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.

Hydrogen generation through indirect biophotolysis in batch cultures of the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum.

The nitrogen-fixing nonheterocystous cyanobacterium Plectonema boryanum was used as a model organism to study hydrogen generation by indirect biophotolysis in nitrogen-limited batch cultures that were continuously illuminated and sparged with argon/CO(2) to maintain anaerobiosis. The highest hydrogen-production rate (i.e., 0.18 mL/mg day or 7.3 micromol/mg day) was observed in cultures with an initial medium nitrate concentration of 1 mM at a light intensity of 100 micromol/m(2) s. The addition of photosystem II (PSII) inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not reduce hydrogen-production rates relative to unchallenged controls for 50 to 150 h, and intracellular glycogen concentrations decreased significantly during the hydrogen generation period. The insensitivity of the hydrogen-production process to DCMU is indicative of the fact that hydrogen was not derived from water splitting at PSII (i.e., direct biophotolysis) but rather from electrons provided by intracellular glycogen reserves (i.e., indirect biophotolysis). It was shown that hydrogen generation could be sustained for long time periods by subjecting the cultures to alternating cycles of aerobic, nitrogen-limited growth and anaerobic hydrogen production.

Dissolution of dead corals by euendolithic microorganisms across the northern Great Barrier Reef (Australia).

Spatial and temporal variabilities in species composition, abundance, distribution, and bioeroding activity of euendolithic microorganisms were investigated in experimental blocks of the massive coral Porites along an inshore-offshore transect across the northern Great Barrier Reef (Australia) over a 3-year period. Inshore reefs showed turbid and eutrophic waters, whereas the offshore reefs were characterized by oligotrophic waters. The euendolithic microorganisms and their ecological characteristics were studied using techniques of microscopy, petrographic sections, and image analysis. Results showed that euendolithic communities found in blocks of coral were mature. These communities were dominated by the chlorophyte Ostreobium quekettii, the cyanobacterium Plectonema terebrans, and fungi. O. quekettii was found to be the principal agent of microbioerosion, responsible for 70-90% of carbonate removal. In the offshore reefs, this oligophotic chlorophyte showed extensive systems of filaments that penetrated deep inside coral skeletons (up to 4.1 mm) eroding as much as 1 kg CaCO3 eroded m(-2) year(-1). The percentage of colonization by euendolithic filaments at the surface of blocks did not vary significantly among sites, while their depths of penetration, especially that of O. quekettii (0.6-4.1 mm), increased significantly and gradually with the distance from the shore. Rates of microbioerosion (0.1-1.4 kg m(-2) after 1 year and 0.2-1.3 kg m(-2) after 3 years of exposure) showed a pattern similar to the one found for the depth of penetration of O. quekettii filaments. Accordingly, oligotrophic reefs had the highest rates ofmicrobioerosion ofup to 1.3 kg m(-2) year(-1), whereas the development of euendolithic communities in inshore reefs appeared to be limited by turbidity, high sedimentation rates, and low grazing pressure (rates < 0.5 kg m(-2) after 3 years). Those results suggest that boring microorganisms, including O. quekettii, have a significant impact on the overall calcium carbonate budget of coral reef ecosystems, which varies according to environmental conditions.

Common freshwater cyanobacteria grow in 100% CO2.

Cyanobacteria and similar organisms produced most of the oxygen found in Earth's atmosphere, which implies that early photosynthetic organisms would have lived in an atmosphere that was rich in CO2 and poor in O2. We investigated the tolerance of several cyanobacteria to very high (>20 kPa) concentrations of atmospheric CO2. Cultures of Synechococcus PCC7942, Synechocystis PCC7942, Plectonema boryanum, and Anabaena sp. were grown in liquid culture sparged with CO2-enriched air. All four strains grew when transferred from ambient CO2 to 20 kPa partial pressure of CO2 (pCO2), but none of them tolerated direct transfer to 40 kPa pCO2. Synechococcus and Anabaena survived 101 kPa (100%) pCO2 when pressure was gradually increased by 15 kPa per day, and Plectonema actively grew under these conditions. All four strains grew in an anoxic atmosphere of 5 kPa pCO2 in N2. Strains that were sensitive to high CO2 were also sensitive to low initial pH (pH 5-6). However, low pH in itself was not sufficient to prevent growth. Although mechanisms of damage and survival are still under investigation, we have shown that modern cyanobacteria can survive under Earth's primordial conditions and that cyanobacteria-like organisms could have flourished under conditions on early Mars, which probably had an atmosphere similar to early Earth's.

Macromolecule metabolism and photosynthetic functions in blue-green algae treated with virginiamycin, an inhibitor of protein synthesis.

The M component of virginiamycin inhibited growth of Plectonema boryanum under both photoautotrophic and heterotrophic conditions. Though the S component of this antibiotic had no apparent activity per se, it enhanced the inhibitory action of its partner. Cells incubated with suitable concentrations of either M or M + S stopped growing and lysed. Loss of the colony-forming capacity occurred quickly in the presence of M + S and slowly in the presence of M alone. Virginiamycin M inhibited protein synthesis in autotrophically and heterotrophically growing Plectonema. This effect was very rapid and could be reversed by removing the antibiotic. The S component did not block the incorporation of amino acids into proteins, but prevented the reversibility of the inhibitory effect of M. Virginiamycin M or S did not affect the photosynthetic oxygen development (Hill's reaction) in Plectonema. Moreover, carbon dioxide photoassimilation and formation of chlorophyll were inhibited only after an appreciable lag. Deoxyribonucleic acid synthesis was blocked virtually without delay by virginiamycin M. Since virginiamycin inhibited protein synthesis in a similar fashion in the unicellular Anacystis nidulans, as well as in the filamentous P. boryanum, the mechanism of action of this antibiotic is probably the same in all blue-green algae.