatment did not significantly alter flour moisture content (which could affect beetle fitness independently of the microbiome). Alternatively, we mixed fresh wheat flour with dry, powdered forms of three different broad-spectrum antibiotics: Ampicillin, Streptomycin, and Kanamycin (0.005% w/w each). To test the impact of varying the antibiotic dosage, we mixed fresh wheat flour with varying concentrations of Ampicillin (0.005% to 0.05% w/w). While the antibiotics should not be active in dry flour, we expected that they would be active after contact with fluids in the beetles’ body.To minimize microbial transmission via the pupal surface, we first removed excess flour on the surface with a fine brush. We then gently cleaned the pupal surface using an autoclaved #0 nylon brush dipped first in 10% sodium hypochlorite and then in 70% ethanol. In pilot experiments, we observed high mortality and dehydration of pupae; thus, we added a washing step with ultra-pure double-distilled water between the bleach and alcohol treatments. A final wash with water eliminated residual bleach on the pupal surface, minimizing toxicity via surface absorption. We used brushes for these steps because submerging pupae in alcohol and bleach  caused To characterize the flour microbiome, after our usual freeze-thaw treatment we used UV or antibiotics to create microbe depleted treatments of wheat, finger millet, corn or sorghum flour; and collected ~0.07 g samples in Eppendorf tubes using a sterilized spoon (n = 4). To test the effect of beetle infestation on the flour microbiome, we collected ‘conditioned’ wheat flour samples from stock population boxes that had been occupied by all beetle life stages for 4 weeks (n = 2). To each sample, we added 600 μl Lysis buffer from the Promega DNA extraction kit, before proceeding with DNA extraction.To determine the beetle microbiome across sexes and life stages, we collected individuals  directly from stock populations maintained on wheat, ensuring <1-week age variation within each life stage (n = 3–5). To test the effect of isolation (vs. group living in the stock populations described above), we isolated eggs from stocks in wells of a 96 well plate and reared them to adulthood with ~0.4 g flour/egg, obtaining 1 week old adult females (n = 6). To determine the microbiome of beetles reared on different flour resources, we collected eggs from wheat stock populations and isolated them in the respective flour . From the successfully eclosed adults, we randomly chose females for microbiome analysis. To test the impact of treating flour with UV (or antibiotic) on the beetle microbiome, we obtained eggs from wheat stock populations and distributed them in wells of 96 well plates containing ~0.4 g of untreated or treated flour. We stored 1 week old adults for microbiome analysis. We placed each insect at -80°C for at least 30 min, cleaned its body surface with 70% ethanol, and stored at -80°C until DNA extraction.To determine the bacterial community associated with flour and individual insects, we sequenced part of the 16S rRNA gene of bacterial taxa in each sample. We extracted DNA using the Promega DNA extraction kit following manufacturers’ instructions, with the following modifications. For insect samples, we first surface-sterilised individuals by dipping them in 70% molecular grade ethanol, washed in double-distilled autoclaved water to remove residual ethanol, and then crushed the whole insect with a micropestle in 600 μL Nuclei Lysis solution (from the DNA extraction kit). To minimize protein contamination, we increased the incubation time with Proteinase K from 3 h to 12 h, and washed the pelleted DNA twice in 70% ethanol. We dissolved DNA in double-distilled autoclaved water. Next, we amplified the V3-V4 hypervariable region of the 16S rRNA gene using standard Illumina primers, and prepared sequencing libraries according to the Illumina metagenomic sequencing library preparation guide . We sequTo determine bacterial community composition in each sample, we analysed amplicon sequencing data using QIIME v.1 . We geneTo visualize changes in the abundance of dominant bacteria, we focused on the five most abundant bacterial OTUs across replicate samples of a treatment , with flour. We then paired adult males and females in 1 g wheat flour for 48 h to mate; removed the male; and allowed the female to oviposit for 24 h in 5 g sifted wheat flour. Sifting through a #50 sieve removes large flour particles and facilitates identifying and counting eggs. After 24 h, we removed each female, counted eggs , and returned the females to their Petri dish. After 3 weeks, we counted the number of surviving offspring  and the fraction of pupae (measuring offspring development rate). At each step, we used either sterilized or untreated flour, according to the corresponding treatment. We followed this protocol for all measurements of reproductive fitness, unless specified otherwise. Sample sizes for each experiment are noted in the respective figures.To test the effect of competition on offspring survival and development in UV-treated flour, we controlled egg density during offspring development. We collected pupae and allowed them to eclose, mature, and mate in the appropriate flour (untreated or UV-treated wheat or sorghum) for 14 days (~200 pupae per treatment). We transferred all eclosed adults to 50 g sifted wheat flour. After 24 h of oviposition, we collected and distributed 20 eggs each in 5 g of untreated or UV-treated flour (wheat or sorghum) in sterile Petri dishes (60 mm diameter). We measured offspring survival and development rate as described above. In a separate experiment, we altered the amount of resource provided to developing offspring. We isolated eggs in microplate wells, provided each egg with either 0.2 g flour or 0.4 g of flour, and measured offspring survival and development rate.To test the impact of microbes on female fecundity across time, we isolated individual mated females (n = 25 per resource per treatment) from stock populations in ~0.7 g sifted flour , allowed them to oviposit for a fixed period , and counted the number of eggs laid per female. Since eggs hatch in 2–3 days, for the 96 h and 7 day treatments, we included larvae in the counts (i.e. eggs + larvae). We tested 7 d fecundity only in wheat, in two independent experimental blocks. For antibiotic treatments, we only tested fecundity after 48 h of oviposition, when fecundity is highest and is not confounded by hatched eggs and small larvae.To test the impact of microbes on offspring survival in different resources , we isolated individual eggs in single wells in a 96 well plate with ~0.4 g of either control or UV-treated flour, and counted the proportion of surviving offspring after 3 weeks.To test whether variation in female fecundity in normal wheat flour could be explained by individual variation in the microbiome, we allowed 24 females to oviposit in isolation (in 1.5 ml Eppendorf tubes with a hole in the lid) with ~0.7 g sifted wheat flour for 48 h. We counted the eggs, and stored each female for microbiome analysis.To test whether the microbiome altered larval and adult cannibalism rates, we used 2-week old larvae or adults (obtained from stock populations) to measure egg cannibalism rate in untreated vs. UV-treated flour. We mixed 20 eggs with 1 g sifted flour in a 30 mm Petri plate, and added a single larva or adult. After 24 h, we counted the number of remaining eggs and estimated the cannibalism rate as: (Number of eggs provided—Number of surviving eggs)/ Number of eggs provided. To easily differentiate eggs from flour and improve accuracy of egg counts, we stained eggs pink using neutral red dye (1% w/w mixed in flour). This is important for measuring egg cannibalism in adult females, because females can lay new eggs during the experiment, confounding estimates of cannibalism rate. Providing dyed eggs for cannibalism allowed us to differentiate them from freshly laid (white) eggs. Thus, for adult females, we calculated cannibalism rate only for dyed eggs. For larvae, we used a second experimental block to test whether the dye alters cannibalism rates.To test the impact of microbes on beetle lifespan, we isolated eggs from wheat stock populations in 96 well microplates with 0.25–3 g wheat flour per egg . After ~25 days, 68–75 individuals had pupated in each microplate. To test the impact of a lack of microbes during adulthood (vs. throughout life), we moved pupae from two microplates with untreated flour into a fresh plate with UV-treated flour. To test for effects of moving pupae into fresh flour at this stage, we moved pupae from one microplate into fresh untreated flour. Thus, we had a total of four treatments, with individuals reared (1) entirely in untreated wheat flour (“control”) (2) in untreated wheat flour until pupation, and then moved to untreated flour (3) in untreated flour until pupation and then moved to UV-treated flour, and (4) entirely in UV-treated wheat flour (“UV”). We monitored the number of surviving adults in each plate every day for 80 days after eclosion, providing fresh flour every 7 days. We repeated the experiment in a second block, with 2 microplates (n = 192 eggs) each for individuals reared entirely in untreated or UV-treated wheat flour.Bacillus thuringiensis , following previously described protocols [To determine the impact of the microbiome on survival under pathogen attack, we isolated eggs, and let them develop for 2 weeks in either UV-treated or untreated wheat flour. We infected each larva with either insect Ringer solution , or live culture of rotocols . We moniWe conducted all statistical analyses in R version 3.2.2 . To compErwinia). However, in conditioned flour as well as beetle samples, a different OTU from the family Enterobacteriaceae was dominant . In addition, an OTU from the genus Enterococcus  was also much more dominant in conditioned flour and beetle samples . Interestingly, these two OTUs were less abundant in females reared in isolation . Visualizing OTUs at a higher taxonomic level  and beetle samples had ~450 bacterial OTUs. Notably, fresh wheat flour had distinct dominant OTUs from conditioned flour (used by beetles for weeks), which was in turn more similar to the dominant taxa in beetles , conditioned flour and beetles had similar microbiomes p = 0.1; . BacteriErwinia, whereas OTUs from the family Sphingomonadales were not abundant. In contrast, corn flour was strongly