Molecular signature of excessive female aggression: study of stressed mice with genetic inactivation of neuronal serotonin synthesis.

Aggression is a complex social behavior, critically involving brain serotonin (5-HT) function. The neurobiology of female aggression remains elusive, while the incidence of its manifestations has been increasing. Yet, animal models of female aggression are scarce. We previously proposed a paradigm of female aggression in the context of gene x environment interaction where mice with partial genetic inactivation of tryptophan hydroxylase-2 (Tph2+/- mice), a key enzyme of neuronal 5-HT synthesis, are subjected to predation stress resulting in pathological aggression. Using deep sequencing and the EBSeq method, we studied the transcriptomic signature of excessive aggression in the prefrontal cortex of female Tph2+/- mice subjected to rat exposure stress and food deprivation. Challenged mutants, but not other groups, displayed marked aggressive behaviors. We found 26 genes with altered expression in the opposite direction between stressed groups of both Tph2 genotypes. We identified several molecular markers, including Dgkh, Arfgef3, Kcnh7, Grin2a, Tenm1 and Epha6, implicated in neurodevelopmental deficits and psychiatric conditions featuring impaired cognition and emotional dysregulation. Moreover, while 17 regulons, including several relevant to neural plasticity and function, were significantly altered in stressed mutants, no alteration in regulons was detected in stressed wildtype mice. An interplay of the uncovered pathways likely mediates partial Tph2 inactivation in interaction with severe stress experience, thus resulting in excessive female aggression.

Gut microbiome responses to dietary intervention with hypocholesterolemic vegetable oils.

Hypercholesterolemia is becoming a problem with increasing significance. Dietary vegetable oils may help to improve this condition due to presence of phytonutrients with potentially synergistic cholesterol-lowering effects. The objective of this 8-week double-blinded randomized clinical trial was to investigate the effects of consuming 30 g of two different blended cooking oils, rich in omega-3 alpha-linolenic acid and phytonutrients, or refined olive oil on the intestinal microbiota in 126 volunteers with borderline hypercholesterolemia. Multi-factor analysis of relationships between the gut microbiota composition at various taxonomic ranks and the clinical trial parameters revealed the association between beneficial effects of the dietary intervention on the blood lipid profile with abundance of Clostridia class of the gut microbiota. This microbiota feature was upregulated in the course of the dietary intervention and associated with various plasma markers of metabolic health status, such as Triglycerides, Apolipoprotein B and Total Cholesterol to HDL ratio in a beneficial way. The relative abundance of a single species-Clostridium leptum-highly increased during the dietary intervention in all the three study groups. The oil blend with the highest concentration of omega-3 PUFA is associated with faster and more robust responses of the intestinal microbiota, including elevation of alpha-diversity. Butyrate production is being discussed as a plausible process mediating the observed beneficial influence on the plasma lipid profile. Causal mediation analysis suggested that Clostridium genus rather than the higher rank of the phylogeny-Clostridia class-may be involved in the diet-induced improvements of the blood lipid profile.

UM171 expands distinct types of myeloid and NK progenitors from human pluripotent stem cells.

Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34+CD41aloCD45+ phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34+CD45+CD7+ lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.

GATA2 Is Dispensable for Specification of Hemogenic Endothelium but Promotes Endothelial-to-Hematopoietic Transition.

The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2-/- hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43-CD73+ non-HE or VE-cadherin+CD43-CD73- HE generation and subsequent HE diversification into DLL4+ arterial and DLL4- non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.

Activation of the Arterial Program Drives Development of Definitive Hemogenic Endothelium with Lymphoid Potential.

Understanding the pathways guiding the development of definitive hematopoiesis with lymphoid potential is essential for advancing human pluripotent stem cell (hPSC) technologies for the treatment of blood diseases and immunotherapies. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries but not veins. Here, we show that activation of the arterial program through ETS1 overexpression or by modulating MAPK/ERK signaling pathways at the mesodermal stage of development dramatically enhanced the formation of arterial-type HE expressing DLL4 and CXCR4. Blood cells generated from arterial HE were more than 100-fold enriched in T cell precursor frequency and possessed the capacity to produce B lymphocytes and red blood cells expressing high levels of BCL11a and ß-globin. Together, these findings provide an innovative strategy to aid in the generation of definitive lymphomyeloid progenitors and lymphoid cells from hPSCs for immunotherapy through enhancing arterial programming of HE.

Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032.

BACKGROUND: Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. RESULTS: In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. CONCLUSIONS: The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.

NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

Zebrafish Rfx4 controls dorsal and ventral midline formation in the neural tube.

