Isolation of cyanobacterial mutants exhibiting growth defects under microoxic conditions by transposon tagging mutagenesis of Synechocystis sp. PCC 6803.

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis by extracting electrons from water, with the generation of oxygen as a byproduct. Cyanobacteria use oxygen not only for respiration to produce energy in the dark but also for biosynthesis of various metabolites, such as heme and chlorophyll. Oxygen levels dynamically fluctuate in the field environments, from hyperoxic at daytime to almost anaerobic at night. Thus, adaptation to anaerobiosis should be important for cyanobacteria to survive in low-oxygen and anaerobic environments. However, little is known about the molecular mechanisms of cyanobacterial anaerobiosis because cyanobacteria have been regarded as aerobic organisms. As a first step to elucidate cyanobacterial adaptation mechanisms to low-oxygen environments, we isolated five mutants, T-1-T-5, exhibiting growth defects under microoxic conditions. The mutants were obtained from a transposon-tagged mutant library of the cyanobacterium Synechocystis sp. PCC 6803, which was produced by in vitro transposon tagging of cyanobacterial genomic DNA. Southern blot analysis indicated that a kanamycin resistance gene was inserted in the genome as a single copy. We identified the chromosomal transposon-tagged locus in T-5. Two open reading frames (sll0577 and sll0578) were partially deleted by the insertion of the kanamycin resistance gene in T-5. A reverse transcription polymerase chain reaction suggested that these co-transcribed genes are constitutively expressed under both aerobic and microoxic conditions. Then, we isolated two mutants in which one of the two genes was individually disrupted. Only the mutants partially lacking an intact sll0578 gene showed growth defects under microoxic conditions, whereas it grew normally under aerobic conditions. sll0578 is annotated as purK encoding N5-carboxy-aminoimidazole ribonucleotide synthetase involved in purine metabolism. This result implies the unexpected physiological importance of PurK under low-oxygen environments.

[Anesthetic Management of Two Patients with Chilaiditi Syndrome].

Chilaiditi syndrome is assosiated with hepatodiaphragmatic interposition of the colon and the small intestines. We anesthetized 2 patients with Chilaiditi syndrome. A 62-year-old woman with interposition of the intestine was scheduled for right femoral fibrosarcoma resection. She had been medicated for schizophrenia. Total intravenous anesthesia was induced and maintained. Another patient an 81-year-old man with interposition of the colon, was scheduled for transurethral resection of a bladder tumor. He was anesthetized with spinal anesthesia. In both cases, there was no cardiovascular complication or digestive disorder during the perioperative period.

Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments.

Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes.

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.

MarR-type transcriptional regulator ChlR activates expression of tetrapyrrole biosynthesis genes in response to low-oxygen conditions in cyanobacteria.

Oxygen is required for three enzyme reactions in chlorophyll and bilin biosynthesis pathways: coproporphyrinogen III oxidase (HemF), heme oxygenase (HO1), and Mg-protoporphyrin IX monomethylester cyclase (ChlA(I)). The cyanobacterium Synechocystis sp. PCC 6803 has alternative enzymes, HemN, HO2, and ChlA(II), to supply chlorophyll/bilins even under low-oxygen environments. The three genes form an operon, chlA(II)-ho2-hemN, that is induced in response to low-oxygen conditions to bypass the oxygen-dependent reactions. Here we identified a transcriptional regulator for the induction of the operon in response to low-oxygen conditions. A pseudorevertant, Δho1R, was isolated from a HO1-lacking mutant Δho1 that is lethal under aerobic conditions. Δho1R grew well even under aerobic conditions. In Δho1R, HO2 that is induced only under low-oxygen conditions was anomalously expressed under aerobic conditions to complement the loss of HO1. A G-to-C transversion in sll1512 causing the amino acid change from aspartate 35 to histidine was identified as the relevant mutation by resequencing of the Δho1R genome. Sll1512 is a MarR-type transcriptional regulator. An sll1512-lacking mutant grew poorly under low-oxygen conditions with a remarked decrease in Chl content that would be caused by the suppressed induction of the chlA(II) and hemN genes in Chl biosynthesis under low-oxygen conditions. These results demonstrated that Sll1512 is an activator in response to low-oxygen environments and that the D35H variant becomes a constitutive activator. This hypothesis was supported by a gel shift assay showing that the Sll1512-D35H variant binds to the DNA fragment upstream of the operon. We propose to name sll1512 chlR.