dominated by the genera Enterococcus and Delftia; and sorghum flour was enriched in the genera Enterococcus and Bacillus. At the phylum level, Proteobacteria were more abundant in finger millet, whereas sorghum had the opposite skew favouring Firmicutes  was abundant only in females reared in corn.Next, we tested whether beetles can maintain a stable microbiome despite consuming distinct resources. If the microbiome is environment (i.e. flour)-derived, we expected the beetle microbiome to change in resources with distinct resident flora. Indeed, different flours had distinct microbiomes  beetles acquire microbes from the flour (2) a few taxa are enriched in beetles, ultimately altering the flour microbiome (likely via beetle feces) (3) this may set up a positive feedback for further enrichment of the dominant taxa via repeated passage of the flour (mixed with feces) through the beetle gut and (4) two major bacterial clades dominate both the flour and beetle microbiomes, although their relative abundance varies across flours and beetles reared on different resources.Next, we established experimental protocols to deplete or disrupt the beetle microbiome using two methods: UV treatment of flour , and administering antibiotics via flour. As expected, both treatments reduced the bacterial load in flour .In all four flours tested, UV treatment significantly altered the flour microbiome  to maintain consistent sibling competition across replicates. Again, fewer offspring survived in UV-treated wheat; but also in sorghum, and even with pupal sterilization . In thisFinally, additional experiments corroborated the broad patterns noted above: depleting flour microbes reduced fecundity in wheat flour, but not in novel resources , regardless of the time allowed for oviposition . SimilarThe strong and repeated impact of the flour microbiome  on female fecundity prompted us to ask whether the females’ own microbiome may also influence their fecundity. Hence, we tested whether more fecund females were especially enriched in specific bacteria . However, despite wide variation in fecundity , all females had a similar microbiome dominated by Proteobacteria; specifically, a single OTU belonging to the family Enterobacteriacae . Thus, aCannibalism plays a major role in flour beetle biology –29, and As expected, larval cannibalism increased in UV-treated flour  or a buffer ; and reared each group on either UV-treated or untreated (control) wheat flour. As expected, sham-infected larvae had higher survival than infected larvae; and overall, larvae reared in UV-treated flour had lower survival than control larvae . We find that beetles have a flour-derived microbiome that is shared across sexes and life stages—most likely via the flour—and is enriched in specific bacterial taxa. Establishing protocols to manipulate the beetle microbiome, we show that all life stages of flour beetles derive multiple fitness benefits from their wheat flour-acquired microbiome, leading to an ~2 fold increase in fecundity and offspring survival, and ~1.5 fold increase in lifespan. When reared in sibling groups (with resource competition), offspring also develop more slowly in UV-treated flour. However, when reared in isolation (without competition), all larvae develop faster, with no effect of UV treatment. In grain and flour warehouses, eggs likely do not develop in isolation; hence, we speculate that the former results are more instructive about the true impact of the microbiome on development rate. Notably, although depleting the wheat flour microbiome clearly reduces female fecundity, the fecundity of individual females in normal wheat flour was not correlated with their microbiome. In contrast to the strong consequences of the wheat flour microbiome, removing microbes from other  flour types did not reduce beetle fitness. Finally, we find that in the absence of an intact flour microbiome, adult beetles and larvae increase egg cannibalism. Since flour beetle cannibalism typically increases in poor resources , 30, 31,T. castaneum, which likely sample diverse resources even within a single generation. The lack of microbiome impacts on fitness in three different novel resources supports this hypothesis. In these resources (used commonly in India), the beetles would encounter distinct flour microbiomes compared to wheat. Further experiments are necessary to test these ideas, and to determine factors that can facilitate the evolution of a strong obligate host-microbe association akin to that observed in many other insects.Despite these overarching impacts, the microbiome does not appear to be essential for any of the fitness parameters that we measured, suggesting that the host-microbiome interaction is not an obligate association. The facultative nature of the relationship  suggests that it may be recently established. Alternatively, the flexible nature of the relationship may itself be selectively favoured due to environmental heterogeneity. For instance, if flour beetles experience substantial spatial or temporal habitat heterogeneity and cannot reliably access appropriate microbes, the ability to maintain relatively high reproductive fitness without microbes may be beneficial. This is especially relevant for generalist species such as In contrast to other insect species , 34, we Interestingly, we found that the flour beetle microbiome varies across individuals, with some individuals showing almost no bacterial reads. We speculate that this is likely an artifact of the large fraction of reads from chloroplast and mitochondria—largely derived from cereal flour—and may not necessarily reflect biological differences. Due to the resulting paucity of bacterial reads , some of our treatments also had low sample sizes, weakening some of our conclusions. Nonetheless, the individual variation is also biologically interesting, and is not uncommon in other insects . All elsEnterococcus were enriched in beetles reared on new suboptimal flours. Hence, we speculate that one or both of these taxa may explain a large fraction of the microbiome’s fitness impact on host beetles. The genus Enterococcus includes species that frequently live in invertebrate guts and are facultative pathogens that use various mechanisms such as secreted toxins, quorum sensing and cell surface adhesion to cause host damage [Enterococcus OTU that we observed in beetles may be harmful, especially in high abundance, and in the absence of other beneficial OTUs. On the other hand, the family Enterobacteriaceae encompasses a large and very diverse set of rod-shaped bacteria, including well-known animal commensals and pathogens such as Escherichia coli and Salmonella, as well as obligate or facultative insect mutualists . Many members of this family produce toxins called microcins [Despite the substantial individual variation in bacterial OTUs, the dominant bacterial phyla were largely consistent across hosts, suggesting that host reliance on bacteria is fairly general at higher levels of bacterial taxonomy. The major bacterial phyla that we observed—Proteobacteria and Firmicutes—also dominate the microbiomes of most other insects, accounting for ~64% and ~7% of bacterial communities [t damage . Thus, wicrocins , which microcins , 40. In Our work identifies some avenues for further refining methods to manipulate the microbial communities of flour and flour beetles. First, the relatively weak effects of UV treatment and antibiotics on bacterial community composition may reflect the fact that neither treatment is foolproof, allowing some bacteria to persist. Therefore, to completely eliminate the beetle microbiome, stronger doses or exposure times may be necessary. Second, the flour beetle system appears to be especially prone to problems with amplification of chloroplast and mitochondrial DNA. The resulting problem of insufficient bacterial reads could potentially be resolved by using methods to reduce amplification from mitochondrial and chloroplast DNA (e.g. ), or by In summary, our work showcases flour beetles as a tractable model system allowing manipulation of host and microbial genotype, microbial community composition, microbial transmission and its effects, and analysis of the impacts of the association on host and microbial community members alike. Thus, we suggest that flour beetles are an excellent model to understand the evolution, establishment, and maintenance of host-microbial associations; especially in the context of animal guts, where such associations have important ecological and evolutionary impacts . Such anS1 FigBoxplots show the total number of reads and reads mapping to mitochondria and chloroplasts, for (A) Flour samples (B) Flour and beetle samples (C) Beetles reared on untreated vs. UV-treated flour (D) Beetles reared on flour mixed with antibiotics.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig Relative abundance of the top five dominant bacterial OTUs in individual samples of flour, classified to (A) the lowest possible taxonomic level or (C) phylum level.  Average abundance across replicates shown in panels A and C. (E) Linear Discriminant (LD) analysis and (F) Principal Coordinate Analysis (PCoA) of the complete bacterial community of flour samples; axes labels indicate % variation explained.(TIF)Click here for additional data file.S4 Fig Relative abundance of the top five dominant bacterial OTUs in individual beetle samples , classified to (A) the lowest possible taxonomic level or (C) phylum level.  Average abundance across replicates shown in panels A and C. (E) Linear Discriminant (LD) analysis and (F) Principal Coordinate Analysis (PCoA) of the complete bacterial community of beetle samples; axes labels indicate % variation explained.(TIF)Click here for additional data file.S5 FigFlour samples plated onto nutrient agar plates.(TIF)Click here for additional data file.S6 Fig Relative abundance of the top five dominant bacterial OTUs in individual samples of flour, classified to (A) the lowest possible taxonomic level or (B) phylum level. (C) Principal Coordinate Analysis (PCoA) of the complete bacterial community of flour samples; axes labels indicate % variation explained.(TIF)Click here for additional data file.S7 Fig Relative abundance of the top five dominant bacterial OTUs in beetle samples , classified to (A) the lowest possible taxonomic level or (B) phylum level. (C) Principal Coordinate Analysis (PCoA) of the complete bacterial community of beetle samples; axes labels indicate % variation explained.