Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

Complete loss of RelA and SpoT homologs in Arabidopsis reveals the importance of the plastidial stringent response in the interplay between chloroplast metabolism and plant defense response.

The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used the CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases, and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type (WT) under normal growth conditions, and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense.

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

Arabidopsis mutants lacking DLDG1 and non-photochemical quenching-related proteins reveal the regulatory role of DLDG1 in chloroplast pH homeostasis.

The control of pH in chloroplasts is important to regulate photosynthesis, although details of the precise regulatory mechanisms of H+ homeostasis in chloroplasts are not fully understood. We recently found that the cyanobacterial PxcA homolog DLDG1 is involved in plastidial pH control. PxcA and DLDG1 have been thought to control light-dependent H+ extrusion across the cyanobacterial cytoplasmic and chloroplast envelope membranes, respectively. To investigate DLDG1-dependent pH control in chloroplasts, we crossed the dldg1 mutant with various mutants lacking known non-photochemical quenching (NPQ)-related proteins, such as fluctuating-light acclimation protein 1 (FLAP1), PsbS/NPQ4, and proton gradient regulation 5 (PGR5). Phenotypes of these double mutants revealed that PsbS works upstream of DLDG1, PGR5 affects NPQ independently from DLDG1, and the ΔpH regulation by FLAP1 and DLDG1 are independent of each other.

Polysulfide metabolizing enzymes influence SqrR-mediated sulfide-induced transcription by impacting intracellular polysulfide dynamics.

Sulfide plays essential roles in controlling various physiological activities in almost all organisms. Although recent evidence has demonstrated that sulfide is endogenously generated and metabolized into polysulfides inside the cells, the relationship between polysulfide metabolism and polysulfide-sensing mechanisms is not well understood. To better define this interplay between polysulfide metabolism and sensing in cells, we investigated the role of polysulfide-metabolizing enzymes such as sulfide:quinone oxidoreductase (SQR) on the temporal dynamics of cellular polysulfide speciation and on the transcriptional regulation by the persulfide-responsive transcription factor SqrR in Rhodobacter capsulatus. We show that disruption of the sqr gene resulted in the loss of SqrR repression by exogenous sulfide at longer culture times, which impacts the speciation of intracellular polysulfides of Δsqr vs. wild-type strains. Both the attenuated response of SqrR and the change in polysulfide dynamics of the Δsqr strain is fully reversed by the addition to cells of cystine-derived polysulfides, but not by glutathione disulfide (GSSG)-derived polysulfides. Furthermore, cysteine persulfide (CysSSH) yields a higher rate of oxidation of SqrR relative to glutathione persulfide (GSSH), which leads to DNA dissociation in vitro. The oxidation of SqrR was confirmed by a mass spectrometry-based kinetic profiling strategy that showed distinct polysulfide-crosslinked products obtained with CysSSH vs. GSSH. Taken together, these results establish a novel association between the metabolism of polysulfides and the mechanisms for polysulfide sensing inside the cells.

The Sulfide-Responsive SqrR/BigR Homologous Regulator YgaV of Escherichia coli Controls Expression of Anaerobic Respiratory Genes and Antibiotic Tolerance.

Compositions and activities of bacterial flora in the gastrointestinal tract significantly influence the metabolism, health, and disease of host humans and animals. These enteric bacteria can switch between aerobic and anaerobic growth if oxygen tension becomes limited. Interestingly, the switching mechanism is important for preventing reactive oxygen species (ROS) production and antibiotic tolerance. Studies have also shown that intracellular and extracellular sulfide molecules are involved in this switching control, although the mechanism is not fully clarified. Here, we found that YgaV, a sulfide-responsive transcription factor SqrR/BigR homolog, responded to sulfide compounds in vivo and in vitro to control anaerobic respiratory gene expression. YgaV also responded to H2O2 scavenging in the enteric bacterium Escherichia coli. Although the wild-type (WT) showed increased antibiotic tolerance under H2S-atmospheric conditions, the ygaV mutant did not show such a phenotype. Additionally, antibiotic sensitivity was higher in the mutant than in the WT of both types in the presence and absence of exogenous H2S. These results, therefore, indicated that YgaV-dependent transcriptional regulation was responsible for maintaining redox homeostasis, ROS scavenging, and antibiotic tolerance.

Live Cell Imaging of ATP Dynamics in Plant Cells.

Adenosine triphosphate (ATP) is a central metabolite that functions as the energy currency in a living cell. Therefore, visualizing cellular ATP dynamics provides the fundamental information necessary to understand the molecular events involving life phenomena. Live cell imaging technologies using fluorescence (FL)-based indicators have been developed to analyze the dynamics of various biological processes, such as intracellular ATP synthesis and consumption. However, the application of FL-based indicators to plant cells is limited due to the presence of strong chlorophyll autofluorescence, which drastically worsen the signal-to-noise ratio. The bioluminescent (BL) indicators that do not require excitation light could overcome this problem. In this chapter, we introduce a methodology to analyze ATP dynamics in plant cells using BL ATP indicators.