A heme oxygenase isoform is essential for aerobic growth in the cyanobacterium Synechocystis sp. PCC 6803: modes of differential operation of two isoforms/enzymes to adapt to low oxygen environments in cyanobacteria.

Heme oxygenase (HO) catalyzes the oxygen-dependent cleavage of heme to produce biliverdin IXα in phycobilin biosynthesis. In the genome of the cyanobacterium Synechocystis sp. PCC 6803 there are two genes, ho1 (sll1184) and ho2 (sll1875), encoding HO isoforms. Reverse transcription-PCR indicated that ho1 is constitutively expressed, and ho2 is induced under micro-oxic conditions. A mutant lacking ho1 (Δho1) failed to grow under aerobic conditions while it did grow at a significantly slower rate than the wild type under anaerobic (micro-oxic) conditions. When micro-oxically grown Δho1 was incubated under aerobic conditions, the cells underwent chlorosis with a significant decrease in phycocyanin accompanied by anomalous accumulation of protoporphyrin IX. These results suggested that HO1 is essential for aerobic growth as the sole HO and is dispensable under micro-oxic conditions. A mutant lacking ho2 (Δho2) grew under both aerobic and micro-oxic conditions like the wild type at low light intensity (50 µmol(photon) m⁻² s⁻¹). At higher light intensity (120 µmol(photon) m⁻² s⁻¹) the Δho2 mutant showed significant growth retardation under micro-oxic conditions. It is suggested that HO2 operates as a dominant HO under high light and micro-oxic environments and acts as an accessory HO at low light intensity. Constitutive expression of HO2 in a neutral site of the chromosome restored aerobic growth of Δho1, suggesting that HO2 has an activity high enough to substitute for HO1 under aerobic conditions. The differential operation of two isoforms/enzymes in cyanobacterial tetrapyrrole biosynthesis to adapt to low oxygen environments is discussed, including three other reactions.

Functional differentiation of two analogous coproporphyrinogen III oxidases for heme and chlorophyll biosynthesis pathways in the cyanobacterium Synechocystis sp. PCC 6803.

Coproporphyrinogen III oxidase (CPO) catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX in heme biosynthesis and is shared in chlorophyll biosynthesis in photosynthetic organisms. There are two analogous CPOs, oxygen-dependent (HemF) and oxygen-independent (HemN) CPOs, in various organisms. Little information on cyanobacterial CPOs has been available to date. In the genome of the cyanobacterium Synechocystis sp. PCC 6803 there is one hemF-like gene, sll1185, and two hemN-like genes, sll1876 and sll1917. The three genes were overexpressed in Escherichia coli and purified to homogeneity. Sll1185 showed CPO activity under both aerobic and anaerobic conditions. While Sll1876 and Sll1917 showed absorbance spectra indicative of Fe-S proteins, only Sll1876 showed CPO activity under anaerobic conditions. Three mutants lacking one of these genes were isolated. The Deltasll1185 mutant failed to grow under aerobic conditions, with accumulation of coproporphyrin III. This growth defect was restored by cultivation under micro-oxic conditions. The growth of the Deltasll1876 mutant was significantly slower than that of the wild type under micro-oxic conditions, while it grew normally under aerobic conditions. Coproporphyrin III was accumulated at a low but significant level in the Deltasll1876 mutant grown under micro-oxic conditions. There was no detectable phenotype in Deltasll1917 under the conditions we examined. These results suggested that sll1185 encodes HemF as the sole CPO under aerobic conditions and that sll1876 encodes HemN operating under micro-oxic conditions, together with HemF. Such a differential operation of CPOs would ensure the stable supply of tetrapyrrole pigments under environments where oxygen levels fluctuate greatly.