(TIF)Click here for additional data file.S8 Fig Relative abundance of the top five dominant bacterial OTUs in beetle samples , classified to (A) the lowest possible taxonomic level or (B) phylum level. (C) Principal Coordinate Analysis (PCoA) of the complete bacterial community of beetle samples; axes labels indicate % variation explained.(TIF)Click here for additional data file.S1 TableThe table shows detailed statistics for comparisons of fecundity, survival, and development rate in control (untreated) or UV-treated flour (UV), with or without surface sterilization of pupae, and varying egg density during survival and development . SS = Surface-sterilised pupae; Untreated = not surface sterilized; Conf. = confidence interval. The model specified for each analysis is indicated. Significant effects are highlighted in bold.(DOCX)Click here for additional data file.S2 Table(I) The table shows detailed statistics for comparisons of egg survival and development rate in control (untreated) or UV-treated flour (UV), with or without surface sterilization of pupae; and with a fixed egg density (20 eggs per replicate). SS = Surface-sterilised pupae. The model specified for each analysis is indicated. (II) The table shows detailed statistics for comparisons of egg survival in control (untreated) or UV-treated flour (UV). Significant effects are highlighted in bold.(DOCX)Click here for additional data file.S3 TableThe table shows detailed statistics for comparisons of fecundity in control (untreated) or microbe-depleted flour (via UV treatment or antibiotics with the indicated concentration). Conf. = confidence interval; Amp = ampicillin. The model specified for each analysis is indicated. Significant effects are highlighted in bold.(DOCX)Click here for additional data file.S4 TableThe table shows detailed statistics for comparisons of cannibalism rates of larvae and adult females in control (untreated) or UV-treated flour (UV). Eggs were either dyed or not; and females were provided with their own eggs (self eggs) or another female’s eggs (non-self eggs) to cannibalise. The model specified for each analysis is indicated. Significant effects are highlighted in bold.(DOCX)Click here for additional data file.S5 TableThe table shows detailed statistics for comparisons of survival rates of beetles reared in untreated wheat flour (control) or UV-treated flour (UV). As pupae, some control individuals were transferred to either fresh untreated flour ((ii) in (DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Zika virus has recently emerged as an important human pathogen that has spread to more than 60 countries. Infection of a pregnant woman with Zika virus can cause severe brain malformations in the child such as microcephaly and other birth defects. Despite the medical importance of Zika virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. Stem-loop A (SLA), located in the 5′ untranslated region of the viral genome, acts as a promotor for viral replication and thus is critical for recognition of the viral genome by the viral polymerase NS5. However, how NS5 engages SLA is not clear. We have quantitatively examined the intrinsic affinities between Zika virus SLA and NS5, and identified the SLA-binding site on NS5. Amino acid substitutions in the thumb subdomain of the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) domain reduced SLA-binding affinity, indicating that they each are part of the SLA-binding site. Furthermore, stopped-flow kinetic analysis of Zika NS5-, RdRp- and MTase–SLA interactions identified distinct intermediates during NS5 and SLA complex formation. These data suggest a model for SLA recognition and the initiation of flaviviral replication by NS5. Flaviviridae, and since 2007 has emerged as a major threat to global health, causing a series of epidemics in Micronesia, the South Pacific and both Americas1. Currently, the virus has been reported in 65 countries3. ZIKV is closely related to human pathogens such as tick-borne encephalitis virus, dengue virus (DENV), West Nile virus, yellow fever virus, and Japanese encephalitis virus5. ZIKV can be transmitted sexually and through blood transfusions, and spread from a pregnant woman to her fetus. ZIKV infection of a pregnant woman can cause severe brain malformations in her child including microcephaly and other birth defects7. Moreover, in adults, ZIKV has been linked to Guillain–Barré syndrome, a condition wherein a person’s immune system attacks their peripheral nerves9. In severe cases, this can lead to near-total paralysis and death.Zika virus (ZIKV) is a member of the family 12. The ORF is translated into a polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is subsequently cleaved into three structural  and seven non-structural (NS) proteins. The viral NS proteins, along with viral RNA and unidentified host proteins, self-assemble on the cytoplasmic side of the ER membrane to form a viral replication complex that carries out genome replication. Although all NS proteins are required to form a viral replication complex, only NS3 and NS5 enzyme activities are involved in viral genome replication. NS3 consists of an N-terminal serine protease domain, which requires NS2B as a cofactor, and a C-terminal helicase domain. The helicase domain also has nucleoside-triphosphatase (NTPase) activity and 5′-RNA triphosphatase activity, which hydrolyzes the γ-phosphate of RNA for RNA capping13. NS5 is the viral replicative polymerase responsible for genome synthesis15. Full-length NS5 encompasses approximately 900 amino acids and is composed of two functionally different domains21. The smaller N-terminal domain functions as a methyltransferase (MTase), whose activity is necessary for the 5′-RNA cap formation and methylation that are important for viral RNA recognition by the host cell translational apparatus23. The larger C-terminal domain possesses RNA-dependent RNA polymerase (RdRp) activity, containing the active site for RNA synthesis24.The structure of the ZIKV genome is similar to other flaviviral genomes. The 11 kb long positive sense RNA contains an open reading frame (ORF) as well as 5′ and 3′ untranslated regions (UTRs) that regulate viral replication26. Although NS5 can synthesize RNA from short RNA templates, NS5 cannot synthesize RNA from a sub-genomic RNA template that lacks SLA11. A predicted ‘Y’-shaped secondary structure of SLA is conserved in all dengue virus serotypes (DENV1-4) as well as in other flaviviruses such as ZIKV and West Nile virus28, suggesting that flaviviruses share a common promoter structure and initiation mechanism. Additionally, SLA is required for 5′ cap methylation at the N7 position, indicating that the MTase domain of NS5 recognizes structural features of the SLA to carry out the methylation reaction29. Despite the importance of SLA in initiating both of these critical aspects of viral replication (RNA synthesis and RNA capping), the exact roles each of the NS5 domains play in recognizing and binding SLA are not well understood. In particular, the SLA-binding site on NS5 has not been identified. Thus, it is not clear whether both NS5 domains are involved in SLA binding or whether one of the two domains regulates promotor binding.In flaviviruses, the 5′-UTR of the viral genome includes a structured stem-loop, called stem-loop A SLA, nt 1- 70) . The formation of the complex between the ZIKV NS5 proteins and SLA-F causes a decrease in nucleic acid fluorescence . The binding constants for the RdRp domain and MTase domain are KRdRp = 1.9 × 106 M−1 (Kd = 0.53 μM) and KMTase = 1.7 × 106 M−1 (Kd = 0.59 μM), respectively. Thus, both MTase and RdRp domains individually interact with SLA-F with  approximately  two-fold lower affinities than the full-length NS5.Flavivirus replication is initiated when the viral polymerase NS5 identifies the viral genome via specific recognition of the SLAnce Fig.  to the ence Fig. ” section19 to look for positively charged patches that would be complementary to the negatively charged SLA , a 1.8-fold reduction from the wild-type affinity   Fig. d. The CDμM) Fig. . For exa19, εA(pεA)19  and Kmut = 1.5 × 106 M−1 (Kd = 0.67 μM). Thus, the mutations that affect SLA binding in NS5 do not significantly change nonspecific ssRNA interaction, indicating that the positively charged patch in the thumb subdomain  specifically interacts with SLA.To determine if the SLA-binding site on the thumb subdomain is specific for SLA interaction, we next measured NS5 protein interactions with a nonspecific single-stranded RNA (ssRNA). Because the identified SLA-binding site does not overlap with the template-binding site 19 Fig. e. Wild-t35. The structures also suggest a putative RNA-binding site, consisting of K28, K29, R37, R41, R42, R57, and R213  at 20 °C are shown in Fig. wt = 3.2 × 106 M−1 (Kd = 0.31 μM). This value measured by anisotropy is similar to the binding constant determined by the fluorescence titration in the same buffer B2, 2.5 × 106 M−1 (Kd = 0.40 μM) , i.e., it decreased fourfold compared to the wild-type MTase  Fig. . The aff19 in buffer B2 . The binding constant of the mutant MTase K28E-K29E-R41E-R42E for the ssRNA is Kmut = 0.4 × 106 M−1 (Kd = 2.5 μM), a 2.8-fold reduction compared to the wild-type. The SLA-binding site identified in the MTase  partially overlaps with the capped RNA-binding site  determined by the crystal structures of MTase in complex with capped RNA37. Thus, it is likely that the SLA-binding site mutant in the MTase domain also reduces the ssRNA interaction. Nevertheless, the mutant MTase (K28E-K29E-R41E-R42E) reduced the SLA binding affinity more significantly than the ssRNA binding affinity (four-fold vs. 2.8-fold), indicating that the positively charged patch containing K28, K29, R41, and R42 is involved in SLA binding.To determine if the decreased affinity is specific for SLA compared to non-specific ssRNA, the associations of the wild-type and mutant MTase were measured by fluorescence anisotropy with the fluorescently labeled ssRNA, εA(pεA) B2 Fig. c. The wii or 1/τi, i = 1, 2, 3) and the fractional individual amplitudes  of the NS5–SLA complex formation with nonlinear least-squares fits  in these steps  and the maximum fluorescence change (ΔFmax = 0.15) that are independently determined  followed by two structural rearrangement steps .The partial equilibrium constants for each step of the above reaction are related to the