Chloroplast pH Homeostasis for the Regulation of Photosynthesis.

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin-Benson-Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.

Regulation of ppGpp Synthesis and Its Impact on Chloroplast Biogenesis during Early Leaf Development in Rice.

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2, the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice.

Metabolic changes contributing to large biomass production in the Arabidopsis ppGpp-accumulating mutant under nitrogen deficiency.

MAIN CONCLUSION: The Arabidopsis ppGpp-overproducing mutant indicates a larger biomass than wild type by modulated amino-acid metabolism under nitrogen-limiting conditions. The regulatory nucleotide, guanosine 3', 5'-bis(pyrophosphate; ppGpp)-originally identified in Escherichia coli-controls gene expression and enzyme activities in the bacteria and plastids of plant cells. We recently reported that the ppGpp over-producing mutant of Arabidopsis thaliana had a larger shoot weight than wild type (WT), especially under nutrient-deficient conditions. However, the mechanisms behind the influence of ppGpp on plant growth and biomass remain elusive. To understand the impact of the ppGpp accumulation on plant growth, we characterized metabolic changes in the ppGpp-overproducing mutant upon transition from nitrogen-rich to nitrogen-limiting concentrations. We found that the fresh weight of the mutant was significantly larger than WT when the total nitrogen source (KNO3 and NH4NO3) concentration was below 0.9 mM. When the nitrogen content in the medium decreased, aromatic and branched-chain amino acids increased in WT due to accelerated protein degradation and/or attenuated protein synthesis. These amino-acid levels in the ppGpp over-accumulating mutant decreased upon nitrogen deficiency. The results suggest that the ppGpp-overaccumulation affects amino-acid and protein homeostasis and facilitates growth under nitrogen-limiting conditions.

Lack of plastid-encoded Ycf10, a homolog of the nuclear-encoded DLDG1 and the cyanobacterial PxcA, enhances the induction of non-photochemical quenching in tobacco.

pH homeostasis in the chloroplast is crucial for the control of photosynthesis and other metabolic processes in plants. Recently, nuclear-encoded Day-Length-dependent Delayed Greening1 (DLDG1) and Fluctuating-Light Acclimation Protein1 (FLAP1) that are required for the light-inducible optimization of plastidial pH in Arabidopsis thaliana were identified. DLDG1 and FLAP1 homologs are specifically conserved in oxygenic phototrophs, and a DLDG1 homolog, Ycf10, is encoded in the chloroplast genome in plant cells. However, the function of Ycf10 and its physiological significance are unknown. To address this, we constructed ycf10 tobacco Nicotiana tabacum mutants and characterized their phenotypes. The ycf10 tobacco mutants grown under continuous-light conditions showed a pale-green phenotype only in developing leaves, and it was suppressed in short-day conditions. The ycf10 mutants also induced excessive non-photochemical quenching (NPQ) compared with those in the wild-type at the induction stage of photosynthesis. These phenotypes resemble those of Arabidopsis dldg1 mutants, suggesting that they have similar functions. However, there are distinct differences between the two mutant phenotypes: The highly induced NPQ in tobacco ycf10 and the Arabidopsis dldg1 mutants are diminished and enhanced, respectively, with increasing duration of the fluctuating actinic-light illumination. Ycf10 and DLDG1 were previously shown to localize in chloroplast envelope-membranes, suggesting that Ycf10 and DLDG1 differentially control H+ exchange across these membranes in a light-dependent manner to control photosynthesis.

Femtosecond Time-Resolved Absorption Study of Signaling State of a BLUF Protein PixD from the Cyanobacterium Synechocystis: Hydrogen-Bond Rearrangement Completes during Forward Proton-Coupled Electron Transfer.

Femtosecond time-resolved absorption measurements were carried out for the dark and signaling states of a BLUF (Blue Light Using FAD) protein, PixD, from the cyanobacterium Synechocystis. When the dark state was excited, FAD semiquinone radical (FADH•) was produced from the S1 state, and FADH• led to the signaling state. On the other hand, photoexcitation of the signaling state generated FADH• and FAD anion radical (FAD•-), and they decayed back to the original signaling state. In both cases, FADH• was formed and decayed with a proton-coupled electron transfer (PCET) via the hydrogen-bond network that involves FAD, Gln50, and Tyr8, and hence the kinetics of FADH• directly reflects the hydrogen-bond structure in the FAD-binding sites. It was found that the formation rate of FADH• was significantly different between the dark and signaling states, whereas the decay rate was the same. This indicates that the hydrogen-bond network of FAD-Gln50-Tyr8 in the dark and signaling states is initially different but it becomes indistinguishable after FADH• is formed, implying that the FAD-Gln50-Tyr8 hydrogen-bond network is rearranged during the PCET to generate FADH•. The present results best agree with the model in which the Gln tautomerizes without rotation in the signaling-state formation.

