Accelerating strain phenotyping with desorption electrospray ionization-imaging mass spectrometry and untargeted analysis of intact microbial colonies.

Reading and writing DNA were once the rate-limiting step in synthetic biology workflows. This has been replaced by the search for the optimal target sequences to produce systems with desired properties. Directed evolution and screening mutant libraries are proven technologies for isolating strains with enhanced performance whenever specialized assays are available for rapidly detecting a phenotype of interest. Armed with technologies such as CRISPR-Cas9, these experiments are capable of generating libraries of up to 1010 genetic variants. At a rate of 102 samples per day, standard analytical methods for assessing metabolic phenotypes represent a major bottleneck to modern synthetic biology workflows. To address this issue, we have developed a desorption electrospray ionization-imaging mass spectrometry screening assay that directly samples microorganisms. This technology increases the throughput of metabolic measurements by reducing sample preparation and analyzing organisms in a multiplexed fashion. To further accelerate synthetic biology workflows, we utilized untargeted acquisitions and unsupervised analytics to assess multiple targets for future engineering strategies within a single acquisition. We demonstrate the utility of the developed method using Escherichia coli strains engineered to overproduce free fatty acids. We determined discrete metabolic phenotypes associated with each strain, which include the primary fatty acid product, secondary products, and additional metabolites outside the engineered product pathway. Furthermore, we measured changes in amino acid levels and membrane lipid composition, which affect cell viability. In sum, we present an analytical method to accelerate synthetic biology workflows through rapid, untargeted, and multiplexed metabolomic analyses.

Targeted modification of the Per2 clock gene alters circadian function in mPer2luciferase (mPer2Luc) mice.

Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii.

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.

Peroxiredoxins are conserved markers of circadian rhythms.

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth's rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.

A post-translational oscillator (PTO) composed of the proteins KaiA, KaiB and KaiC is at the heart of the cyanobacterial circadian clock. KaiC interacts with KaiA and KaiB over the daily cycle, and CII domains undergo rhythmic phosphorylation/dephosphorylation with a 24 h period. Both the N-terminal (CI) and C-terminal (CII) rings of KaiC exhibit ATPase activity. The CI ATPase proceeds in an input-independent fashion, but the CII ATPase is subject to metabolic input signals. The crystal structure of KaiC from Thermosynechococcus elongatus allows insight into the different anatomies of the CI and CII ATPases. Four consecutive arginines in CI (Arg linker) that connect the P-loop, CI subunits and CI and CII at the ring interface are primary candidates for the coordination of the CI and CII activities. The mutation of linker residues alters the period or triggers arhythmic behavior. Comparison between the CI and CII structures also reveals differences in loop regions that are key to KaiA and KaiB binding and activation of CII ATPase and kinase. Common packing features in KaiC crystals shed light on the KaiB-KaiC interaction.

Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.

The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.

Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/ß heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.

Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB-KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.

RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level.

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

A guanidine-degrading enzyme controls genomic stability of ethylene-producing cyanobacteria.

Recent studies have revealed the prevalence and biological significance of guanidine metabolism in nature. However, the metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity have not been widely studied. Here, via comparative proteomics and subsequent experimental validation, we demonstrate that Sll1077, previously annotated as an agmatinase enzyme in the model cyanobacterium Synechocystis sp. PCC 6803, is more likely a guanidinase as it can break down guanidine rather than agmatine into urea and ammonium. The model cyanobacterium Synechococcus elongatus PCC 7942 strain engineered to express the bacterial ethylene-forming enzyme (EFE) exhibits unstable ethylene production due to toxicity and genomic instability induced by accumulation of the EFE-byproduct guanidine. Co-expression of EFE and Sll1077 significantly enhances genomic stability and enables the resulting strain to achieve sustained high-level ethylene production. These findings expand our knowledge of natural guanidine degradation pathways and demonstrate their biotechnological application to support ethylene bioproduction.

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are  located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other  luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.

CikA, an Input Pathway Component, Senses the Oxidized Quinone Signal to Generate Phase Delays in the Cyanobacterial Circadian Clock.

The circadian clock is a timekeeping system in most organisms that keeps track of the time of day. The rhythm generated by the circadian oscillator must be constantly synchronized with the environmental day/night cycle to make the timekeeping system truly advantageous. In the cyanobacterial circadian clock, quinone is a biological signaling molecule used for entraining and fine-tuning the oscillator, a process in which the external signals are transduced into biological metabolites that adjust the phase of the circadian oscillation. Among the clock proteins, the pseudo-receiver domain of KaiA and CikA can sense external cues by detecting the oxidation state of quinone, a metabolite that reflects the light/dark cycle, although the molecular mechanism is not fully understood. Here, we show the antagonistic phase shifts produced by the quinone sensing of KaiA and CikA. We introduced a new cyanobacterial circadian clock mixture that includes an input component in vitro. KaiA and CikA cause phase advances and delays, respectively, in this circadian clock mixture in response to the quinone signal. In the entrainment process, oxidized quinone modulates the functions of KaiA and CikA, which dominate alternatively at day and night in the cell. This in turn changes the phosphorylation state of KaiC-the central oscillator in cyanobacteria-ensuring full synchronization of the circadian clock. Moreover, we reemphasize the mechanistic input functionality of CikA, contrary to other reports that focus only on its output action.

Revealing Oft-cited but Unpublished Papers of Colin Pittendrigh and Coworkers.

Among the scientific resources that Colin Pittendrigh passed on to his colleagues after his death in 1996 were two unpublished papers. These manuscripts, developed first in the mid-1960s and continually updated and refined through the late 1970s, centered on the development and experimental exploration of a model of circadian entrainment combining aspects of the well-known parametric (continuous) and nonparametric (discrete) models of entrainment. These texts reveal the experimental work surrounding Pittendrigh's determination of the limits of entrainment and the explanation of the bistability phenomenon. These manuscripts are being made publicly available in their final format (February 1978) as supplementary material to this introduction.

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942.

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.

CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates that KaiB monomers interact with KaiC and block ATP binding clefts.

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB-KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.

The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.

Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.

BACKGROUND: The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS-->pTS-->pTpS-->TpS-->TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC. METHODOLOGY AND PRINCIPAL FINDINGS: The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP gamma-phosphate. T432 is phosphorylated first because it lies consistently closer to Pgamma. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation. CONCLUSIONS AND SIGNIFICANCE: We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.