Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Extracellular vesicle-mediated secretion of chlorophyll biosynthetic intermediates in the cyanobacterium Leptolyngbya boryana.

Extracellular vesicles (EVs) are derived from the outer membrane (OM) in Gram-negative bacteria and have diverse physiological functions. EV-mediated secretion of monovinyl protochlorophyllide (MV-Pchlide), the chlorophyll a (Chl) biosynthetic intermediate, was previously reported in a mutant lacking dark-operative Pchlide reductase in the cyanobacterium Leptolyngbya boryana. This study showed a detailed characterization of EVs from the wild-type (WT) of L. boryana grown under photoautotrophic and dark heterotrophic conditions, focusing on the accumulation of Chl intermediates. WT L. boryana cells produce two types of EVs, low-density EVs (L-EVs) and high-density EVs (H-EVs), both under light and dark conditions. L-EVs and H-EVs showed distinct morphological features and protein compositions. L-EVs from cells grown under both light and dark conditions commonly contained carotenoids, ketomyxol glycoside, and zeaxanthin, as major pigments. Based on the protein compositions of EVs and other cellular membrane fractions, L-EVs and H-EVs are probably derived from low-density OM and high-density OM interacting with cell walls, respectively. Fluorescence detection of pigments was applied to EVs, and the two Chl intermediates, protoporphyrin IX and protoporphyrin IX monomethyl ester, were commonly detected in both L-EVs from light- and dark-grown cells, whereas L-EVs from dark-grown cells contained additional MV-Pchlide, MV-protopheophorbide, and pheophorbide. The pigment ratios of L-EVs to the total culture medium of the Chl intermediates were much higher than those of carotenoids, suggesting an active transport of the Chl intermediates from the thylakoid membrane to L-EVs. Cyanobacterial EVs may play a novel role in alleviating the accumulation of Chl intermediates in cells. (248 words).

Indocyanine green fluorescence visualizes landmark arteries for endoscopic sinus and skull base surgery.

OBJECTIVE: Landmark arteries during endoscopic sinus surgery are currently identified on the basis of anatomy, CT imaging and navigation, and Doppler flowmetry. However, the advantage of intraoperative fluorescence imaging during endoscopic sinus surgery has not been demonstrated. This study aimed to investigate whether Indocyanine Green (ICG) is useful for visualizing landmark arteries during endoscopic sinus and skull base surgery. METHODS: Eight patients who underwent endoscopic sinus and pituitary surgeries and consented to study participation were included. After planned procedures were performed as usual, landmark arteries were examined by ICG endoscope. Recorded video and preoperative CT images were analyzed for identification of five landmark arteries: anterior ethmoidal artery (AEA), posterior ethmoidal artery (PEA), internal carotid artery (ICA), sphenopalatine artery (SPA), and postnasal artery (PNA). Identification of arteries was evaluated three grades: identifiable, locatable, unrecognizable. RESULTS: Eight patients and eleven sides were evaluated. The ICG dose was 2.5 mg/body and a single shot was sufficient for evaluation. 100 % of AEA was identified (9/9 sides), 86 % of PNA (6/7 sides), 56 % of ICA (5/9 sides), and 25 % of PEA and SPA (2/8 sides). CONCLUSION: ICG could visualize landmark arteries, even thin arteries like AEA, during endoscopic sinus and skull base surgeries. Visualization was affected by thickness of bone or soft tissue above arteries, blood clots, sensitivity setting, and angle and distance of near-infrared light irradiation. ICG visualization of landmark arteries may help avoid vascular injuries during endoscopic sinus and skull base surgeries, particularly of AEA, PNA and ICA.

Heterologous complementation systems verify the mosaic distribution of three distinct protoporphyrinogen IX oxidase in the cyanobacterial phylum.

The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.

Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

Differences in specific concerns perceived by parents of children with cleft lip and/or palate based on the types of cleft.

