A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae.

CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions, and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for the industrial conversion of lignocellulosic biomasses. Tolerance to acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of >9,000 strains, where >98% of all essential and respiratory growth-essential genes were targeted with multiple guide RNAs (gRNAs). The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where high-resolution CRISPRi library screening paves the way to understanding and bioengineering the robustness of yeast against acetic acid stress. IMPORTANCE Acetic acid is inhibitory to the growth of the yeast Saccharomyces cerevisiae, causing ATP starvation and oxidative stress, which leads to the suboptimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth-essential genes that regulate tolerance to acetic acid were identified, providing a new understanding of the stress response of yeast and new targets for the bioengineering of industrial yeast. Our findings on the fine-tuning of the expression of proteasomal genes leading to increased tolerance to acetic acid suggest that this could be a novel strategy for increasing stress tolerance, leading to improved strains for the production of biobased chemicals.

Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions.

Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of ~ 740 Mbp. A pilot genome assembly rendered a total assembly size of ~ 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (π) in coding regions (average π ≈ 5% and average π in fourfold degenerate sites ≈ 20%), and an overall high repeat content (at least 40%). We also report on high variation in the α-octopamine receptor OctA (average π = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

The complex barnacle perfume: identification of waterborne pheromone homologues in Balanus improvisus and their differential expression during settlement.

A key question in barnacle biology is the nature of cues that induce gregarious settlement. One of the characterised cues is the waterborne settlement pheromone (WSP). This study aimed to identify WSP homologues in Balanus improvisus and to investigate their expression during settlement. Six WSP homologues were identified, all containing an N-terminal signal peptide, a conserved core region, and a variable C-terminus comprising several -GR- and -HDDH- motifs. The B. improvisus WSP homologues were expressed in all settlement stages but showed different expression patterns. The homologue most similar to the B. amphitrite WSP was the most abundant and was constantly expressed during settlement. In contrast, several of the other WSP homologues showed the greatest expression in the juvenile stage. The presence of several WSP homologues suggests the existence of a pheromone mix, where con-specificity might be determined by a combination of sequence characteristics and the concentration of the individual components.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Importance of plasticity and local adaptation for coping with changing salinity in coastal areas: a test case with barnacles in the Baltic Sea.

BACKGROUND: Salinity plays an important role in shaping coastal marine communities. Near-future climate predictions indicate that salinity will decrease in many shallow coastal areas due to increased precipitation; however, few studies have addressed this issue. The ability of ecosystems to cope with future changes will depend on species' capacities to acclimatise or adapt to new environmental conditions. Here, we investigated the effects of a strong salinity gradient (the Baltic Sea system--Baltic, Kattegat, Skagerrak) on plasticity and adaptations in the euryhaline barnacle Balanus improvisus. We used a common-garden approach, where multiple batches of newly settled barnacles from each of three different geographical areas along the Skagerrak-Baltic salinity gradient were exposed to corresponding native salinities (6, 15 and 30 PSU), and phenotypic traits including mortality, growth, shell strength, condition index and reproductive maturity were recorded. RESULTS: We found that B. improvisus was highly euryhaline, but had highest growth and reproductive maturity at intermediate salinities. We also found that low salinity had negative effects on other fitness-related traits including initial growth and shell strength, although mortality was also lowest in low salinity. Overall, differences between populations in most measured traits were weak, indicating little local adaptation to salinity. Nonetheless, we observed some population-specific responses--notably that populations from high salinity grew stronger shells in their native salinity compared to the other populations, possibly indicating adaptation to differences in local predation pressure. CONCLUSIONS: Our study shows that B. improvisus is an example of a true brackish-water species, and that plastic responses are more likely than evolutionary tracking in coping with future changes in coastal salinity.

Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.

Detection of ligand-receptor binding using microfluidic frontal affinity chromatography on proteoliposomes derived directly from native cell membranes.

A method for characterization of ligand binding to membrane receptors in their native cell membrane is presented. The methodology is based on microfluidic frontal affinity chromatography coupled to mass spectrometry (FAC-MS). Proteoliposomes with receptor of interest are prepared directly from cell membranes and serve as a stationary phase in a microfluidic flow cell for frontal analysis. The G-Protein-Coupled Receptor (GPCR) Ste2 involved in the pheromone-induced yeast mating pathway is used as a model receptor for proof of principle characterization. The ligand affinity of the natural pheromone peptide, the α-factor, is compared to a set of pheromone analogs having different receptor affinities. With short preparation time, preserved lipid composition and the ability to immobilize proteoliposomes from any cell membrane, we propose that our methodology with immobilized proteoliposomes together with microfluidics FAC-MS can be an important improvement for ligand-receptor studies in native membranes.

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.

Down regulation of TRPC1 by shRNA reduces mechanosensitivity in mouse dorsal root ganglion neurons in vitro.

Mechanosensitivity is a crucial but poorly understood property of the sensory nervous system. Transient receptor potential (TRP) channels, which have been found to be responsible for the detection of other sensory stimuli such as temperature and pungent chemicals, have been suggested to also recognize stretch or pressure to cell membranes. TRPC1 is one candidate from studies in oocytes but evidence in native sensory neurons has been lacking. Therefore, we have measured an increase in intracellular Ca(2+) levels upon mechanical activation of native mouse dorsal root ganglion (DRG) neurons in culture using hypoosmolar buffer. Our results show that down regulation of TRPC1 with short hairpin RNA results in a 65% reduction of neurons with stretch activated responses. These results implicate a direct or indirect involvement of TRPC1 in the mechanosensitivity of DRG neurons.

Gene expression analysis suggests that EBF-1 and PPARgamma2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics.

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.

Identification of the human ApoAV gene as a novel RORalpha target gene.

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORalpha regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORalpha also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORalpha increased the endogenous expression of ApoAV in HepG2 cells and RORalpha also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORalpha transactivation, one of which overlaps with a previously identified binding site for PPARalpha. Together, these results suggest a novel mechanism whereby RORalpha modulates lipid metabolism and implies RORalpha as a potential target for the treatment of dyslipidemia and atherosclerosis.

Early B-cell factor (O/E-1) is a promoter of adipogenesis and involved in control of genes important for terminal adipocyte differentiation.

Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.