Kmentclass2pt{minimThe stopped-flow kinetic traces of SLA-F fluorescence after mixing with ZIKV RdRp were analogously analyzed. The best fit for the RdRp-SLA kinetic traces were obtained using a double-exponential function Fig. e. Howeve1, we can determine its amplitude, A1 by Eq.  and normalized amplitudes, A1, A2, and A3 of three observed relaxation processes on the concentration of the ZIKV RdRp domain are shown in Fig. T, is dominated by A2. With increasing protein concentrations, the contribution of A1 to AT increases while A3 remains negative throughout the process. The partial equilibrium constants and kinetic rate constants characterizing the intermediates for the three steps are related to the overall binding constant Koverall and the maximum fluorescence change ΔFmax, as described above (Table 1 = 1.0 × 109 M−1 s−1 and k-1 = 2000s−1), which is followed by two structural rearrangement steps . Note the initial bimolecular association and dissociation (k1 and k−1) of RdRp and SLA are 10 times faster than the initial association of NS5 and SLA.The dependences of the reciprocal relaxation times 1/τ and 1/τ3Lastly, a stopped-flow kinetic trace for the MTase domain and SLA interaction is shown in Fig. 1 and 1/τ2, characterizing the observed kinetic steps as a function of the total MTase domain concentration are shown in Fig. 1) versus MTase concentrations showed a linear dependence, indicating a MTase–SLA complex is formed after the bimolecular event  is followed by only one structural rearrangement step (k2 = 15 s−1 and k−2 = 14 s−1)  to 500 and 710 nM, respectively 29. These two functionally different NS5 and SLA interactions would engage the same binding site, since it seems unlikely that NS5 possesses two separate SLA-binding sites. In such a case, both MTase and RdRp domains are likely involved in SLA binding. The positively charged patches in RdRp and MTase are separated by ~ 55–60 Å. Based on the secondary structure prediction 1−RdRp = 1.0 × 109 M−1 s−1 vs k1−NS5 = 9.5 × 107 M−1 s−1). On the other hand, the RdRp has a large reverse rate constant (k−1−RdRp = 2000s−1), indicating that the RdRp-SLA complex dissociates more easily than the NS5–SLA complex where the reverse rate constant is much lower (k−1−NS5 = 360 s−1). This indicates that the MTase domain in the intact NS5 slows the recognition step but increases the stability of the formed complex. Accordingly, binding of the MTase domain to SLA is characterized by only a two-step mechanism and the initial recognition step is much slower than the RdRp domain (k1−MTase = 9.0 × 107 M−1 s−1 vs. k1−RdRp = 1.0 × 109 M−1 s−1). Therefore, NS5 likely recognizes SLA first through the RdRp domain. Taken together, we propose an NS5–SLA interaction model wherein the first fast bimolecular association between NS5 and SLA is carried out via the RdRp domain involving the thumb subdomain 19, εA(pεA)19 was obtained by modification of the oligomer with chloroacetaldehyde as described previously31. The concentration of εA(pεA)19 was determined using the extinction coefficient, −1 M−1 (nucleotide)31.A Zika SLA construct containing the first 80 nucleotides with 5′ triphosphate and a 3′ fluorescein tag was obtained from Midland Certified Reagents . SLA concentration was determined using the extinction coefficient of SLA 19. Full-length ZIKV NS5 and the RdRp domain were expressed with an N-terminal hexahistidine tag and a Small Ubiquitin-like Modifier (SUMO) tag. The gene encoding the MTase domain (GenBank: KU955593.1) was synthesized with an N-terminal hexahistidine tag. All three proteins, the full-length NS5, the RdRp and the MTase domain, were expressed in Rosetta (DE3) Escherichia coli cells. Briefly, the cells were grown at 37 °C in Luria Broth (LB) containing 100 μg/ml ampicillin to an OD600 of 0.6–0.7. Protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside and cell growth continued overnight at 18 °C. For protein purification, the cells were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 500 mM NaCl, 2 mM β-mercaptoethanol, and 1 tablet of EDTA-free protease inhibitor , and lysed by sonication. After centrifugation to clear the insoluble debris, the protein in the soluble fraction was purified by Talon metal affinity chromatography . NS5 was eluted with a 10–200 mM gradient of imidazole in 50 mM Tris (pH 7.0) buffer containing 500 mM NaCl, 2 mM β-mercaptoethanol and 10% glycerol. The fractions containing NS5 protein were collected and concentrated using an Ultrafree centrifugal filter device . For the full-length NS5 and RdRp domain, the SUMO and hexahistidine tag was cleaved from the protein by incubation with SUMO protease (1:100 w/w ratio). Subsequently, the cleaved fractions were loaded on a Superdex 200 size exclusion column , equilibrated with buffer containing 20 mM Tris–HCl (pH 7.0), 500 mM NaCl, 2 mM β-mercaptoethanol and 20% glycerol. The MTase domain was purified similarly using Talon metal affinity and size-exclusion chromatography.Full-length ZIKV NS5 (GenBank: KY559015.1) and its RdRp domain constructs were obtained from Dr. Cheng Kao (Indiana University)Mutations within the ZIKV NS5 and the MTase domain were introduced by site-directed mutagenesis using the PfuUltra II Fusion HS DNA Polymerase kit . The primers used for mutagenesis are listed in Table 2, 2 mM β-mercaptoethanol, and 10% glycerol, was used in fluorescence experiments. Each experiment was carried out in triplicate. The binding of ZIKV NS5 to SLA was monitored by measuring changes in the signal originating from the fluorescein attached to SLA . Titration curves were fit using Kaleida Graph software . In these experiments, the relative fluorescence change (ΔFobs) is defined as (Fi—F0)/F0, where Fi is the fluorescence of the RNA at a given titration point and F0 is the initial value of the fluorescence of the sample. Both F0 and Fi were corrected for background fluorescence at the applied excitation wavelength. Interactions between the NS5 proteins (wild-type and R771A-R772A-R775A-K843A-R844A) and ssRNA were measured with etheno-labeled εA(pεA)19 in buffer B1 . The εA(pεA)19 samples were excited at λex = 325 nm and emission was recorded at λem = 410 nm31.Steady-state fluorescence titrations were performed using an ISS PC1 spectrofluorometer . Polarizers were placed in excitation and emission channels and set at 90° and 55° (magic angle), respectively, in order to avoid possible artifacts due to fluorescence anisotropy of the sample. Experiments were carried out by adding Zika NS5 proteins or the RdRp/MTase domain to a solution containing fluorescein-labeled Zika SLA. A standard buffer B1, containing 50 mM Tris adjusted to pH 8.0 with HCl at 20 °C, 150 mM NaCl, 1 mM MgCl2, 2 mM β-mercaptoethanol, and 10% glycerol, was used for anisotropy measurement. Fluorescence anisotropy (r) is defined as:VV and IVH are fluorescence intensities where the first and second subscripts refer to vertical (V) polarization of the excitation and vertical (V) or horizontal (H) polarization of the emitted light. The factor, G = IHV/IHH, corrects for the different sensitivity of the emission monochromator for vertically and horizontally polarized light. The samples in the fluorescence anisotropy experiments were excited at 480 nm and the emission recorded at 520 nm . Each experiment was carried out in triplicate. Interactions between the MTase domain proteins (wild-type and K28E-K29E-R41E-R42E) and etheno-labeled εA(pεA)19 were measured in buffer B2 using fluorescence anisotropy. The fluorescence signal of εA(pεA)19 were monitored with λex = 325 nm and λem = 410 nm31. Each experiment was carried out in triplicate.Interactions between the MTase domain proteins (wild-type and K28E-K29E-R41E-R42E) and fluorescein-labeled SLA were analyzed using fluorescence anisotropy (r), because fluorescence intensity changes were too small to accurately determine the binding constant for the mutant. Buffer B2, containing 50 mM Tris adjusted to pH 8.0 with HCl at 20 °C, 100 mM NaCl, 1 mM MgCl1, characterizing the SLA association with NS5, is defined as1]F is the concentration of the formed complex.The binding constant, KF and FC are the molar fluorescence intensities of the free SLA and the formed complex C1, respectively. Thus,The observed fluorescence of the sample at any point of the titration is defined asT, in the sample isThe mass conservation equation for the total SLA concentration, [SLA]obs, is thenmax = FC/FF, is the maximum value of the observed relative fluorescence quenching. Fluorescence titration curves were fitted using Eq. (Using Eqs.  and 10)10), the ex = 480 nm and λem = 520 nm. Generally, 10–12 traces were collected and averaged for each concentration of proteins. The kinetic curves were fitted to extract relaxation times and corresponding amplitudes using nonlinear least-squares fitting software provided by the manufacturer, with the exponential function defined asi is the amplitude corresponding to ith relaxation process, λi is the time constant  characterizing the relaxation process, and n is the number of relaxation processes. Global analyses of the stopped-flow kinetic data were performed using the matrix projection operator technique 39. All analyses of the data were performed using Mathematica  and Kaleida Graph  as previously described39.All fluorescence stopped-flow kinetic experiments were performed using SX.MV20 stopped-flow instrument . Fluorescein-labeled SLA (15 nM) was mixed with 7–8 concentrations of NS5 (300 nM—3 µM), RdRp (300 nM—3 µM) or MTase (500 nM—5 µM) under pseudo-first-order conditions ([protein] >  > [RNA]), and the fluorescence signal upon complex formation was monitored as a function of time with λ2. CD spectra were collected at 20 °C in a 0.2-cm path-length cuvette from 195 to 260 nm. Three scans were averaged in the range of 200–250 nm. Data were converted from millidegrees to mean residue ellipticity (MRE) using the formula:θ] is the molar residue ellipticity, θ is the CD signal in millidegrees, c is the protein concentration in µM, l is the path length in mm, and n is the number of amino acid residues. The CD data analysis program BeStSel was used to analyze the data and estimate the secondary structure41.The far-UV circular-dichroism measurements for wild-type and mutant proteins were performed on a model No. J-815 spectrometer . The protein concentration was ~ 0.1 mg/ml in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 10% glycerol and 1 mM MgClSupplementary information.


SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity.SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.The online version of this article (10.1007/s11103-020-01067-y) contains supplementary material, which is available to authorized users. Biological membranes define two milieus by selectively regulating the exchange of substances and flow of information. Membrane proteins dynamically interact with membrane lipids to monitor and elicit regulatory steps at membranes and in between different membranes, such as during secretion and endocytosis.Saccharomyces cerevisiae) by screening for secretory mutants, share their characteristic SEC14 domain, also known as CRAL-TRIO domain  superfamily. SEC14L-PITPs, first identified in yeast , not only have high numbers of SEC14L-PITPs, but also many of them with modular complex composition. These complex SEC14L-PITPs carry at their C- or N-terminus additional stretches, mostly with recognizable domains, also found in other types of proteins , containing an additional Golgi dynamics (GOLD) domain at their C-terminus , and PM Na+/H+ antiporter SALT OVERLY-SENSITIVE1 (SOS1), and contributes to the plant tolerance to cold and salt stress  and protein BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) using as query yeast Sec14p and the SEC14 domain of AtPATL2, and via InterPro using the CRAL-TRIO domain. Sequences were retrieved from selected species of green algae, a moss, a lycophyte, and of selected flowering plants, and analyzed for the presence of multiple domains. The positions of domains were determined using NCBI Conserved Domain Search NIH (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), PROSITE (https://prosite.expasy.org/) and InterPro (https://www.ebi.ac.uk/interpro). InterPro domain accession numbers are GOLD-IPR009038, SEC14/CRAL-TRIO-IPR001251, CTN-IPR011074, GDAP2/Macro domain-IPR035793, DDT domain-IPR018501, PHD domain-IPR019787, WHIM1 domain-IPR028942, WHIM2 domain-IRO28941. Nodulin and nodulin-like domains were defined according to Kapranov et al. ; AT2G21540 (AtSfH3); At4G36490 (AtSfH12); At1g72150 (PATL1); At1g22530 (PATL2); At1g72160 (PATL3); At1g30690 (PATL4); At4g09160 (PATL5); At3g51670(PATL6); At4g39180; At1g01630; At1g05370; At1g14820; At1g19650; At1g22180; At1g55840; At1g75170; At1g75370; At2g15670; At2g16380; At2g18180; At2g21520; At3g22410; At3g24840; At3g46450; At4g08690; At4g36640; At4g39170; At5G47730; At5g56160; At5g63060; At1g69340; At4g35750; At3g10210; At1g55690.Chlamydomonas reinhardtii v5.5: Cre10.g444250; Cre12.g503950; Cre12.g527050.t1.2; Cre10.g448051; Cre02.g141950; Cre03.g166201; Cre02.g147800; Cre02.g101200; Cre17.g718100; Cre17.g703200; Cre11.g467563.Volvox carteri v2.1: Vocar.0070s0030; Vocar.0004s0276; Vocar.0005s0399; Vocar.0008s0014; Vocar.0036s0128; Vocar.0037s0062.Chara braunii ; Solyc11g027880; Solyc09g025230; Solyc04g080690; Solyc06g064940; Solyc09g060090; Solyc11g012790; Solyc03g112640; Solyc07g065700; Solyc04g005490.Zea mays PH207 v1.1: Zm00008a000784; Zm00008a007108; Zm00008a021866; Zm00008a034613; Zm00008a000376; Zm00008a008524; Zm00008a018020; Zm00008a031256; Zm00008a016003; Zm00008a033937; Zm00008a031138; Zm00008a013174; Zm00008a027397; Zm00008a019107; Zm00008a032518; Zm00008a032332; Zm00008a025849; Zm00008a025849; Zm00008a039685; Zm00008a008448; Zm00008a022847; Zm00008a022585; Zm00008a025654; Zm00008a018864; Zm00008a017887; Zm00008a003138; Zm00008a012744; Zm00008a037114; Zm00008a037115; Zm00008a019021; Zm00008a004286; Zm00008a022784; Zm00008a012865; Zm00008a016847; Zm00008a028031; Zm00008ă80; Zm00008a005640; Zm00008a025640; Zm00008a026335; Zm00008a009102; Zm00008a017141; Zm00008a033936; Zm00008a012432; Zm00008a013571; Zm00008a025498; Zm00008a028943; Zm00008a008572; Zm00008a033935; Zm00008a032805; Zm00008a034926; Zm00008a020872; Zm00008a020873; Zm00008a014049; Zm00008a021294.PATL1, PATL2, PATL3, PATL4 and PATL6 genes were obtained using the ePlant Heatmap Viewer module (https://bar.utoronto.ca/eplant/)   were generated as follows: The PATL2 coding sequence was amplified from cDNA, obtained from wild-type Col-0 roots, using primers PATL2B1F and PATL2B2stopR (Table S3) and transferred via Gateway cloning into pDONR207 . This plasmid was used as a template for generating all different PATL2 deletion mutant coding sequences by PCR (Table S3 for primers), subsequently transferred into pDONR207. Then, PATL2 and deletion-mutant forms were transferred into pH7WGY2 vector via Gateway cloning , allowing constitutive expression of N-terminally tagged YFP proteins in plant cells via pCaMV35S H containing recombinant pH7WGY2 with PATL2 or PATL2-mutant inserts was used for transforming tobacco (Nicotiana benthamiana) by leaf infiltration, as previously described  supplemented with 100 μM acetosyringone. The suspension was infiltrated into young tobacco leaves. After 24–48 h, leaves were used for confocal microscopy. For co-localization studies, the PATL2 vector-containing bacteria were mixed with bacteria containing plant vectors for expression of PM marker AHA1-mRFP . YFP signals were excited at 514 nm, and emission was detected at 520–550 nm. mRFP and mOFP were excited at 561 nm, and emission was detected at 580–630 nm.600 of 0.08). The temperature was lowered from 37 to 30 °C when the culture reached an OD600 of 0.3–0.4. Protein expression was induced 30 min after the temperature shift with 1 mM isopropyl-ϐ-d-thiogalactopyranosid (IPTG) and cells were harvested 3 h later. StrepII-tagged protein of interest was purified using the Strep-tag®/Strep-Tactin® system (IBA Lifesciences) according to the manufacturer’s protocol for Strep-Tactin® Macroprep® or via Strep-Tactin®XT Superflow® cartridge using ÄKTA prime plus . Protein expression and purification were assessed following standard SDS–polyacrylamide gel electrophoresis, either followed by Coomassie Brilliant Blue staining or by blotting on Amersham™ Protran™ 0.2 µm nitrocellulose membranes  and affinity detection via enhanced chemiluminescence  of StrepII-tagged protein using Strep-Tactin®-horseradish peroxidase (HRP) conjugate (Iba Lifesciences).BL21 (DE3) pLysS-containing pET-52b(+) vectors with recombinant PATL2 or PATL2 deletion mutants were grown in 50 ml LB  were blocked with 3% (w/v) BSA in TBST  for 2 ½ h at room temperature in a Petri plate. The protein–lipid overlay incubation was performed with 2 ml Strep-tagged protein (0.5 µg/ml protein in 3% (w/v) BSA in TBST) at 4 °C overnight. The strip was washed five times for 8 min with TBST and incubated with 1:1000 diluted custom-made α-PATL2-1 IgG antibody, generated against the peptide TKKEETPVAPAPVEC (GenScript), in 3% (w/v) BSA in TBST for one hour at room temperature. The strip was washed four times for ten minutes with TBST and incubated for 1 h with 1:2000 Goat anti-rabbit IgG-HRP-conjugated antibody (Agrisera) in 3% (w/v) BSA in TBST. After four washing steps with TBST for 10 min, the protein–lipid binding was detected by enhanced chemiluminescence  and quantified.2 and PIP3 or from PI and all phosphorylated derivatives .For signal quantification of lipid dot binding, the Multiplex Band Analysis feature of AlphaView®Software (ProteinSimple) was used. Circular regions of same sizes were selected for each dot, and by using multi-regional background subtraction the corrected signal was quantified as background-corrected sum. Relative signal intensities were calculated by dividing each background corrected signal coming from one lipid drop by the sum of corrected signals from PI, PI(4)P, PIPsn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) (PI), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate) (PI(4)P) and 1,2-dioleoyl-sn-glycero-3-phospho- P2) were each dissolved 20:9:1 in chloroform:methanol:water, mixed to a total of 300 nmol with 7% PI, and pelleted by vacuum centrifugation. The resulting pellet was rehydrated in extrusion buffer and sonicated, and then extruded over a 0.2 µm polycarbonate membrane using a mini-extruder (Avanti®). Liposomes were harvested at 50,000×g for 15 min and suspended in 25 µl binding buffer. The size and homogeneity of liposome samples was checked by dynamic light scattering using a Zetasizer Nano S . 12 pmol of protein in 25 µl of binding buffer were incubated with the 25 µl liposomes for 30 min at room temperature. Supernatant (s) and pelleted membrane fractions (m) were separated by centrifugation at 18,000×g (30 min) and both analyzed by SDS-PAGE followed by immunoblot using the α-PATL2-1 IgG antibody and detection, as described above. Band intensities were quantified using the Multiplex Band Analysis feature of AlphaView®Software (ProteinSimple). Relative membrane fraction intensity was calculated in % as signal in the liposome fraction (m) versus the 100% total signal (s + m). For statistical analysis, data were processed using analysis of variance (ANOVA) and Fisher’s least significant difference post hoc test.All lipids used in the assay were purchased from Avanti® Polar Lipids, Inc. The liposome-binding assays were performed as described in Julkowska et al.  with theChlamydomonas reinhardtii and multicellular Volvox carteri, both belong to Chlorophyta, have a low degree of cell differentiation. In the representatives of this ancestral green algae taxon, we identified six and eleven single-domain SEC14 proteins (SEC14-only proteins) . In most of them, a CTN domain is associated with the SEC14 domain. This resembles the organization of the yeast SEC14-PITP family. Charophyta have a complex morphology, are evolutionarily advanced and among closest living algal relatives of land plants. The charophytic algal model Chara braunii has six SEC14-only proteins, one of them devoid of the CTN, and one multi-domain SEC14L-PITP . The latter, (CBR_g38007), has a N-terminal plant homeodomain (PHD) and a DNA-binding homeobox-DDT domain along with WHIM motifs 1 and 2 involved in packing the DDT domain . Seven were SEC14-only proteins, three with and four without CTN. The remaining three proteins were SEC14-GOLD domain proteins, similar to Arabidopsis PATLs. The lycophyte Selaginella moellendorffii, an ancient vascular plant, has 14 SEC14L-PITPs . Ten of them are Sec14-only proteins, one without CTN. Three are SEC14-GOLD proteins. We found one new multidomain protein with an N-terminal domain similar to the human Ganglioside-induced differentiation-associated protein 2 (GDAP2) domain. ADP-ribose is a potential substrate for the GDAP2 domain  and the monocot maize , 32 and 54 SEC14L-PITPs are present, comprising 11 and 20 multi-domain proteins, again with either GOLD, GDAP, nodulin or nodulin-related domains . The plant-specific SEC14-nodulin protein family is only present in investigated flowering plants  SEC14L-PITPs, summing up to 35 proteins  and adaptive responses (via N).PATL genes reflect the three different clades . Since PIP3 is likely not produced in plant cells P and PIPC14 Fig. d. This s2-containing liposomes (this work) and the PM, enriched in PIP2   P2, since it aligns well to the PIP2-binding motif of two proteins, AP180 and µ2-adaptin, which are involved in clathrin-coated vesicle formation  P. The above-mentioned amino acid similarities among C regions of SEC14-GOLD proteins suggest conserved domain functions for them . Also in the cellular context, the N region alone lacking the CTN-SEC14-GOLD module (PATL2ΔC), does not bind membranes. However, the N region confers binding to immobilized phospholipid units, and we explain this with charges and ionic interactions involving the positively-charged  repeats of PATL2-N. Perhaps the N regions of SEC14-GOLD proteins contribute to membrane interactions in cellular contexts by binding to membrane proteins, such as shown in the case of PATL1 binding the salt transporter SOS1 via the N region Below is the link to the electronic supplementary material.


C. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections. Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics.Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of  Clostridium perfringens is a major cause of histotoxic and intestinal infections in humans and other animals , C. perfringens beta toxin (CPB), C. perfringens enterotoxin (CPE), and epsilon (ETX), iota (ITX), and NetB toxins. This scheme assigns C. perfringens isolates to one of seven types (A to G)  . Of impootoxemia –3, 5.C. perfringens toxin production is frequently regulated by both TCRS and QS systems (C. perfringens are the VirS/VirR (VirS/R) TCRS and the accessory gene-like regulator (Agr) QS system (–Toxin production by pathogenic bacteria is often regulated by quorum sensing (QS) systems and two-component regulatory systems (TCRS), which can work in tandem , 8. Cons systems . The twoS system –11.virR virS (virR/S) operon, becomes activated by a signal that induces autophosphorylation of VirS, which is followed by phosphotransfer to VirR , by binding to VirR boxes located upstream of the promoters for those genes (–C. perfringens toxin genes (such as cpa) by inducing expression of a small regulatory RNA named VR-RNA  is encoded by the agrD gene and modified and secreted by the AgrB membrane protein. In the same S. aureus operon encoding AgrB/AgrD (AgrB/D) are genes encoding the AgrC/AgrA (AgrC/A) TCRS. In this system, AgrC is a sensor receptor protein for the AIP and acts by phosphorylating the transcriptional regulator AgrA to induce expression of a gene, located upstream of the agr operon, that encodes a small regulatory RNA named RNAIII. RNAIII then regulates toxin production -based peptides. For both strains, their agrB mutants respond to 5R, which likely corresponds to the natural 5-mer SP of C. perfringens .Previous studies , 28, 29 cpb gene transcript levels had increased by 3 h postinoculation and then reached maximum levels at 5 h postinoculation (during the early stationary phase). Levels of the cpb gene transcript were barely detected in 8-h (stationary-phase) or overnight  cultures  with the 5R versus the 8R peptide, consistent with our previous study , but onus study .agrB null mutant strains respond differently to the 5R or 8R peptides, we compared the VirS proteins made by these two strains using virS sequencing. Differences detected between the deduced VirS sequences of these two strains are shown in virS open reading frames (ORFs) of these two strains is that the virS ORF in CN3685 encodes a 6-amino-acid insertion at the 152nd amino acid, producing a VirS protein of 446 amino acids compared to the 440-amino-acid VirS protein made by CN1795.Since it has been hypothesized  that theThe transmembrane prediction algorithm (TMHMM) predicts that the VirS protein has 4 extracellular regions that might interact with SP if VirS is an SP receptor . FurthervirS gene in our existing agrB mutants and then complemented those double mutants to swap which VirS protein they expressed in order to test if this swap affects 8R sensitivity, as might be anticipated if VirS is an SP receptor.Given the differences in 8R signaling sensitivity observed between CN1795 and CN3685  and the Clostridium-modified TargeTron insertional mutagenesis method  or virS ORF sequences. Compared to the PCR products amplified from the agrB or virS genes in wild-type strains, PCR of DNA from the double mutants amplified larger bands from both genes due to insertion of an intron into the wild-type agrB and virS genes  two complementing strains named CN3685DKOc1795virR/S and CN1795DKOc3685virR/S that now expressed the virR/S operon of the other strain , the r strain . PCR resanalysis  demonstr results  demonstr results  confirmeagrB knockout (KO) but unlike CN1795 agrB KO. However, the CN3685 complementing strain expressing CN1795 VirS responded only to the 5R peptide, unlike CN3685 agrB KO but resembling CN1795 agrB KO. While this complementing strain also expressed trace amounts of CPB even without addition of a signaling peptide, likely attributable to VirR overproduction due to complementation with a multicopy plasmid  pulldown experiment were unsuccessful. Therefore, we used a CN3685 strain producing HisvirS gene in CN3685 was inactivated by insertion of an intron using the Clostridium-modified TargeTron insertional mutagenesis method  with an intron insertion in virS and a complementing strain producing the CN1795 VirS for studies , confirming that the presence of the His6 tag did not interfere with VirS function.For use in the pulldown experiment, we also transformed the CN3685 agrB null mutant strains (Before using a biotin-labeled 5R peptide (B-5R) in a pulldown experiment to test for its interactions with VirS, it was important to evaluate whether this biotin-modified peptide retains biologic activity, i.e., signaling function. Western blot analysis showed that B-5R induced CPB production by both the CN1795 and CN3685  strains .virSc3685virR/Shis cells .With positive results from those control experiments, we then performed a pulldown experiment to evaluate if VirS can physically bind to SP. This involved preincubating streptavidin-coated beads with B-5R or 5R before mixing those beads with B-PER buffer cell extracts from 4-h TY cultures of CN3685::is cells , lane 1.6 tag antibody detected (lane 3) no immunoreactive material in the crude extract of a control complementing strain (CN3685::virSc3685virS/R) that does not produce His6-tagged VirS, confirming that the immunoreactive material present in CN3685::virSc3685virR/Shis extracts was attributable to His6-tagged VirS. In addition, no immunoreactive material was detected by His6 antibody using extracts of CN3685::virSc3685virR/S after pulldown by beads preincubated with either 5R or B-5R (lanes 6 and 7). However, an ∼50-kDa immunoreactive species was pulled down from extracts of CN3685::virSc3685virR/Shis using beads preincubated with B-5R, which contains biotin and binds well to streptavidin-coated beads, but not from extracts of those cells preincubated with 5R, which lacks biotin and thus does not bind well to streptavidin-coated beads. This Western blot result analysis supports the ability of C. perfringens VirS to physically bind SP.As a specificity control, Western blotting with HisThe ability of B-5R-coated beads to pulldown VirS supports the binding of SP to VirS as a receptor. To confirm binding interactions between 5R and VirS by an independent approach and to discern a region of VirS involved in SP binding, we used the VirS structural model  to identagrB null mutant strains. CPB Western blot analysis  to CN1795 and CN3685 to determine if this peptide can also block Agr-like QS signaling by those two wild-type strains. Results  showed tS. aureus, sensing of the AIP causes the receptor AgrC to undergo histidine autophosphorylation and then phosphorylate AgrA, which initiates a positive feedback loop to make more AgrB and AgrD  are located within the same operon, the agrD gene is not expressed in null mutant strains with an insertion in their agrB gene  in the VirS made by CN1795 but to be 19 amino acids long (extending from amino acids 110 to 128) in the VirS made by CN3685. We speculate that, relative to the VirS made by CN1795, the larger size of its ECL2 allows the CN3685 VirS to functionally dock and accommodate the larger 8R  for signaling. Our results with ECL2 peptides (discussed in further detail below) implicated the Asp residue present at residue 117 in the CN1795 VirS as being important for SP binding. Detailed analysis of this loop should be performed in the future, since it represents a potential therapeutic target.C. perfringens types and origins indicated (data not shown) that only 2 of 53 strains resemble CN3685 in possessing a 6-amino-acid insertion at amino acid 152. This analysis also revealed that the KINSLVNVSELLGK ECL2 sequence of the CN3685 and CN1795 VirS was reasonably conserved among all 53 strains, with only 3 strains having 4 mutations and 26 strains having a single mutation. More common ECL2 sequence variations in these 26 strains were 16 strains producing an ECL2 with an S (versus a V) at position 6 and 10 strains producing an ECL2 with a D (versus an E) at position 10. Whether those ECL2 variations affect SP signaling should be investigated in the future.The VirS variant made by CN3685 is unusual. GenBank analysis of deduced VirS sequences in 53 other strains representing many Those findings also extend understanding of the VirS structure versus function relationship. Consistent with the modeling shown in C. perfringens. Several lines of evidence were obtained that now directly support VirS as an SP receptor. First, we exploited the previous observation (agrB null mutants of CN3685 while 5R (but not 8R) signals agrB null mutants of CN1795, which suggested that there may be differences between the VirS of CN3685 versus CN1795 if VirS is a major SP receptor. As discussed earlier, sequencing confirmed significant differences between the second ECL of VirS of CN3685 versus CN1795; therefore, we reasoned that, if VirS is an SP receptor, then swapping expression of these virS variants between virR/S and agrB/D double-null mutants of CN3685 versus those of CN1795 should change their sensitivity to 8R signaling. This hypothesis was supported when the CN1795 double mutant complemented to express the CN3685 VirS variant was shown to increase its CPB production in the presence of 8R, while the CN3685 double mutant complemented to express the CN1795 VirS variant lost the ability to increase CPB production in the presence of 8R. There was some CPB production by these complementing strains even in the absence of 8R; as noted previously, this effect is likely due to overexpression of VirR from the multicopy plasmid used for complementation  also implicate the second ECL of VirS in this binding. Collectively, these ECL2 peptide results not only further support VirS:SP binding