Regulation of Seed Dormancy Genes in Triticeae Species.

Wild progenitors of Triticeae crops generally have long dormancy periods. Domesticated crops inherited these longer dormancy alleles from their wild progenitors, which have since been modified and selected during cultivation and utilization by humans. Thus, allelic combinations at different seed dormancy loci are currently represented in Triticeae germplasm preserved in seed repositories and gene banks as accessions and materials of breeding programs. Methods to evaluate seed dormancy are key to explore, analyze, and exploit optimal alleles in dormancy genes. Recent developments in genomics have accelerated the identification and analysis of seed dormancy loci in Triticeae species. Transgenic experiments have been conducted to validate if candidate genes affect seed dormancy and more recently have yielded an array of mutations derived from genome editing for practical applications. The information gathered on these seed dormancy loci provides a deeper knowledge of germplasm diversity and offers strategies to control seed dormancy in breeding programs in Triticeae crops.

Evaluation Methods for Preharvest Sprouting Resistance in Japanese Wheat Breeding Programs.

Different methodologies have been applied for the selection of preharvest sprouting resistance in cereal breeding programs. We describe here a series of methods used in practical wheat breeding programs in Japan, including phenotyping based on germination score after artificial rain treatments and genotyping using DNA markers. These methods can be modified and applied to breeding programs in which preharvest sprouting is a problem during cereal cultivation.

Evolution of wheat blast resistance gene Rmg8 accompanied by differentiation of variants recognizing the powdery mildew fungus.

Wheat blast, a devastating disease having spread recently from South America to Asia and Africa, is caused by Pyricularia oryzae (synonym of Magnaporthe oryzae) pathotype Triticum, which first emerged in Brazil in 1985. Rmg8 and Rmg7, genes for resistance to wheat blast found in common wheat and tetraploid wheat, respectively, recognize the same avirulence gene, AVR-Rmg8. Here we show that an ancestral resistance gene, which had obtained an ability to recognize AVR-Rmg8 before the differentiation of Triticum and Aegilops, has expanded its target pathogens. Molecular cloning revealed that Rmg7 was an allele of Pm4, a gene for resistance to wheat powdery mildew on 2AL, whereas Rmg8 was its homoeologue on 2BL ineffective against wheat powdery mildew. Rmg8 variants with the ability to recognize AVR-Rmg8 were distributed not only in Triticum spp. but also in Aegilops speltoides, Aegilops umbellulata and Aegilops comosa. This result suggests that the origin of resistance gene(s) recognizing AVR-Rmg8 dates back to the time before differentiation of A, B, S, U and M genomes, that is, ~5 Myr before the emergence of its current target, the wheat blast fungus. Phylogenetic analyses suggested that, in the evolutionary process thereafter, some of their variants gained the ability to recognize the wheat powdery mildew fungus and evolved into genes controlling dual resistance to wheat powdery mildew and wheat blast.

Breeding of a near-isogenic wheat line resistant to wheat blast at both seedling and heading stages through incorporation of Rmg8.

Wheat blast caused by Pyricularia oryzae pathotype Triticum (MoT) has been transmitted from South America to Bangladesh and Zambia and is now spreading in these countries. To prepare against its further spread to Asian countries, we introduced Rmg8, a gene for resistance to wheat blast, into a Japanese elite cultivar, Chikugoizumi (ChI), through recurrent backcrosses, and established ChI near-isogenic lines, #2-1-10 with the Rmg8/Rmg8 genotype and #4-2-10 with the rmg8/rmg8 genotype. A molecular analysis suggested that at least 96.6% of the #2-1-10 genome was derived from the recurrent parent ChI. The #2-1-10 line was resistant to MoT not only in primary leaves at the seedling stage but also in spikes and flag leaves at the heading stage. The strength of the resistance in spikes of this Rmg8 carrier was comparable to that of a carrier of the 2NS segment which has been the only genetic resource released to farmer's field for wheat blast resistance. On the other hand, the 2NS resistance was not expressed on leaves at the seedling stage nor flag leaves at the heading stage. Considering that leaf blast has been increasingly reported and regarded as an important inoculum source for spike blast, Rmg8 expressed at both the seedling and heading stages, or more strictly in both leaves and spikes, is suggested to be useful to prevent the spread of MoT in Asia and Africa.

The master regulator for entry into sporulation in Bacillus subtilis becomes a mother cell-specific transcription factor for forespore engulfment.