BACKGROUND: Rfx winged-helix transcription factors, best known as key regulators of core ciliogenesis, also play ciliogenesis-independent roles during neural development. Mammalian Rfx4 controls neural tube morphogenesis via both mechanisms. RESULTS: We set out to identify conserved aspects of rfx4 gene function during vertebrate development and to establish a new genetic model in which to analyze these mechanisms further. To this end, we have generated frame-shift alleles in the zebrafish rfx4 locus using CRISPR/Cas9 mutagenesis. Using RNAseq-based transcriptome analysis, in situ hybridization and immunostaining we identified a requirement for zebrafish rfx4 in the forming midlines of the caudal neural tube. These functions are mediated, least in part, through transcriptional regulation of several zic genes in the dorsal hindbrain and of foxa2 in the ventral hindbrain and spinal cord (floor plate). CONCLUSIONS: The midline patterning functions of rfx4 are conserved, because rfx4 regulates transcription of foxa2 and zic2 in zebrafish and in mouse. In contrast, zebrafish rfx4 function is dispensable for forebrain morphogenesis, while mouse rfx4 is required for normal formation of forebrain ventricles in a ciliogenesis-dependent manner. Collectively, this report identifies conserved aspects of rfx4 function and establishes a robust new genetic model for in-depth dissection of these mechanisms. Developmental Dynamics 247:650-659, 2018. © 2017 Wiley Periodicals, Inc.

Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis.

The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development.

Specification and Diversification of Pericytes and Smooth Muscle Cells from Mesenchymoangioblasts.

Elucidating the pathways that lead to vasculogenic cells, and being able to identify their progenitors and lineage-restricted cells, is critical to the establishment of human pluripotent stem cell (hPSC) models for vascular diseases and development of vascular therapies. Here, we find that mesoderm-derived pericytes (PCs) and smooth muscle cells (SMCs) originate from a clonal mesenchymal progenitor mesenchymoangioblast (MB). In clonogenic cultures, MBs differentiate into primitive PDGFRß+CD271+CD73- mesenchymal progenitors, which give rise to proliferative PCs, SMCs, and mesenchymal stem/stromal cells. MB-derived PCs can be further specified to CD274+ capillary and DLK1+ arteriolar PCs with a proinflammatory and contractile phenotype, respectively. SMC maturation was induced using a MEK inhibitor. Establishing the vasculogenic lineage tree, along with identification of stage- and lineage-specific markers, provides a platform for interrogating the molecular mechanisms that regulate vasculogenic cell specification and diversification and manufacturing well-defined mural cell populations for vascular engineering and cellular therapies from hPSCs.

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling.

Many aspects of bacterial physiology and behavior, including motility, surface attachment, and the cell cycle, are controlled by cyclic di-GMP (c-di-GMP)-dependent signaling pathways on the scale of seconds to minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing the expression of genes encoding c-di-GMP-synthetic (diguanylate cyclases) and -degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared-light-regulated diguanylate cyclase, BphS, was engineered previously, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue-light-activated phosphodiesterase EB1 can be used in combination with the red/near-infrared-light-regulated diguanylate cyclase BphS for the bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility, attachment to surfaces, the cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occur at a fast (seconds-to-minutes) pace. Interrogation of these processes at high temporal and spatial resolution using chemicals is difficult or impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue-light-activated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared-light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.

Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research.

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification.

Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides.

Previously, the RubisCO-compromised spontaneous adaptive Rhodobacter sphaeroides mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, glnA and nifA, whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the nifK gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

Salt stress-induced changes in the transcriptome, compatible solutes, and membrane lipids in the facultatively phototrophic bacterium Rhodobacter sphaeroides.

Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides.

Rhodobase, a meta-analytical tool for reconstructing gene regulatory networks in a model photosynthetic bacterium.

We present Rhodobase, a web-based meta-analytical tool for analysis of transcriptional regulation in a model anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The gene association meta-analysis is based on the pooled data from 100 of R. sphaeroides whole-genome DNA microarrays. Gene-centric regulatory networks were visualized using the StarNet approach (Jupiter, D.C., VanBuren, V., 2008. A visual data mining tool that facilitates reconstruction of transcription regulatory networks. PLoS ONE 3, e1717) with several modifications. We developed a means to identify and visualize operons and superoperons. We designed a framework for the cross-genome search for transcription factor binding sites that takes into account high GC-content and oligonucleotide usage profile characteristic of the R. sphaeroides genome. To facilitate reconstruction of directional relationships between co-regulated genes, we screened upstream sequences (-400 to +20bp from start codons) of all genes for putative binding sites of bacterial transcription factors using a self-optimizing search method developed here. To test performance of the meta-analysis tools and transcription factor site predictions, we reconstructed selected nodes of the R. sphaeroides transcription factor-centric regulatory matrix. The test revealed regulatory relationships that correlate well with the experimentally derived data. The database of transcriptional profile correlations, the network visualization engine and the optimized search engine for transcription factor binding sites analysis are available at http://rhodobase.org.

Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications.

Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and up-regulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.

The PpaA/AerR regulators of photosynthesis gene expression from anoxygenic phototrophic proteobacteria contain heme-binding SCHIC domains.

The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides.

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.