being a mechanistic basis for interactions between the Agr-like QS system and VirS/R TCRS during C. perfringens pathogenesis but could also be instructive for developing peptide therapeutics to inhibit QS signaling and reduce toxin production to control type C or type B diseases in which CPB is, respectively, proven or likely to be important for virulence.A third and final line of evidence supporting VirS as an SP receptor came from experiments using synthetic peptides corresponding to either the predicted ECL2 of VirS or to this ECL sequence with a single N to D substitution. The rationale behind this experiment was that if the second ECL of VirS is important for SP binding, as suggested by the VirS sequencing results described earlier, then preincubating this ECL2 peptide with 5R may cause ECL2:5R binding and thus inhibit subsequent Agr-like QS signaling by C. perfringens pathogenesis beyond diseases caused by type B or C strains. Both the Agr-like QS system and VirS/R TCRS positively regulate production of CPA and PFO  production by type F strains  than VirS (∼50 kDa) (data not shown). Whether that band reflects nonspecific binding or a second SP receptor will require further study.While this study provides compelling support for VirS as an SP receptor, it remains possible that ent TCRS . SupportS. aureus Agr system, SP signaling upregulates expression of the agr operon encoding itself and the AgrC receptor in a positive feedback loop  were incubated at 37°C under anaerobic growth conditions in GasPak jars to screen the knockout mutants constructed in this study. A CN3685::agrB null mutant named BMJV10, a CN1795::agrB null mutant, and complementing strains of both mutants had been previously constructed and characterized  activation in dimethylforamide (DMF). FMOC-protected amino acids (EMD Millipore) were used for the stepwise assembly of these linear sequences on preloaded FMOC-Lys(Boc) Wang resin . A cleavage cocktail  was used for 4 h at room temperature, while shaking, to cleave KIGK and KIGK_D from the Wang resin as well as to scavenge side chain-protecting groups. These peptides were first precipitated in cold diethyl ether and then washed and centrifuged 3 times with additional diethyl ether, resulting in a pellet of crude peptide. Crude KIGK and KIGK_D were allowed to air dry and were then dissolved in 0.1% TFA, which was then frozen and lyophilized overnight to remove remaining organics. Each crude peptide was dissolved in 0.1% TFA and then directly loaded onto a Waters 2555 Quaternary Gradient Module with a Waters 2489 UV-visible (UV-Vis) detector and purified on a Phenomenex Gemini (250 mm × 21.20 mm) 10-μm C18 column using standard acetonitrile (ACN)/0/1% TFA gradient conditions. Final analytical determinations of peptide purity for KIGK and KIGK_D were performed on a Waters e2695 separations module with a Waters 2489 UV-Vis detector using a Phenomenex Gemini-NX (250 mm × 4.6 mm) 5 μm C18 column using standard acetonitrile (ACN)/0.1% TFA gradient conditions. Mass spectrometry analysis using an Applied Biosystems Voyager DE-STR matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) was used to confirm the expected mass of each peptide, as follows: KIGK expected, 1,513.80; observed, 1,513.04; KIGK_D expected, 1,514.78; observed 1,514.06.The synthesis of all peptides used in this study was carried out by the Peptide and Peptoid Synthesis Core Facility Division of the Health Sciences Core Research Facilities (HSCRF) at the University of Pittsburgh. SP-based synthetic peptides used included (i) 5R, a 5-mer cyclic ring likely corresponding to the wild-type ngens SP ; (ii) 8Rngens SP . Synthesngens SP  were perEach synthetic peptide was resuspended in dimethyl sulfoxide  at 50 mM before use. Aliquots  of this suspension of synthetic peptide in DMSO were then added to 1 ml of culture medium, resulting in a final peptide concentration of 0.1, 0.5, or 1 mM. After a 5-h or overnight (16-h) culture, CPB or His-tagged Western blots were performed using culture supernatants or pelleted cells .2O) was then added to reach a total reaction volume of 50 μl. The reaction mixtures were placed in a thermal cycler (Techne) and then subjected to the following amplification conditions: 1 cycle of 98°C for 30 sec; 35 cycles of 98°C for 10 s, 50 to 72°C (depending on the primers used) for 20 s, and 72°C for 2 min, followed by a single extension of 72°C for 2 min. The resultant PCR products were cleaned up using a QIAquick PCR purification kit (Qiagen) and sent for DNA sequencing to the University of Pittsburgh Core Sequencing Facility.DNA was isolated from CN1795 or CN3685 using the MasterPure Gram-positive DNA purification kit (Epicentre). The primers used in a PCR to amplify this operon are listed in virS sequencing results were used to model the predicted structure of VirS made by CN1795 or CN3685 using the TMHMM server v.2.0  transmembrane prediction algorithms.The Clostridium-modified group II TargeTron technology . These primers were specific for 16S RNA (as a control housekeeping gene) (virS gene (qvirSF and qvirSR), or the agrD gene (qagrDF and qagrDR).A plasmid named pJIR750virSi was constructed to inactivate, using chnology , the virng gene) , the virvirR/S operon complementation vector named pJIR750virR/Scomp was constructed as follows. A region of CN1795 or CN3685 DNA spanning from ∼200 bp upstream of the virR/S operon to ∼200 bp downstream of the virR/S operon was PCR amplified using primers VirR/ScompF and VirR/ScompR . Transformants were then selected on BHI agar plates containing 15 μg · ml−1 of chloramphenicol with anaerobic incubation in GasPak jars. For expression of His6-tagged VirS, the virR/S operon was PCR amplified using primers VirR/ScompF and VirR/ShisR  that expresses VirS protein labeled with a His6 tag.A R/ScompR . The resrR/ShisR , which avirS gene was disrupted in CN1795 or CN3685 to generate single-null mutant strains that do not produce VirS. The virS gene was also inactivated in existing CN1795::agrB or CN3685::agrB strains  into the virS gene, generating virS single-null mutants or virS agrB double-null mutants of CN1795 or CN3685. For this purpose, the intron donor plasmid pJIR750virSi, which carries a virS-targeted intron, was electroporated into CN1795, CN3685, CN1795::agrB, or CN3685::agrB. Transformants were selected by plating onto BHI agar plates containing 15 μg · ml−1 of chloramphenicol, followed by overnight anaerobic growth in a GasPak jar. Colony PCR was carried out for screening using internal virS primers virSKOF and virSKOR , a CN3685 virS null mutant (CN3685::virS), a CN1795 virS agrB double-null mutant (CN1795DKO), and a CN3685 virS agrB double-null mutant (CN3685DKO). Each mutant was then further characterized by PCR, RT-PCR, and Southern blotting analyses, as described below.The  strains , 30 to c virSKOR , which avirR/S1795comp or pJIR750virR/S3685comp. Transformants were selected on chloramphenicol, as described earlier.VirS complementing strains of the single and double mutants were prepared by transformation with pJIR750C. perfringens vegetative growth, a 0.2-ml aliquot of an overnight FTG culture of a wild-type or null mutant strain was inoculated into 10 ml of TY medium. The cultures were incubated at 37°C; thereafter, at 0-, 1-, 3-, 5-, 8-, and 24-h culture times, 1 ml of each culture was removed for measurement of optical density at 600 nm (OD600) using a Bio-Rad Smart spectrometer.For analysis of C. perfringens RNA isolation, RT-PCR, and qRT-PCR analyses.”For the two wild-type strains, another 1-ml aliquot of culture was removed and centrifuged at 15,000 rpm for 3 min. Equal volumes of each resultant culture supernatant were then mixed with 5× SDS loading buffer and boiled for 5 min. An aliquot (30 μl) of each boiled sample was electrophoresed on a 10% SDS-PAGE gel and then subjected to a CPB Western blot analysis . Using the same cultures, total RNA was isolated from the pellets at each time point, and qRT-PCR were performed as described in “C. perfringens strains using the MasterPure Gram-positive DNA purification kit. PCR for the virS or agrB genes was performed using the primers described in the previous section. For the wild-type strains, the sizes of PCR products are listed in DNA was extracted from all For Southern blot analysis to detect an intron insertion, aliquots (3 μg each) of wild-type, single-null, or double-null mutant strain DNA were digested overnight with EcoRI at 37°C according to the manufacturer’s instructions (New England Biolabs). The digested DNA samples were then electrophoresed on a 1% agarose gel before transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe . The intvirS expression) or 5 h  at 37°C. Cultures were then pelleted at the indicated times, and RNA was extracted using saturated phenol and purified by TRIzol and chloroform (Life Technologies and Sigma), as previously described , avian myeloblastosis virus (AMV) reverse transcriptase , ddH2O, and primers specific for the virS or agrB genes  was used in a one-step RT-PCR containing 10 μl of 2× rB genes , 16S RNArB genes . Reactio−ΔΔCT) method  of purified RNA in a total 10-μl reaction mixture was first synthesized to cDNA using a Maxima first-strand cDNA synthesis kit (Thermo Scientific), according to the manufacturer’s instructions. The cDNA synthesis programming was as follows: 25°C for 10 min, 50°C for 30 min, and 85°C for 5 min. All qRT-PCR primers were designed using the Integrated DNA Technologies (IDT) website and are listed in ) method .agrB mutants of CN1795 and CN3685 were each grown in 10 ml of FTG overnight at 37°C. An aliquot (0.2 ml) of each culture was inoculated into 10 ml of fresh TY broth, and those cultures were incubated overnight (about 16 h) at 37°C. A 15-μl aliquot of each TY overnight culture was then inoculated into 1 ml of TY medium in a 1.5-ml microcentrifuge tube with a screw cap (Fisher Scientific) that contained a final concentration of 