The Spo0A transcription factor is activated by phosphorylation in starving Bacillus subtilis cells. The activated Spo0A (Spo0A~P) regulates genes controlling entry into sporulation and appears to control mother-cell-specific gene expression after asymmetric division, but the latter remains elusive. Here, we found that Spo0A~P directly binds to three conserved DNA sequences (0A1-3) in the promoter region of the mother cell-specific lytic transglycosylase gene spoIID, which is transcribed by σE -RNA polymerase (RNAP) and negatively controlled by the SpoIIID transcription factor and required for forespore engulfment. Systematic mutagenesis of the 0A boxes revealed that the 0A1 and 0A2 boxes located upstream of the promoter positively control the transcription of spoIID. In contrast, the 0A3 box located downstream of the promoter negatively controls the transcription of spoIID. The mutated SpoIIID binding site located between the -35 and -10 promoter elements causes increased expression of spoIID and reduced sporulation. When the mutations of 0A1, 0A2, and IIID sites are combined, sporulation is restored. Collectively, our data suggest that the mother cell-specific spoIID expression is precisely controlled by the coordination of three factors, Spo0A~P, SpoIIID, and σE -RNAP, for proper sporulation. The conservation of this mechanism across spore-forming species was discussed.

The Slowdown of Growth Rate Controls the Single-Cell Distribution of Biofilm Matrix Production via an SinI-SinR-SlrR Network.

In Bacillus subtilis, master regulator Spo0A controls several cell-differentiation pathways. Under moderate starvation, phosphorylated Spo0A (Spo0A~P) induces biofilm formation by indirectly activating genes controlling matrix production in a subpopulation of cells via an SinI-SinR-SlrR network. Under severe starvation, Spo0A~P induces sporulation by directly and indirectly regulating sporulation gene expression. However, what determines the heterogeneity of individual cell fates is not fully understood. In particular, it is still unclear why, despite being controlled by a single master regulator, biofilm matrix production and sporulation seem mutually exclusive on a single-cell level. In this work, with mathematical modeling, we showed that the fluctuations in the growth rate and the intrinsic noise amplified by the bistability in the SinI-SinR-SlrR network could explain the single-cell distribution of matrix production. Moreover, we predicted an incoherent feed-forward loop; the decrease in the cellular growth rate first activates matrix production by increasing in Spo0A phosphorylation level but then represses it via changing the relative concentrations of SinR and SlrR. Experimental data provide evidence to support model predictions. In particular, we demonstrate how the degree to which matrix production and sporulation appear mutually exclusive is affected by genetic perturbations. IMPORTANCE The mechanisms of cell-fate decisions are fundamental to our understanding of multicellular organisms and bacterial communities. However, even for the best-studied model systems we still lack a complete picture of how phenotypic heterogeneity of genetically identical cells is controlled. Here, using B. subtilis as a model system, we employ a combination of mathematical modeling and experiments to explain the population-level dynamics and single-cell level heterogeneity of matrix gene expression. The results demonstrate how the two cell fates, biofilm matrix production and sporulation, can appear mutually exclusive without explicitly inhibiting one another. Such a mechanism could be used in a wide range of other biological systems.

The ridge line of left pulmonary vein isolation from left atrial appendage can subsequently increase the completion rate of the mitral isthmus block line.

BACKGROUND AND OBJECTIVES: Mitral isthmus (MI) ablation for mitral flutter is technically difficult, and incomplete block line is not uncommon. The objective of this study is to investigate the effect of the ridge line of left pulmonary vein isolation (LPVI) from left atrial appendage (LAA) on completion rate of mitral isthmus (MI) block line and recurrence rate of atrial tachycardia (AT) or atrial flutter (AFL) after the first MI ablation. METHODS: We identified 611 patients who underwent first MI ablation for mitral flutter during the study period. Finally, 559 patients were enrolled and divided into two groups according to the method of ridge line ablation of LPVI (LAA group, n = 467, conventional group, n = 92). Outcome measures were the completion of MI block line by first MI ablation, the recurrence of AT/AFL, and repeat MI ablation after the first MI ablation. RESULTS: The first MI block line completion rate was significantly higher in the LAA group than the conventional group (95% vs. 85%, p < 0.001). The recurrence rate of AT/AFL after 3 months from first MI ablation was significantly lower in the LAA group. The requirement of additional MI ablation tended to be lower in the LAA group. CONCLUSIONS: Our novel approach of ablating LPV-LAA ridge from the LAA side during PVI can increase the success rate of MI block line completion, and reduce the recurrence rate of AT/AFL and the need for additional MI block line ablation. Graphical abstract Ablation of the left pulmonary vein-left atrial appendage ridge from the left atrial appendage side during PVI increased the success rate of mitral isthmus block line completion.

