SGRN: A Cas12a-driven Synthetic Gene Regulatory Network System.

Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the "Synthetic Gene Regulatory Network" (SGRN, pronounced "sojourn").

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

Regulation of the Ppr histidine kinase by light-induced interactions between its photoactive yellow protein and bacteriophytochrome domains.

Ppr is a unique bacteriophytochrome that bleaches rather than forming a far-red-shifted Pfr state upon red light activation. Ppr is also unusual in that it has a blue light photoreceptor domain, PYP, which is N-terminally fused to the bacteriophytochrome domain (Bph). When both photoreceptors are activated by light, the fast phase of Bph recovery (1 min lifetime) corresponds to the formation of an intramolecular long-lived complex between the activated PYP domain and the Bph domain (lifetime of 2-3 days). Since this state is unusually long-lived as compared to other intermediates in the photocycle of both PYP and Bph, we interpret this as formation of a metastable complex between activated PYP and Bph domains that takes days to relax. In the metastable complex, the PYP domain is locked in its activated UV absorbing state and the Bph domain is in a slightly red-shifted state (from 701 to 702 nm), which is photochemically inactive to red or white light. The amount of metastable complex formed increases with the degree of prior activation of PYP, reaching a maximum of 50% when PYP is fully activated compared to 0% when no PYP is activated. The saturation of complex formation at 50% is believed to be due to light-induced heterogeneity within the Ppr dimer. UV irradiation (365 nm) of the metastable complex state photoreverses the activated PYP and the red-shifted Bph to the initial dark state within seconds. We therefore postulate that Ppr functions as a UV-red light sensor and describe the different Ppr states that can be obtained depending on the light quality. Both red and white light upregulate the autokinase activity, while it is downregulated in the dark. The physiological state of Ppr is most likely a mixture of three different states, dark, metastable complex, and red light-activated, with fractional populations whose amounts depend on the light quality of the environment and that regulate the extent of phosphorylation by the kinase.

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination.

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.

Structural role of tyrosine 98 in photoactive yellow protein: effects on fluorescence, gateway, and photocycle recovery.

We have recently shown that the Y98Q mutant of PYP has a major effect on the photocycle kinetics ( approximately 40 times slower recovery). We have now determined the crystal structure of Y98Q at 2.2 A resolution to reveal the role of residue Y98 in the PYP photocycle. Although the overall structure is very similar to that of WT, we observed two major effects of the mutation. One obvious consequence is a conformational change of the beta4-beta5 loop, which includes a repositioning of residue M100. It had previously been shown that the photocycle is slowed by as much as 3 orders of magnitude when residue M100 is substituted or when the conformation is altered as in Rhodocista centenaria PYP. To investigate whether the altered photocycle of Y98Q is due to this repositioning of M100 or is caused by an effect unrelated to M100, we determined the dark recovery kinetics of the Y98Q/M100A mutant. We find the recovery kinetics to be very similar to the M100A single mutant kinetics and therefore conclude that the slower recovery kinetics in Y98Q are most likely due to repositioning of M100. In addition, we find that other substitutions at position 98 (Y98W, Y98L, and Y98A) have differing effects on the photocycle recovery, presumably due to a variable distortion of the beta4-beta5 loop. The second effect of the Y98Q mutation is a repositioning of R52, which is thought to interact with Y98 in WT PYP and now forms new interactions with residues Q99 and Q56. To determine the role of R52, we also characterized an R52A/M100A double mutant and found that the effects on the recovery kinetics ( approximately 2000 slower recovery than WT) are due to unrelated events in the photocycle. Since the Y98Q/M100A recovery kinetics are more similar to those of M100 than R52A/M100A, we conclude that the repositioning of R52, caused by the Y98Q mutation, does not affect the dark state recovery. In addition, it has been proposed that Y98 and P68 are "gateway residues" between which the chromophore must pass during isomerization. We tested the recovery kinetics of mutant P68A and found that, although the gateway may be important for photocycle initiation, its role in recovery to the ground state is minimal.

GHP, a new c-type green heme protein from Halochromatium salexigens and other proteobacteria.