BACKGROUND: Parents of children with cleft lip and/or palate (CL/P) have concerns including guilt and worry. Differences in concerns by cleft type is, however, under-researched. AIM: To compare differences in perceived concerns among parents of children with CL/P by cleft type, adjusting for children's gender and age. DESIGN: Cross-sectional study conducted in an outpatient centre, assessing psychological status of 171 parents of children aged <12 with CL/P using 12 items. RESULTS: Across cleft types, 'I am worried about whether the child's teeth will be straight' was the most strongly perceived concern (70.8%). After adjusting for gender and age, logistic regression showed significant differences in type of CL/P for (eg) 'I am worried that the child's appearance will not be beautiful', associated with CL over CP (OR = 0.07, 95% CI: 0.02-0.26) and CLP over CP (8.52, 3.23-22.50);' 'I am concerned about my child being able to speak well', associated with CP (3.12, 1.07-9.11) and CLP (5.69, 2.43-13.33) over CL, and 'I am worried that the child could suffer due to his/her appearance', associated with CL over CP (0.08, 0.02-0.29) and CLP over CP (10.07, 13.78-27.36). CONCLUSIONS: Parents' concerns were influenced by cleft type.

Hyperintense signal on diffusion-weighted imaging for monitoring the acute response and local recurrence after photodynamic therapy in malignant gliomas.

PURPOSE: Photodynamic therapy (PDT) subsequent to surgical tumor removal is a novel localized treatment for malignant glioma that provides effective local control. The acute response of malignant glioma to PDT can be detected as linear transient hyperintense signal on diffusion-weighted imaging (DWI) and a decline in apparent diffusion coefficient values without symptoms. However, their long-term clinical significance has not yet been examined. The aim of this study was to clarify the link between hyperintense signal on DWI as an acute response and recurrence after PDT in malignant glioma. METHODS: Thirty patients (16 men; median age, 60.5 years) underwent PDT for malignant glioma at our institution between 2017 and 2020. We analyzed the signal changes on DWI after PDT and the relationship between these findings and the recurrence pattern. RESULTS: All patients showed linear hyperintense signal on DWI at the surface of the resected cavity from day 1 after PDT. These changes disappeared in about 30 days without any neurological deterioration. During a mean post-PDT follow-up of 14.3 months, 19 patients (63%) exhibited recurrence: 10 local, 1 distant, and 8 disseminated. All of the local recurrences arose from areas that did not show hyperintense signal on DWI obtained on day 1 after PDT. CONCLUSIONS: The local recurrence in malignant glioma after PDT occurs in an area without hyperintense signal on DWI as an acute response to PDT. This characteristic finding could aid in the monitoring of local recurrence after PDT.

Factors Associated with Hospital Discharge Difficulties for Pediatric Patients in Japan - Using the Delphi Technique.

PURPOSE: This study aimed to identify factors associated with discharge difficulties for pediatric patients in Japan in order to develop pediatric discharge screening tools. DESIGN AND METHODS: This study employed the Delphi method by setting consensus level to 80%. A total of 131 acute hospitals were randomly selected by computer from among 261 hospitals. Five nurses with experience in discharge planning for children from each hospital were recruited for this study resulting in 655 participants enrolled. RESULTS: We received 160 responses from participants who expressed consent to participate in the first edition questionnaire, and 112 responses with the second questionnaire. The study identified three factor categories associated with discharge difficulties: patient factors (22 items), family factors (32), and societal factors (12). Among these, 4 patient factors including "Unstable child with congenital disease", 15 family factors including "Abuse or possibility of abuse", "Carer with difficulties in bringing up the child due to a tendency towards depression", and "Carer with a poor opinion of home care", and 5 societal factors including "No alternative pediatric hospitals for transfers" reached consensus showing them to be considered important for the participants. IMPLICATIONS: The findings suggest that the factors associated with discharge difficulties for Japanese pediatric patients may be useful when used for the screening, in providing discharge planning at an early stage. These factors would provide useful ideas when developing discharge screening tools that will enable nurses to provide discharge planning for pediatric patients at the early stages of hospitalization.

Chlorophyllide a oxidoreductase Preferentially Catalyzes 8-Vinyl Reduction over B-Ring Reduction of 8-Vinyl Chlorophyllide a in the Late Steps of Bacteriochlorophyll Biosynthesis.

Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The Km value for 8 V-Chlide was 1.4 µM, which is much lower than the 6.2 µM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.

Super-activator variants of the cyanobacterial transcriptional regulator ChlR essential for tetrapyrrole biosynthesis under low oxygen conditions.