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

Photoreaction Mechanisms of Flavoprotein Photoreceptors and Their Applications.

Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.

In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803.

Phototaxis is a phenomenon where cyanobacteria move toward a light source. Previous studies have shown that the blue-light-using-flavin (BLUF)-type photoreceptor PixD and the response regulator-like protein PixE control the phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. The pixD-null mutant moves away from light, whereas WT, pixE mutant, and pixD pixE double mutant move toward the light. This indicates that PixE functions downstream of PixD and influences the direction of movement. However, it is still unclear how the light signal received by PixD is transmitted to PixE, and then subsequently transmitted to the type IV pili motor mechanism. Here, we investigated intracellular localization and oligomerization of PixD and PixE to elucidate mechanisms of phototaxis regulation. Blue-native PAGE analysis, coupled with western blotting, indicated that most PixD exist as a dimer in soluble fractions, whereas PixE localized in ~250 kDa and ~450 kDa protein complexes in membrane fractions. When blue-native PAGE was performed after illuminating the membrane fractions with blue light, PixE levels in the ~250 kDa and ~450 kDa complexes were reduced and increased, respectively. These results suggest that PixE, localized in the ~450 kDa complex, controls activity of the motor ATPase PilB1 to regulate pilus motility.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

ppGpp functions as an alarmone in metazoa.

Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.

Regulation of light-induced H+ extrusion and uptake by cyanobacterial homologs of the plastidial FLAP1, DLDG1, and Ycf10 in Synechocystis sp. PCC6803.

Upon a dark-to-light transition, multiple species of cyanobacteria release a certain amount of H+ from the inside to the outside of their cells. Previous studies revealed the plasma membrane-localizing Proton exchange A (PxcA) is involved in the light-induced H+ extrusion in the cyanobacterium Synechocystis sp. PCC6803. Among oxygenic phototrophs, two PxcA homologs are conserved; they are the nuclear-encoded Day-length-dependent delayed-greening1 (DLDG1) and the plastid-encoded Ycf10 in Arabidopsis thaliana. We previously identified the putative DLDG1/Ycf10-interacting protein, Fluctuating-light acclimation protein1 (FLAP1), required for pH regulation in Arabidopsis chloroplasts. Synechocystis has PxcA and FLAP1 homologs designated here as PxcA like (PxcL) and FLAP1 homolog A (FlpA). Synechocystis mutants lacking pxcA, pxcL, and flpA were constructed and characterized to gain more insight into regulatory mechanisms of light-induced H+ extrusion in cyanobacteria. pH change kinetics of the extracellular solvent after shifting Synechocystis cells from dark to light indicated that PxcA is essential for the light-induced H+ extrusion, and both PxcA and PxcL were involved in H+ uptake. Mutational loss of flpA resulted in altered PxcA- and PxcL-dependent H+ efflux/influx activities, and the flpA-null mutant showed inhibited growth under dark-light cycles, indicating the importance of FlpA function for photosynthetic growth under fluctuating light. Collectively, these data suggest that PxcA is involved in H+ efflux immediately after light irradiation for the rapid formation of the H+ concentration gradient across the thylakoid membranes, PxcL is involved in H+ influx for activation of the Calvin-Benson-Bassham cycle, and FlpA controls the H+ transport under fluctuating light.

Persulphide-responsive transcriptional regulation and metabolism in bacteria.

Hydrogen sulphide (H2S) impacts on bacterial growth both positively and negatively; it is utilized as an electron donor for photosynthesis and respiration, and it inactivates terminal oxidases and iron-sulphur clusters. Therefore, bacteria have evolved H2S-responsive detoxification mechanisms for survival. Sulphur assimilation in bacteria has been well studied, and sulphide:quinone oxidoreductase, persulphide dioxygenase, rhodanese and sulphite oxidase were reported as major sulphide-oxidizing enzymes of sulphide assimilation and detoxification pathways. However, how bacteria sense sulphide availability to control H2S and sulphide metabolism remains largely unknown. Recent studies have identified several bacterial (per)sulphide-sensitive transcription factors that change DNA-binding affinity through persulphidation of specific cysteine residues in response to highly reactive sulphur-containing chemicals and reactive sulphur species (RSS). This review focuses on current understanding of the persulphide-responsive transcription factors and RSS metabolism regulated by RSS sensory proteins.