Pharmacotherapy in Patients with Alzheimer-type Dementia Presenting with Behavioral and Psychological Symptoms of Dementia: A Retrospective Chart Review of 102 Patients Available for 12-month Follow-up after Initiation of Treatment.

Objectives: Alongside non-pharmacological intervention, pharmacotherapy particularly with atypical antipsychotics is assumed to be effective for behavioral and psychological symptoms of dementia (BPSD). Methods: This retrospective study investigated the effectiveness and safety of pharmacotherapy including antipsychotics in outpatients or inpatients with BPSD. Results: Of all Alzheimer-type dementia (AD) patients with BPSD initiating treatment between March and August 2011, a total of 102 patients available for 12-month follow-up comprised the subjects in this chart review. Of these, 68 (66.7%) continued treatment in the ambulatory or inpatient setting, with their MMSE scores improved from 17.3 ± 3.6 at baseline to 18.3 ± 3.53, 17.9 ± 3.80 and 17.0 ± 4.14 after 3, 6 and 12 months, respectively. In contrast, their NPI scores were significantly different from 11.7 ± 11.2 at baseline to 4.86 ± 5.40, 3.56 ± 4.65 and 2.27 ± 3.77 after 3, 6 and 12 months, respectively. Of the 36 inpatients available for follow-up, 27 (75%) on concurrent antipsychotics (chlorpromazine [CP] equivalent, 162.2 mg) at baseline remained on concurrent antipsychotics (CP equivalent, 212.5 mg) after 12 months, while, of the 66 outpatients available for follow-up, 13 (19.7%) on concurrent antipsychotics (CP equivalent, 93.4 mg) at baseline remained on concurrent antipsychotics (CP equivalent, 113.0 mg) after 12 months. Conclusions: Study results confirmed the effectiveness and safety of the study treatment in Japanese AD patients with BPSD for up to 12 months. How best to incorporate antipsychotics into the treatment of BPSD in clinical settings lies in the hands of us Japanese clinicians.

Comparison of the empirical linear ablation and low voltage area-guided ablation in addition to pulmonary vein isolation in patients with persistent atrial fibrillation: a propensity score-matched analysis.

BACKGROUND: The efficacy of pulmonary vein isolation (PVI) alone is not guaranteed for persistent atrial fibrillation (PeAF), and it is unclear which type of ablation approach should be applied in addition to PVI. This study aimed to compare outcomes and prognosis between empirical linear ablation and low-voltage area (LVA) ablation after PVI for PeAF. METHODS: We enrolled 128 patients with PeAF who were assigned to the linear ablation group (n = 64) and the LVA ablation group (n = 64) using a propensity score-matched model. After PVI and cardioversion, the patients underwent either empirical linear ablation or LVA ablation during sinus rhythm. All patients in the linear ablation group underwent both roof line and mitral valve isthmus (MVI) ablations. An electrical-guided ablation targeting LVA (< 0.5 mV) was performed in the LVA group. When there was no LVA in the LVA group, only PVI was applied. We compared the procedural outcomes and recurrence after ablation between the two groups. RESULTS: The baseline characteristics were well-balanced between the two groups. Fifty patients had LVA (22 and 28 patients in the linear and LVA groups). The roof and MVI lines were completed in 100% and 96.9% of the patients. During the mean follow-up of 279.5 ± 161.3 days, the LVA group had significantly lower recurrence than the linear group (15 patients [23%] vs. 29 patients [45%], p = 0.014). Thirty-five patients were prescribed antiarrhythmic drugs during the follow-up period (linear group, n = 17; LVA group, n = 18); amiodarone and bepridil were administered to most of the patients (15 and 17 patients, respectively). The difference in the prognosis was relevant among the patients with LVA, while this trend was not observed in those without LVA. The LVA ablation group demonstrated significantly lower radiofrequency energy and shorter procedural time compared to the linear ablation group. The recurrence of atrial flutter was more likely to occur in the linear group than in the LVA group (14 [22%] vs. 6 [9.4%], p = 0.052). CONCLUSION: The electrophysiological-guided LVA ablation is more effective than empirical linear ablation in PeAF patients with LVA. Unnecessary empirical linear ablation might have a risk of iatrogenic gap and atrial flutter recurrence.

