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.

Leptolyngbya boryana (L. boryana) (formerly Plectonema boryanum) is a versatile, filamentous cyanobacterium that has the ability to fix nitrogen under microoxic conditions and to grow heterotrophically with glucose in the dark, providing an excellent system to investigate photosynthesis, nitrogen fixation, and their regulatory mechanisms. While L. boryana is not naturally transformable different from the unicellular cyanobacterium Synechocystis sp. PCC 6803, it can be transformed by electroporation. Here we describe the transformation of L.boryana by electroporation to isolate mutants in which a targeted gene is disrupted. Materials and Reagents1. Bottle top filter system (0.22 µm) (Corning, catalog number: 430624) 2. Microcentrifuge tubes (1.5 ml) (Ina-optika corporation, BIO-BIK, catalog number: ST-0150F) or its equivalent, sterilized by autoclave (121 ºC, 20 min) 3. Micropipettes tips (121 ºC, 20 min) 4. Sterile syringe filter (Millex-GV Syringe Filter Unit, 0.22 µm), used for filter sterilization of solutions of antibiotics and glucose (Thermo Fisher Scientific, Millipore, catalog number: SLGV033RV) or its equivalent 5. Petri dish [sterile Petri dishes (90 mm x 15 mm)] (ASONE Corporation, catalog number: 1-7484-01-30) or its equivalent 6. Pulse cuvettes (Gene Pulser cuvette, 0.1 cm) (Bio-Rad Laboratories, AbD Serotec ® , catalog number: 1652089) 7. Hybond N+ filter (disc 82 mm diameter) (GE Healthcare, Amersham, catalog number: RPN82B), sterilized by autoclave (121 ºC, 20 min). 8. Leptolyngbya boryana (wild type or dg5) (grown on a BG-11 agar plate supplemented with 20 mM HEPES-KOH, pH 7.5 and 30 mM glucose)Note: The dg5 strain was isolated from the wild type as a natural mutant that grows much faster heterotrophically in the dark than wild type (Fujita et al., 1996). Recently we identified that the mutation responsible for the dg5 phenotype is one adenine insertion causing a frameshift in the cytM gene encoding cytochrome cM (Hiraide et al., 2015).

Background: ChlR activates the transcription of genes encoding low-oxygen-type enzymes in response to hypoxia in cyanobacteria. Results:The chlR-lacking mutant showed a novel "oxygen-induced" greening process upon exposure to air. Conclusion: The contents of photosystems were correlated well with the chlorophyll contents in the greening process. Significance: Oxygen-induced greening provides a promising alternative system to investigate the biogenesis of photosystems.

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.