We have isolated a minor soluble green-colored heme protein (GHP) from the purple sulfur bacterium, Halochromatium salexigens, which contains a c-type heme. A similar protein has also been observed in the purple bacteria Allochromatium vinosum and Rhodopseudomonas cryptolactis. This protein has wavelength maxima at 355, 420, and 540 nm and remains unchanged upon addition of sodium dithionite or potassium ferricyanide, indicating either an unusually low or high redox potential, respectively. The amino-acid sequence indicates one heme per peptide chain of 72 residues and reveals weak similarity to the class I cytochromes. The usual sixth heme ligand methionine in these proteins appears to be replaced by a cysteine in GHP. Only one known cytochrome has a cysteine sixth ligand, SoxA (cytochrome c-551) from thiosulfate-oxidizing bacteria, which is low-spin and has a high redox potential because of an un-ionized ligand. The native size of GHP is 34 kDa, its subunit size is 11 kDa, and the net charge is -12, accounting for its very acidic nature. A database search of complete genome sequences reveals six homologs, all hypothetical proteins, from Oceanospirillum sp., Magnetococcus sp., Thiobacillus denitrificans, Dechloromonas aromatica, Thiomicrospira crunogena and Methylobium petroleophilum, with sequence identities of 35-64%. The genetic context is different for each species, although the gene for GHP is transcriptionally linked to several other genes in three out of the six species. These genes, coding for an RNAse, a protease/chaperone, a GTPase, and pterin-4a-carbinolamine dehydratase, appear to be functionally related to stress response and are linked in at least 10 species.

Thermochromatium tepidum photoactive yellow protein/bacteriophytochrome/diguanylate cyclase: characterization of the PYP domain.

The purple phototrophic bacterium, Thermochromatium tepidum, contains a gene for a chimeric photoactive yellow protein/bacteriophytochrome/diguanylate cyclase (Ppd). We produced the Tc. tepidum PYP domain (Tt PYP) in Escherichia coli, and found that it has a wavelength maximum at 358 nm due to a Leu46 substitution of the color-tuning Glu46 found in the prototypic Halorhodospira halophila PYP (Hh PYP). However, the 358 nm dark-adapted state is in a pH-dependent equilibrium with a yellow species absorbing at 465 nm (pK(a) = 10.2). Following illumination at 358 nm, photocycle kinetics are characterized at pH 7.0 by a small bleach and red shift to what appears to be a long-lived cis intermediate (comparable to the I(2) intermediate in Hh PYP). The recovery to the dark-adapted state has a lifetime of approximately 4 min, which is approximately 1500 times slower than that for Hh PYP. However, when the Tt PYP is illuminated at pH values above 7.5, the light-induced difference spectrum indicates a pH-dependent equilibrium between the I(2) intermediate and a red-shifted 440 nm intermediate. This equilibrium could be responsible for the sigmoidal pH dependence of the recovery of the dark-adapted state (pK(a) = 8.8). In addition, the light-induced difference spectrum shows that, at pH values above 9.3, there is an apparent bleach near 490 nm superimposed on the 358 and 440 nm changes, which we ascribe to the equilibrium between the protonated and ionized dark-adapted forms. The L46E mutant of Tt PYP has a wavelength maximum at 446 nm, resembling wild-type Hh PYP. The kinetics of recovery of L46E following illumination with white light are slow (lifetime of 15 min at pH 7), but are comparable to those of wild-type Tt PYP. We conclude that Tt PYP is unique among the PYPs studied to date in that it has a photocycle initiated from a dark-adapted state with a protonated chromophore at physiological pH. However, it is kinetically most similar to Rhodocista centenaria PYP (Ppr) despite the very different absorption spectra due to the lack of E46.

Heterologous production of Halorhodospira halophila holo-photoactive yellow protein through tandem expression of the postulated biosynthetic genes.

The photoactive yellow protein (PYP) is a bacterial photoreceptor which is the structural prototype for the PAS domain superfamily of regulators and receptors. PYP is known to have a unique p-hydroxycinnamic acid chromophore, covalently attached to a cysteine. To date, it has not been shown how holo-PYP is formed in vivo. Two genes, nearby pyp, were postulated to encode the biosynthetic enzymes, but only one was previously isolated and shown to have the requisite activity. By using a dual plasmid system, one expressing the PYP from Halorhodospira halophila and the other expressing a two-gene operon, consisting of tyrosine ammonia lyase and p-hydroxycinnamic acid ligase, we are able to present evidence that a functionally active holo-PYP can be synthesized in Escherichia coli. Plasmids containing only one of the two enzymes failed to produce holoprotein. Thus, the two genes have been shown to be both necessary and sufficient for production of holoprotein, although the activating group remains unknown. This expression system not only holds great potential for mutagenesis studies but also opens new possibilities in the search for (a) signaling partner(s) of the PYP.