Bacillus subtilis Histidine Kinase KinC Activates Biofilm Formation by Controlling Heterogeneity of Single-Cell Responses.

In Bacillus subtilis, biofilm and sporulation pathways are both controlled by a master regulator, Spo0A, which is activated by phosphorylation via a phosphorelay-a cascade of phosphotransfer reactions commencing with autophosphorylation of histidine kinases KinA, KinB, KinC, KinD, and KinE. However, it is unclear how the kinases, despite acting via the same regulator, Spo0A, differentially regulate downstream pathways, i.e., how KinA mainly activates sporulation genes and KinC mainly activates biofilm genes. In this work, we found that KinC also downregulates sporulation genes, suggesting that KinC has a negative effect on Spo0A activity. To explain this effect, with a mathematical model of the phosphorelay, we revealed that unlike KinA, which always activates Spo0A, KinC has distinct effects on Spo0A at different growth stages: during fast growth, KinC acts as a phosphate source and activates Spo0A, whereas during slow growth, KinC becomes a phosphate sink and contributes to decreasing Spo0A activity. However, under these conditions, KinC can still increase the population-mean biofilm matrix production activity. In a population, individual cells grow at different rates, and KinC would increase the Spo0A activity in the fast-growing cells but reduce the Spo0A activity in the slow-growing cells. This mechanism reduces single-cell heterogeneity of Spo0A activity, thereby increasing the fraction of cells that activate biofilm matrix production. Thus, KinC activates biofilm formation by controlling the fraction of cells activating biofilm gene expression. IMPORTANCE In many bacterial and eukaryotic systems, multiple cell fate decisions are activated by a single master regulator. Typically, the activities of the regulators are controlled posttranslationally in response to different environmental stimuli. The mechanisms underlying the ability of these regulators to control multiple outcomes are not understood in many systems. By investigating the regulation of Bacillus subtilis master regulator Spo0A, we show that sensor kinases can use a novel mechanism to control cell fate decisions. By acting as a phosphate source or sink, kinases can interact with one another and provide accurate regulation of the phosphorylation level. Moreover, this mechanism affects the cell-to-cell heterogeneity of the transcription factor activity and eventually determines the fraction of different cell types in the population. These results demonstrate the importance of intercellular heterogeneity for understanding the effects of genetic perturbations on cell fate decisions. Such effects can be applicable to a wide range of cellular systems.

Allelic variations of Vrn-1 and Ppd-1 genes in Japanese wheat varieties reveal the genotype-environment interaction for heading time.

The timing of heading is largely affected by environmental conditions. In wheat, Vrn-1 and Ppd-1 have been identified as the major genes involved in vernalization requirement and photoperiod sensitivity, respectively. To compare the effects of Vrn-1 and Ppd-1 alleles on heading time under different environments, we genotyped Vrn-1 and Ppd-1 homoeologues and measured the heading time at Morioka, Tsukuba and Chikugo in Japan for two growing seasons. A total of 128 Japanese and six foreign varieties, classified into four populations based on the 519 genome-wide SNPs, were used for analysis. Varieties with the spring alleles (Vrn-D1a or Vrn-D1b) at the Vrn-D1 locus and insensitive allele (Hapl-I) at the Ppd-D1 locus were found in earlier heading varieties. The effects of Vrn-D1 and Ppd-D1 on heading time were stronger than those of the other Vrn-1 and Ppd-1 homoeologues. Analysis of variance revealed that heading time was significantly affected by the genotype-environment interactions. Some Vrn-1 and Ppd-1 alleles conferred earlier or later heading in specific environments, indicating that the effect of both alleles on the timing of heading depends on the environment. Information on Vrn-1 and Ppd-1 alleles, together with heading time in various environments, provide useful information for wheat breeding.

Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds.

TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB-ExbB-ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2-6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1-exbB1-exbD1-exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of ß-O-4-, ß-5-, ß-1-, ß-ß-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.

Longer Bread Shelf-Life and Improved Noodle-Making Properties Imparted by a Novel Wheat Genotype Are Stable in Different Genetic Backgrounds.

