Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.     

Flow regimes in a two phase reversed flow jet loop bioreactor

Reversed flow jet loop bioreactors (RFJLB) have been used extensively for 2 or 3 phase biochemical reactions. From visual observations and gas holdup data, 3 distinct flow regimes are identified in RFJLB, namely: (1) Bubble free regime (BFR), where bubbles are observed in the draft tube only; (2) Transition regime (TR), where bubbles are observed in both the draft tube and the annulus, but without circulation; and (3) Complete bubble circulation regime (CBCR), where bubbles circulate in both the draft tube and annulus. CBCR is the most desirable regime, since the reactor operation in this regime gives a higher gas holdup and mass transfer rate than in the other two regimes. In the present study, the hydrodynamic behavior of RFJLB was investigated under various operational and geometrical conditions, such as gas and liquid velocity and nozzle configuration. Factors affecting the critical liquid circulation velocity (CLCV) above which the CBCR is established were identified and evaluated quantitatively.     

Femtosecond photodynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 1. Forward dynamics

Phytochromes are well-known red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the ratio of red to far-red light. Cyanobacteriochromes (CBCRs) are related photosensory proteins with a bilin-binding GAF domain, but much more diverse spectral sensitivity, with five recognized subfamilies of CBCRs described to date. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct (P(g)). Here, we examine the ultrafast forward photodynamics of the red/green CBCR NpR6012g4 from the NpR6012 locus of the nitrogen-fixing cyanobacterium Nostoc punctiforme. Using transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics that induces the forward reaction (red-absorbing to green-absorbing), which we interpret as arising from ground-state heterogeneity. Excited-state decays with lifetimes of 55 and 345 ps generate the primary photoproduct (Lumi-R), and the fastest decay (5 ps) did not produce Lumi-R. Although the photoinduced kinetics of Npr6012g4 is comparable with that of the Cph1 phytochrome isolated from Synechocystis cyanobacteria, NpR6012g4 exhibits a ≥2-3-fold higher photochemical quantum yield. Understanding the structural basis of this enhanced quantum yield may prove to be useful in increasing the photochemical efficiency of other bilin-based photosensors.     

Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily

Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.     

Femtosecond photodynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 2. reverse dynamics

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P(r) and P(fr), respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15Z ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct ((15E)P(g)). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium Nostoc punctiforme to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily.     

mTOR kinase domain phosphorylation promotes mTORC1 signaling, cell growth, and cell cycle progression

The mammalian target of rapamycin complex 1 (mTORC1) functions as an environmental sensor to promote critical cellular processes such as protein synthesis, cell growth, and cell proliferation in response to growth factors and nutrients. While diverse stimuli regulate mTORC1 signaling, the direct molecular mechanisms by which mTORC1 senses and responds to these signals remain poorly defined. Here we investigated the role of mTOR phosphorylation in mTORC1 function. By employing mass spectrometry and phospho-specific antibodies, we demonstrated novel phosphorylation on S2159 and T2164 within the mTOR kinase domain. Mutational analysis of these phosphorylation sites indicates that dual S2159/T2164 phosphorylation cooperatively promotes mTORC1 signaling to S6K1 and 4EBP1. Mechanistically, S2159/T2164 phosphorylation modulates the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1-associated mTOR S2481 autophosphorylation. Moreover, mTOR S2159/T2164 phosphorylation promotes cell growth and cell cycle progression. We propose a model whereby mTOR kinase domain phosphorylation modulates the interaction of mTOR with regulatory partner proteins and augments intrinsic mTORC1 kinase activity to promote biochemical signaling, cell growth, and cell cycle progression.     

Natural diversity provides a broad spectrum of cyanobacteriochrome-based diguanylate cyclases

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves—affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field.

Natural diversity of light-regulated diguanylate cyclases outperforms rational design for generating promising candidates for robust, broad spectrum optogenetic applications in live cells.     

Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor

Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and 0.068 microM for the homologous human and mouse apical transporters, respectively. Hybrid human-mouse and mouse-human transporters were engineered to identify regions involved in this 150-fold difference in the inhibition constant for 2164U90. A mouse-human chimera with only the most C-terminal hydrophobic domain and the C-terminus of the transporter originating from the human variant was found to have a sensitivity to 2164U90 inhibition similar to that of the human transporter. Conversely, a human-mouse hybrid transporter encompassing the same C-terminal region from the mouse sequence but now inserted into the human sequence demonstrated the greater inhibition seen with the mouse wild type ASBT. Amino acid substitutions, individually or in combinations, of six candidate nonconserved residues between mouse and human transporters in this C-terminal domain showed replacements of Thr294 by Ser and Val295 by Ile to be responsible for the difference in the sensitivity toward 2164U90 seen between the species. The hamster apical SBAT encompassing Ser/Ile in these positions shared the lower sensitivity to 2164U90, as seen with the human ASBT, even though it is identical to the mouse SBAT in the remaining four positions of this region. In addition, the rat ASBT which is identical to the mouse ASBT in this domain also had the high sensitivity to 2164U90 inhibition found for the mouse ASBT. Methanethiosulfonates (MTS) are known to inactivate the sodium/bile acid transporters through alkylation of a cysteine in the most C-terminal hydrophobic domain (1). Inactivation of the human ASBT due to MTS modification of cysteine 270 was shown to be largely abolished when the transporter was preincubated with 2164U90, suggesting that the binding of this benzothiazepine is in the vicinity of position 270. Thus, the domain containing the two most C-terminal putative transmembrane regions of the SBATs, H8-H9, previously shown to constitute part of the binding pocket for bile acids, interacts also with the bile acid transport competitive inhibitor, 2164U90.     

Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC 6803

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.     

An ATP-grasp ligase involved in the last biosynthetic step of the iminomycosporine shinorine in Nostoc punctiforme ATCC 29133

We investigated the genetic basis for mycosporine sunscreen biosynthesis by the cyanobacterium Nostoc punctiforme ATCC 29133. Heterologous expression in Escherichia coli of three contiguous N. punctiforme genes (NpR5600, NpR5599, and NpR5598, here named mysA, mysB, and mysC, respectively) led to the production of mycosporine-glycine, an oxomycosporine. Additional expression of gene NpF5597 (mysD) led to the conversion of mycosporine-glycine into iminomycosporines (preferentially shinorine but also others like mycosporine-2-glycine and porphyra-334). This represents a new mode of enzymatic synthesis for iminomycosporines, one that differs in genetic origin, mechanism, and apparent substrate specificity from that known in Anabaena variabilis ATCC 29413. These results add to the emerging profile of the protein family of ATP-dependent ligases, to which the mysC product belongs, as important condensation enzymes in microbial secondary metabolism.     

Matrix techniques applied to CML-typedH-2 mutants

Techniques associated with the Labeled Reaction Matrix model are used to provide several assignments of discrete labels to some serologically identical, CML-typedH-2 haplotypes. It is shown how this model can utilize the internal consistency of a labeling scheme to aid in making decisions on assigning values of positive or negative to reactions of questionable strength. A few examples of labeling schemes that may suggest further experimentation are derived.     

Novel and unique domains in aminoacyl-tRNA synthetases from human fungal pathogens Aspergillus niger,Candida albicans and Cryptococcus neoformans

Background

Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents.

Results

Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans – A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs.

Conclusions

Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1069) contains supplementary material, which is available to authorized users.     

甲基营养菌MP688萄糖脱氢酶基因分离鉴定及性质研究

目的：鉴定甲基营养菌MP688中的葡萄糖脱氢酶基因。方法：对甲基营养菌MP688基因组序列进行比对和分析,找到与已知细菌葡萄糖脱氢酶同源性最高的基因序列mpq_2164,且该基因所编码蛋白经分析具有跨膜结构域。设计51物扩增mpq_2164和缺失跨膜区域序列的s-mpq_2164,将PCR产物克隆到表达载雄pET-15b上,在大肠杆菌BL21中完成异源重组表达,然后通过组氨酸标签镍柱亲和层析纯化,采用DCIP法测定葡萄糖脱氢酶的活力。结果：分离了甲基营养菌MP688中的葡糖糖脱氢酶基因,并实现了s-mpq_2164的高效异源重组表达;MPQ2164的氯基酸序列与已知的葡萄糖脱氢酶相似性很低,但酶活测定结果表明S-MPQ-2164具有很高的葡糖糖脱氢酶活性。结论：MPQ_2164是-个依赖于吡咯喹啉醌的葡萄糖脱氢酶,去掉跨膜结构域有利于该蛋白的异源嘉{大,     