100 μM and SP-based peptide  suspended in DMSO. As a control, an equal volume of DMSO alone (no peptide) was added to some cultures. These cultures were incubated for 5 h or overnight at 37°C as specified in the experiments.Wild-type or agrB null mutant strains were each grown in 10 ml of FTG medium overnight at 37°C. An aliquot (0.2 ml) of each culture was inoculated into 10 ml of fresh TY broth, and those cultures were incubated overnight (about 16 h) at 37°C. Before the addition of a 15-μl aliquot of each TY overnight culture, 5R , together with a 500 μM final concentration of the 14-amino-acid peptide (KIGK) corresponding to the VirS ECL2 sequence, the variant peptide (KIGK_D), or a control peptide, was dissolved in DMSO and the mixed-in 1 ml TY medium and incubated at 37°C for 30 min. A 15-μl aliquot of the overnight agrB null mutant culture was inoculated into this medium and incubated for 5 h. At that time, the culture supernatants were collected and used for CPB Western blot analysis.CN1795 and CN3685 In a second experiment, wild-type CN1795 and CN3685 cultures were each grown in 10 ml of FTG overnight at 37°C. An aliquot (0.2 ml) of each culture was inoculated into 10 ml of fresh TY broth, and those cultures were incubated overnight (about 16 h) at 37°C. A 15-μl aliquot of each TY overnight culture was then inoculated into 1 ml of TY medium in a 1.5-ml microcentrifuge tube with a screw cap that contained a final concentration of 500 μM or 1 mM KIGK or KIGK_D dissolved in DMSO. These cultures were then incubated for 5 h. At that time, the culture supernatants were collected and used for CPB Western blot analysis.600 values and then centrifuged. The supernatants were mixed with 5× SDS-PAGE loading buffer and boiled for 5 min. Aliquots (20 μl) of each sample were electrophoresed on a 10% SDS-PAGE gel, and the separated proteins were then transferred onto a nitrocellulose membrane. The membrane was blocked with TTBS, i.e., Tris-buffered saline-Tween 20 (0.05% vol/vol), and nonfat dry milk (5% wt/vol) for 1 h at room temperature, followed by probing with a rabbit poly-anti-CPB antibody  overnight at 4°C. Finally, bound antibody was detected with a horseradish peroxidase-conjugated secondary anti-rabbit antibody (Sigma-Aldrich), followed by addition of SuperSignal West Pico chemiluminescent substrate (Fisher Scientific).Aliquots of each culture were adjusted to equal ODvirS was transformed with a complementation vector named pJIR750virR/Shiscomp, which encodes a His6-tagged VirS (see “Plasmids and primers”), to create CN3685::virSc3685virR/Shis. As an antibody specificity control, CN3685::virS was also transformed to create a strain named CN3685::virSc3685virR/S that expresses untagged VirS. A 0.2-ml aliquot of a CMM stock of each culture was transferred to 10 ml of FTG medium and then cultured overnight at 37°C. A 0.2-ml aliquot of each FTG culture was transferred to 10 ml of TY medium for another overnight culture at 37°C. A 2-ml aliquot of this overnight TY culture was transferred to 100 ml of fresh TY medium and cultured for 4 h at 37°C. Those cultures were then centrifuged and the pellets frozen at −80°C for at least 2 h. The frozen pellets were resuspended in 1 ml of B-PER buffer (Fisher Scientific) with proteinase inhibitor  at room temperature for 30 min with gentle shaking. At the same time, a 100-μl aliquot of streptavidin MagBeads (GenScript) was washed twice with B-PER buffer. The washed beads were then incubated with either biotinylated 5R (Bio-5R) or control 5R in 500 μl of B-PER buffer (200 μM) for 1 h at room temperature with slow end-over-end mixing. These pretreated beads were then washed twice with B-PER buffer. The bacterial supernatants, prepared as described above, were applied to the washed MagBeads, and the mixture was incubated at 4°C overnight with slow end-over-end mixing. The MagBeads were washed three times and then 80 μl of 2× SDS loading dye was added, followed by boiling for 5 min. A 40-μl aliquot of each sample was loaded on a 10% SDS gel, followed by a His6 tag detection Western blot. For this purpose, a His6 tag antibody (Aviva Systems Biology OAEA00010) was purchased from Fisher Scientific. The antibody was used at a 1:1,000 dilution in TTBS with 5% milk. Finally, bound antibody was detected with a horseradish peroxidase-conjugated secondary anti-mouse antibody (Sigma-Aldrich), followed by addition of Clarity and Clarity Max ECL Western blotting substrates (Bio-Rad).CN3685::post hoc analysis by Dunnett’s multiple-comparison test. For comparison of two samples, Student’s t test was applied. Differences were considered significant when the P value was less than 0.05.All statistical analyses were performed using GraphPad Prism 8. For comparison of more than two samples, one-way analysis of variance (ANOVA) was applied with MT597430 and MT597431).The sequence results were submitted to NCBI GenBank (accession numbers


Changes in gene regulation are widely recognized as an important driver of adaptive phenotypic evolution. However, the specific molecular mechanisms that underpin such changes are still poorly understood. Chromatin state plays an essential role in gene regulation, by influencing the accessibility of coding loci to the transcriptional machinery. Changes in the function of chromatin remodellers are therefore strong candidates to drive changes in gene expression associated with phenotypic adaptation. Here, we identify amino acid homoplasies in the chromatin remodeller CHD9, shared between the extinct marsupial thylacine and eutherian wolf which show remarkable skull convergence. CHD9 is involved in osteogenesis, though its role in the process is still poorly understood. We examine whether CHD9 is able to regulate the expression of osteogenic target genes and examine the function of a key substitution in the CHD9 DNA binding domain.RUNX2, Osteocalcin (OC) and ALP in HEK293T cells. We found that overexpression of CHD9 upregulated RUNX2, the master regulator of osteoblast cell fate, but not the downstream genes OC or ALP, supporting the idea that CHD9 regulates osteogenic progenitors rather than terminal osteoblasts. We also found that the evolutionary substitution in the CHD9 DNA binding domain does not alter protein secondary structure, but was able to drive a small but insignificant increase in RUNX2 activation. Finally, CHD9 was unable to activate an episomal RUNX2 promoter-reporter construct, suggesting that CHD9 requires the full chromatin complement for its function.We examined whether CHD9 was able to upregulate its osteogenic target genes, RUNX2. Though we were unable to identify significant functional consequences of the evolutionary substitution in HEK293T cells, our study provides important steps forward in the functional investigation of protein homoplasy and its role in developmental processes. Mutations in coding genes may be a mechanism for driving adaptive changes in gene expression, and their validation is essential towards determining the functional consequences of evolutionary homoplasy.We provide new evidence to the role of CHD9 in osteogenic differentiation through its newly observed ability to upregulate the expression of  Selection targets traits in response to specific environmental pressures, though the molecular basis of these evolutionary changes remains unclear. Phenotypic development is driven by suites of genes in complex regulatory networks –3. MutatThe development of complex traits is controlled by the expression of multiple genes in concert . AccordiBGLAP , ALP  and the master osteogenic regulator RUNX2 (runt-related transcription factor 2)  (Promega).We generated  vulpes) . The cor mammals . The thyHuman embryonic kidney (HEK293T) cells were maintained in DMEM (ThermoFisher) media containing 10% FBS. The media was replaced every 2–3 days. Cells were passaged at 90% confluency. HEK293T cells were seeded in 6 well plates and transfected using lipofectamine 3000 (ThermoFisher) according to the manufacturer’s instructions. Cells were either transfected with 2.5μg of empty vector (pcDNA), or plasmids containing the Thr2384 or Ala2384 alleles and incubated for 24 h. Each transfection experiment was repeated 5 times for reproducibility.Chd9 possessing the Thr2384 and Ala2384 alleles (n = 5). Thr2384 or Ala2384 transfected cell gene expression values were normalized against the housekeeping gene HPRT and the un-transfected vehicle using the ΔΔCT method [RUNX2 directly against the normalized exogenous levels of the Thr2384 and Ala2384 CHD9 alleles and determined their levels as a ratio. Primer sequences are listed in Supplementary Table Transfected cells were harvested, and RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma Aldrich). RNA was treated with Turbo DNA-free DNase (Ambion) to remove all residual traces of gDNA. The resulting RNA was stored at − 80 °C. Total RNA was converted to cDNA using the SuperScript™ III First-Strand Synthesis System (ThermoFisher). RT-qPCR was performed to examine expression levels for each of the genes of interest in cells transfected with empty vehicle, or T method . As exogRUNX2 promoter-luciferase reporter. RUNX2 promoter driven firefly luciferase and Renilla luciferase activity were analysed 48 h later using the Dual-Luciferase Reporter Assay System (Promega) on a FLUOstar OPTIMA dual plate reader . Firefly luciferase values were normalized against Renilla luciferase to obtain relative promoter activity, measured as relative light units (RLU). Each transfection and luciferase assay were repeated 5 times for reproducibility.HEK293T cells were seeded in white flat-bottom 96-well plates and allowed to recover for 24 h. The following day, cells were transfected using Lipofectamine 3000, according to manufacturer instructions, with either empty pcDNA, Thr2384 or Ala2384; as well as either the empty pGL4.10 vector, thylacine or fox Additional file 1. Supplementary Table 1 – Primer sequences used in the study.