ChlR is a MarR-type transcriptional regulator that activates the transcription of the chlAII-ho2-hemN operon in response to low oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Upon exposure to low oxygen conditions, ChlR activates transcription of the operon that encodes enzymes critical to tetrapyrrole biosynthesis under low oxygen conditions. We previously identified a super-activator variant, D35H, of ChlR that constitutively activates transcription of the operon. To gain insight into the low-oxygen induced activation of ChlR, we obtained eight additional super-activator variants of ChlR including D35H from pseudorevertants of a chlAI-disrupted mutant. Most substitutions were located in the N-terminal region of ChlR. Mapping of the substituted amino acid residues provided valuable structural insights that uncovered the activation mechanism of ChlR.

Parental factors predicting unnecessary ambulance use for their child with acute illness: A cross-sectional study.

AIMS: To examine characteristics of parents of children with acute, albeit mild, illnesses who used ambulance transport unnecessarily. DESIGN: A cross-sectional study. METHODS: From 2016 - 2017, we recruited parents who visited the emergency room of a Japanese paediatric hospital and whose children were discharged without hospitalization. Participants whose children arrived by ambulance were classified as using ambulance services unnecessarily. Participants answered a questionnaire consisting of parents' characteristics, including health literacy scales and the Parents' Uncertainty regarding their Children with Acute Illness Scale. We conducted a receiver operating characteristic analysis to convert the Parents' Uncertainty regarding their Children with Acute Illness Scale results to binary scores. We analysed questionnaire responses using logistic regression analysis. RESULTS: Analysed data were from 171 participants. The cut-off score was 59 for the Parents' Uncertainty regarding their Children with Acute Illness Scale. Results of the logistic regression indicated that parents who did not use resources to obtain information regarding their child's illness, had low health literacy, were observing presenting symptoms for the first time in their child, or had high uncertainty, were significantly more likely to unnecessarily use ambulances. CONCLUSION: Publicizing available resources regarding child health information, social healthcare activities to raise parents' health literacy and providing explanations in accordance with parents' uncertainty, especially when confronting new symptoms in their child, might reduce unnecessary ambulance use. IMPACT: Of patients transported to hospitals by ambulance, the rate of paediatric parents with mild conditions has been found to be high. The study findings could contribute to the appropriateness of using ambulances and have implications for policymakers and healthcare providers, particularly in the Japanese paediatric emergency system. In particular, parental uncertainty, one of four significant characteristics, could be resolved in clinical settings. Generalization for global health services requires further research.

PAS-histidine kinases PHK1 and PHK2 exert oxygen-dependent dual and opposite effects on gametophore formation in the moss Physcomitrella patens.

Two-component systems, versatile signaling mechanisms based on phosphate transfer between component proteins, must have played important roles in adaptation and diversification processes in land plant evolution. We previously demonstrated that two Per-Arnt-Sim (PAS)-histidine kinases, PHK1 and PHK2, repress gametophore formation in the moss Physcomitrella patens under aerobic conditions, and that, in eukaryotes, the presence of their homologs is restricted to early-diverging streptophyte linages. We assessed here whether or not PHKs play a role in oxygen signaling. When submerged under water, the double disruption line for PHK1 and PHK2 formed fewer gametophores than the wild-type line (WT) both under light-dark cycles or continuous light, indicating that PHKs promote gametophore formation under an aquatic environment, in contrast to aerobic conditions. Similarly, in an artificial low-oxygen condition, the double disruption line formed fewer gametophores than WT. These results indicate that PHKs exert dual and opposite effects on gametophore formation depending on oxygen status. This study adds important insight into functional versatility and evolutionary significance of two-component systems in land plants.

[Clinical Features and Treatment Strategy of Vertebral Artery Injury Associated with Cervical Spine Trauma].