A recently developed wheat variety, known as 5-5 wheat, which has inactive GBSSI-B1, GBSSI-D1, SSIIa-B1, and SSIIa-D1 isozymes, accumulates a novel type of starch, affecting bread texture and leading to reduction in bread staling. These properties are potentially useful for commercial bakery products; thus, the 5-5 genotype represents a new resource for wheat breeding. In this study, the 5-5 alleles were backcrossed into the hard wheat variety "Minaminokaori" and the soft wheat variety "Shirogane-Komugi", which are both leading Japanese wheat varieties. In comparison to their parental varieties, the two 5-5 near-isogenic lines (NILs) showed a decrease in amylose levels, an increase in the proportion of short chains of amylopectin, a lower gelatinization temperature and enthalpy change, a higher peak viscosity and breakdown viscosity as measured by a Rapid Visco Analyzer, a reduced retrogradation rate, and an increase in grain hardness. Importantly, the 5-5 NILs also showed lower bread crumb firmness and reduced hardening after storage for 2 days at either 20 °C or 7 °C. Considering the results obtained here along with those from the original line, it is clear that the 5-5 genotype can generate specific changes in starch characteristics and staling regardless of its genetic background. Thus, we renamed the 5-5 wheat lines "Slow Staling" (SS) wheat. As expected, our results indicated that the hard wheat SS NIL was more suitable for bread-making. On the other hand, we found that white salted noodle made with the SS NIL of the soft wheat variety had a relatively shorter cooking time, a softer texture, and a reduction in textural changes during storage, all of which are potentially useful for noodle manufacturers.

Genetic mechanisms determining grain number distribution along the spike and their effect on yield components in wheat.

The number of wheat grains is one of the major determinants of yield. Many quantitative trait loci (QTLs) and some causal genes such as GNI-A1 and WAPO-A1 that are associated with grain number per spike (GNS) have been identified, but the underlying mechanisms remain largely unknown. We analyzed QTLs for grain number and other related traits using 188 doubled haploid lines derived from the Japanese high-yield variety, Kitahonami, as a parent to elucidate the genetic mechanism determining grain number. The major QTLs for grain number at the apical, central, and basal parts of the spike were identified in different chromosomal regions. We considered GNI-A1 and WAPO-A1 as candidate genes controlling grain number at the central and basal parts of the spike, respectively. Kitahonami had the favorable 105Y allele of GNI-A1 and WAPO-A1b allele and unfavorable alleles of QTLs for grain number at the apical part of spikes. Pyramiding the favorable alleles of these QTLs significantly increased GNS without significantly reducing thousand-grain weight (TGW). In contrast, the accumulation of favorable alleles of QTLs for TGW significantly decreased GNS, whereas days to heading positively correlated with GNS. Late heading increased the spikelet number per spike, resulting in a higher GNS. Pyramiding of the QTLs for TGW and days to heading also altered the GNS. In conclusion, GNS is a complex trait controlled by many QTLs, and it is essential for breeding to design. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01255-8.

Author Correction: Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

Erratum: Publisher Correction: A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

[This corrects the article DOI: 10.1038/s42003-019-0676-z.].

A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB-ExbB-ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, Sphingobium sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation.

Study design and protocol for evaluating the long-term prognosis of patients receiving his bundle pacing: A multicenter observational study.

BACKGROUND: His bundle pacing (HBP) is a recently developed pacing technique that can achieve an ideal physiological pattern of ventricular activation via stimulation of the native His-Purkinje system. Despite the widespread introduction of HBP in clinical practice, its appropriate indications are yet to be determined clearly. Moreover, the efficacy and safety of HBP and long-term prognosis of patients undergoing such are unknown. METHODS: We conducted a multicenter observational prospective study in patients undergoing HBP in Japan. Patients with atrioventricular block or conduction delay and estimated ventricular pacing of ≥ 40% scheduled for HBP implantation are included. All patients are followed up until 3 years after the implantation. The primary endpoints are all-cause death, heart failure-related hospitalization, and upgrade to cardiac resynchronization therapy. The secondary endpoint is changes in cardiac function based on echocardiographic findings and laboratory data after the implantation. RESULTS: The results are currently under investigation. CONCLUSIONS: This multicenter observational study evaluates the long-term prognosis and changes in cardiac function of patients undergoing HBP implantation in a clinical setting. Considering the large number of patients included, the cumulative results would be helpful in establishing evidence on HBP application in this area and consequently allow accurate management and treatment of patients undergoing HBP.

Optogenetic control of Bacillus subtilis gene expression.

The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.