Ubiquitous nuclear proteins bind to 5′ upstream region of major Kunitz chymotrypsin inhibitor gene in winged bean

Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates abundantly in seeds and tuberous roots of winged bean plant. In seeds, the WCI mRNA is observed transiently during seed maturation period. The WCI is encoded by a multigene family and the major WCI (WCI-3) is encoded by two nearly identical genes (WCI-3a and WCI-3b genes), in which nucleotide sequences in the 1.1 kb 5 flanking regions are about 99% homologous to each other and the transcribed regions are completely identical. Here we report the detection of two types of nuclear proteins which bind to the multiple sites in the 5 upstream region of the WCI-3a gene. One of the proteins, band 1-forming protein, also bound to cauliflower mosaic virus 35S (CaMV35S) promoter, but another protein, band 3-forming protein, did not. DNaseI footprinting analysis showed that these proteins bound to AT-rich upstream regions in the WCI-3a gene. Addition of poly(dA-dT)-poly(dA-dT) to the binding reaction inhibited the formation of the retarded bands, while poly(dI-dC)-poly(dI-dC) did not. In various organs and throughout seed maturation period, proteins with invariable binding specificities were detected, and these binding proteins met some operational criteria for high-mobility-group (HMG) proteins. These results suggest that leguminous seed AT-binding proteins reported on several seed storage protein genes may be HMG-like proteins which are present ubiquitously in plant organs.deceased on September 15, 1992     

Targeted resequencing of GWAS loci reveals novel genetic variants for milk production traits

Background

Genome wide association study (GWAS) has been proven to be a powerful tool for detecting genomic variants associated with complex traits. However, the specific genes and causal variants underlying these traits remain unclear.

Results

Here, we used target-enrichment strategy coupled with next generation sequencing technique to study target regions which were found to be associated with milk production traits in dairy cattle in our previous GWAS. Among the large amount of novel variants detected by targeted resequencing, we selected 200 SNPs for further association study in a population consisting of 2634 cows. Sixty six SNPs distributed in 53 genes were identified to be associated significantly with on milk production traits. Of the 53 genes, 26 were consistent with our previous GWAS results. We further chose 20 significant genes to analyze their mRNA expression in different tissues of lactating cows, of which 15 were specificly highly expressed in mammary gland.

Conclusions

Our study illustrates the potential for identifying causal mutations for milk production traits using target-enrichment resequencing and extends the results of GWAS by discovering new and potentially functional mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1105) contains supplementary material, which is available to authorized users.     

SASI-Seq: sample assurance Spike-Ins,and highly differentiating 384 barcoding for Illumina sequencing

Background

A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples.

Results

By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error.

Conclusion

SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-110) contains supplementary material, which is available to authorized users.     

Genetic variants in BMP8B gene are associated with growth traits in Chinese native cattle

As a signaling molecule, bone morphogenetic protein 8B (BMP8B) plays an essential role in bone metabolism and is able to regulate thermogenesis and energy balance, which suggests that BMP8B gene may be a new candidate for growth traits. Here, to characterize the effects of BMP8B gene on growth traits, we first used three Chinese indigenous cattle breeds (n = 845) to detect single nucleotide polymorphisms (SNPs). Five novel SNPs of BMP8B gene (g.− 242C>T, g.2164C>T, g.2639T>C, g.2900C>G and g.10817C>T) were identified by DNA pool sequencing and forced PCR–RFLP. And then we associated the five SNPs with four growth traits (body weight, body length, heart girth, and hucklebone width). Results from association analysis showed that the SNPs 1, 2, and 3 affected growth trait(s) markedly (P < 0.05). Further, 6 combined haplotypes were constructed to guarantee the reliability of analysis results. There were also significant differences in body length, heart girth and body weight between the 6 combined haplotypes (P < 0.05), but not in hucklebone width (P > 0.05). Collectively, our results suggest a modulatory role of BMP8B gene in cattle growth and development, and 3 SNPs could be used as molecular markers in early marker assisted selection (MAS) in beef cattle breeding program.