OBJECTIVE: Vertebral artery injury(VAI)associated with cervical spine trauma has the potential to cause catastrophic vertebrobasilar stroke. However, there are no well-defined treatment recommendations for VAI. The purpose of this study was to identify an effective treatment strategy for VAI following cervical spine trauma. METHODS: Ninety-seven patients with blunt cervical spine trauma were treated at Hyogo Prefectural Kakogawa Medical Center between January 2013 and September 2017. Of these patients, 49 underwent computed tomographic angiography or magnetic resonance angiography for evaluation of the vertebral artery. Eighteen patients(36.7%)had a diagnosis of VAI. We retrospectively analyzed the clinical features, treatment, and outcomes in these 18 patients. RESULTS: Seven patients(38.9%)had bilateral VAI, 16(88.9%)had cervical dislocation, and 2(11.1%)had transverse process fractures extending into the transverse foramen. Surgical reduction was performed in 14 patients. Five patients with either bilateral or unilateral occlusion underwent parent artery occlusion before reduction. There were no complications after this procedure. Two patients with bilateral VAI had a stroke before treatment. There were no infarctions in the distribution of the vertebrobasilar artery after intervention. The perioperative stroke rate was relatively good, and almost all Glasgow Outcome Scale scores were related to the degree of spinal cord injury. CONCLUSIONS: Aggressive screening for VAI is important in patients with cervical spine trauma in order to ensure adequate treatment. Although the treatment strategy described here could yield good results, it may require modification according to the needs of the individual patient.

Identification of a cis-acting element in nitrogen fixation genes recognized by CnfR in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana.

The filamentous cyanobacterium Leptolyngbya boryana has the ability to fix nitrogen without any heterocysts under microoxic conditions. Previously, we identified the cnfR gene for a master transcriptional activator for nitrogen fixation (nif) genes in a 50-kb gene cluster containing nif and nif-related genes in L. boryana. We showed that CnfR activates the transcription of nif genes in response to low oxygen conditions, which allows the oxygen-vulnerable enzyme nitrogenase to function. However, the regulatory mechanism that underlies regulation by CnfR remains unknown. In this study, we identified a conserved cis-acting element that is recognized by CnfR. We established a reporter system in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 using luciferase genes (luxAB). Reporter analysis was performed with a series of truncated and modified upstream regulatory regions of nifB and nifP. The cis-element can be divided into nine motifs I-IX, and it is located 76 bp upstream of the transcriptional start sites of nifB and nifP. Six motifs of them are essential for transcriptional activation by CnfR. This cis-acting element is conserved in the upstream regions of nif genes in all diazotrophic cyanobacteria, including Anabaena and Cyanothece, thereby suggesting that the transcriptional regulation by CnfR is widespread in nitrogen-fixing cyanobacteria.

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria.

Leptolyngbya boryana (Plectonema boryanum) is a diazotrophic cyanobacterium lacking heterocysts. How nitrogen fixation is regulated in filamentous nonheterocystous cyanobacteria remains unclear. Here we describe a large 50-kb nitrogen fixation (nif) gene cluster in L. boryana containing 50 genes. This gene cluster contains 14 nif genes (nifBSUHDKVZT and nifPENXW), two genes encoding transcriptional regulators showing high similarity to ChlR (chlorophyll regulator) and PatB, three genes encoding ferredoxin, three genes encoding cytochrome oxidase subunits, and 28 genes encoding nif-related proteins and proteins with putative or unknown functions. Eleven mutants lacking one gene or a subset of genes were isolated. Five of them did not grow under diazotrophic conditions, including two mutants lacking the transcriptional regulators. Although the chlR homolog-lacking mutant showed a normal level of nitrogenase activity, various intermediates of chlorophyll biosynthesis were accumulated under micro-oxic conditions. The phenotype suggested that ChlR activates the expression of the genes responsible for anaerobic chlorophyll biosynthesis to support energy supply for nitrogen fixation. In another mutant lacking the patB homolog, no transcripts of any nif genes were detected under nitrogen fixation conditions, which was consistent with no activity. Constitutive expression of patB in a shuttle vector resulted in low but significant nitrogenase activity even under nitrate-replete conditions, suggesting that the PatB homolog is the master regulator of nitrogen fixation. We propose to rename the patB homolog as cnfR, after cyanobacterial nitrogen fixation regulator.

[A Case of Transoral Penetrating Head Injury from a Crossbow-Fired Arrow].

Penetrating head injuries are extremely rare in Japan. The authors describe a case involving a penetrating head injury from an arrow fired from a crossbow. A 52-year-old man who had shot himself transorally in a suicide attempt was admitted to the authors' hospital. On admission, he was conscious and exhibited no neurological deficits. The end of the arrow was visible inside his oral cavity. Computed tomography revealed the arrow had penetrated the right cerebellum and occipital lobe, resulting in a very small hematoma. Digital subtraction angiography revealed no significant vascular injuries. After considering these findings and the nature of the object, the authors decided to remove the arrow from the cranium by pulling it from the patient's oral cavity. To remove the arrow, surgery was performed with several devices, including intraoperative X-ray, endoscopy, and intraoperative angiography. The authors were able to completely remove the arrow, and the patient experienced no new deficits, except mild ataxia and mild dysphasia, and no signs of cerebral infection or cerebrospinal fluid leakage after the surgery. Although most cases of penetrating head injuries require craniotomies, the authors were able to safely remove the foreign object in this case without performing a craniotomy. Because guidelines for the treatment of penetrating head injuries have not been established, the treatment of each case must be modified according to the nature of the foreign object and the findings of preoperative imaging techniques.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN-BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e(-)) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase.

X-ray crystal structure of the light-independent protochlorophyllide reductase.

Photosynthetic organisms adopt two different strategies for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide) to form chlorophyllide a, the direct precursor of chlorophyll a (refs 1-4). The first involves the activity of the light-dependent Pchlide oxidoreductase, and the second involves the light-independent (dark-operative) Pchlide oxidoreductase (DPOR). DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer), which are structurally related to nitrogenase Fe protein and MoFe protein, respectively. Here we report the crystal structure of the NB-protein of DPOR from Rhodobacter capsulatus at a resolution of 2.3A. As expected, the overall structure is similar to that of nitrogenase MoFe protein: each catalytic BchN-BchB unit contains one Pchlide and one iron-sulphur cluster (NB-cluster) coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific Pchlide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit. We propose a unique trans-specific reduction mechanism in which the distorted C17-propionate of Pchlide and an aspartate from BchB serve as proton donors for C18 and C17 of Pchlide, respectively. Intriguingly, the spatial arrangement of the NB-cluster and Pchlide is almost identical to that of the P-cluster and FeMo-cofactor in nitrogenase MoFe-protein, illustrating that a common architecture exists to reduce chemically stable multibonds of porphyrin and dinitrogen.

A novel "oxygen-induced" greening process in a cyanobacterial mutant lacking the transcriptional activator ChlR involved in low-oxygen adaptation of tetrapyrrole biosynthesis.

ChlR activates the transcription of the chlAII-ho2-hemN operon in response to low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Three genes in the operon encode low-oxygen-type enzymes to bypass three oxygen-dependent reactions in tetrapyrrole biosynthesis. A chlR-lacking mutant, ΔchlR, shows poor photoautotrophic growth due to low chlorophyll (Chl) content under low-oxygen conditions, which is caused by no induction of the operon. Here, we characterized the processes of etiolation of ΔchlR cells in low-oxygen conditions and the subsequent regreening of the etiolated cells upon exposure to oxygen, by HPLC, Western blotting, and low-temperature fluorescence spectra. The Chl content of the etiolated ΔchlR cells incubated under low-oxygen conditions for 7 days was only 10% of that of the wild-type with accumulation of almost all intermediates of the magnesium branch of Chl biosynthesis. Both photosystem I (PSI) and photosystem II (PSII) were significantly decreased, accompanied by a preferential decrease of antenna Chl in PSI. Upon exposure to oxygen, the etiolated ΔchlR cells resumed to produce Chl after a short lag (∼2 h), and the level at 72 h was 80% of that of the wild-type. During this novel "oxygen-induced" greening process, the PSI and PSII contents were largely increased in parallel with the increase in Chl contents. After 72 h, the PSI content reached ∼50% of the wild-type level in contrast to the full recovery of PSII. ΔchlR provides a promising alternative system to investigate the biogenesis of PSI and PSII.

Loss of cytochrome cM stimulates cyanobacterial heterotrophic growth in the dark.

Although cyanobacteria are photoautotrophs, they have the capability for heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and the WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp) and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from the WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding Cyt c(M). The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of the WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of the WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of the WT. These results suggest a suppressive role(s) for Cyt cM in regulation of heterotrophic activity.