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Comparative genomic analysis of a novel heat-tolerant and euryhaline strain of unicellular marine cyanobacterium Cyanobacterium sp. DS4 from a high-temperature lagoon.

BACKGROUND: Cyanobacteria have diversified through their long evolutionary history and occupy a wide range of environments on Earth. To advance our understanding of their adaptation mechanisms in extreme environments, we performed stress tolerance characterizations, whole genome sequencing, and comparative genomic analyses of a novel heat-tolerant and euryhaline strain of the unicellular cyanobacterium Cyanobacterium sp. Dongsha4 (DS4). This strain was isolated from a lagoon on Dongsha Island in the South China Sea, a habitat with fluctuations in temperature, salinity, light intensity, and nutrient supply. RESULTS: DS4 cells can tolerate long-term high-temperature up to 50 ℃ and salinity from 0 to 6.6%, which is similar to the results previously obtained for Cyanobacterium aponinum. In contrast, most mesophilic cyanobacteria cannot survive under these extreme conditions. Based on the 16S rRNA gene phylogeny, DS4 is most closely related to Cyanobacterium sp. NBRC102756 isolated from Iwojima Island, Japan, and Cyanobacterium sp. MCCB114 isolated from Vypeen Island, India. For comparison with strains that have genomic information available, DS4 is most similar to Cyanobacterium aponinum strain PCC10605 (PCC10605), sharing 81.7% of the genomic segments and 92.9% average nucleotide identity (ANI). Gene content comparisons identified multiple distinct features of DS4. Unlike related strains, DS4 possesses the genes necessary for nitrogen fixation. Other notable genes include those involved in photosynthesis, central metabolisms, cyanobacterial starch metabolisms, stress tolerances, and biosynthesis of novel secondary metabolites. CONCLUSIONS: These findings promote our understanding of the physiology, ecology, evolution, and stress tolerance mechanisms of cyanobacteria. The information is valuable for future functional studies and biotechnology applications of heat-tolerant and euryhaline marine cyanobacteria.

Cyanobacteria

Purification and primary structure of cytochrome c-552 from the cyanobacterium, Synechococcus PCC 6312.

Cytochrome c-552 (soluble 'cytochrome f') from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence consists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.

Amino Acid Sequence

A glucan from the cell wall of the cyanobacterium Spirulina platensis.

A polysaccharide was isolated from the cell wall of the cyanobacterium Spirulina platensis. Hydrolysis of the polysaccharide only yielded glucose. Curie-point pyrolysis mass spectrometry of the polysaccharide resulted in a spectrum very similar to that of beta-1,2-glucan. Probably the glucan originates from the fibrillar inner layer of the cell wall.

Cell Wall

Sporulation in the filamentous cyanobacterium Anabaena cylindrica. The course of spore formation.

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions employed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides as estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.

Cyanobacteria

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth.

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.

Chloramphenicol

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica.

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the exoplasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 A. The corresponding histogram for heterocyst thylakoids lacks the 100 A size class, but has a very large peak at about 55 A with a shoulder at 75 A. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 A for vegetative cells and 64 A for heterocysts. The thylakoids of both cell types have about 5600 particles/micrometers2 on the P-face. On the E-face, the density drops from 939 particles/micrometers2 on vegetative cell thylakoids to 715 particles/micrometers2 on heterocyst thylakoids. The data suggest that the 100 A E-face particle of vegetative cell thylakoids is a PSII complex. The 55 A EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 A EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.

Cyanobacteria

The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica.

The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism.

Arginine

Canalesolide A, a Structurally Unique Polyhydroxy Macrolide from the Marine Cyanobacterium Okeania sp. with Potent Antitrypanosomal Activity.

The discovery of structurally novel natural products remains central to expanding biologically relevant chemical space, particularly within underexplored marine metabolite classes. Herein, we report the discovery and complete structural elucidation of canalesolide A, a new polyhydroxylated macrolide isolated from the marine cyanobacterium Okeania sp. The compound was identified through an integrated workflow combining phenotypic screening against Trypanosoma brucei and LC-MS/MS-based molecular networking, enabling rapid prioritization of bioactive fractions and dereplication of known metabolite families. Spectroscopic analysis revealed that canalesolide A belongs to the bastimolide-related class of macrolides but exhibits a distinct structural architecture. Its structure was established by integrating ultrahigh-resolution NMR spectroscopy, empirical configurational analysis of polyol systems, targeted model compound synthesis, and controlled chemical degradation and derivatization. This combined strategy resolved stereochemical motifs that were inaccessible by direct analysis of the intact macrolide alone, providing a transferable approach for assigning densely oxygenated marine macrolides. Genome mining identified the putative biosynthetic gene cluster and proposed biosynthetic pathway for a bastimolide-related macrolide. Canalesolide A displays potent, low nanomolar antitrypanosomal activity against human-infective subspecies of T. brucei with rapid elimination of parasites within 1 h at 1 μM. Although moderate mammalian cytotoxicity was observed, preliminary in vivo efficacy/toxicity studies in infected mice suggest a narrow therapeutic window highlighting the need for improved selectivity. This study expands the structural and biosynthetic diversity of polyhydroxylated macrolides and establishes a generalizable framework for resolving stereochemically complex natural products.

Macrolides

Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.

Amino Acid Sequence

Purification and primary structure of cytochrome f from the cyanobacterium, Plectonema boryanum.

The amino acid sequence of the soluble c-type cytochrome, cytochrome f, from the cyanobacterium Plectonema boryanum (also called Phormidium luridum or Schizothrix calcicola) has been determined. The proposed sequence consists of one polypeptide chain of 85 residues and has three Asn-Gly linkages. Partly due to the presence of these Asn-Gly bonds, which readily undergo rearrangement, proteolytic digestion on the small amount of protein available was unsatisfactory. The structure was determined partly by a combination of chemical cleavage and automatic sequencing techniques. A new technique for conserving material by cyanogen bromide cleavage of residual polypeptide after automatic degradation is described. The possible evolutionary significance of primary structure comparisons with other cytochromes f is discussed.

Amino Acid Sequence

Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica.

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaema cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective election donors to nitro genase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum.

Amino Acids

Isolation and preliminary characterization of auxotrophs of a filamentous Cyanobacterium.

Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.

Ammonium Chloride

Induction of anaerobic, photoautotrophic growth in the cyanobacterium Oscillatoria limnetica.

Anaerobic photoautotrophic growth of the cyanobacterium Oscillatoria limnetica was demonstrated under nitrogen in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (5micron), a constant concentration of Na2S (2.5 mM), and constant pH (7.3). The photoanaerobic growth rate (2 days doubling time) was similar to that obtained under oxygenic photoautotrophic growth conditions. The potential of oxygenic photosynthesis is constitutive in the cells; that of anoxygenic photosynthesis is rapidly (2 h) induced in the presence of Na2S in the light in a process requiring protein synthesis. The facultative anaerobic phototrophic growth physiology exhibited by O. limnetica would seem to represent an intermediate physiological pattern between the obligate anaerobic one of photosynthetic bacteria and the oxygenic one of eucaryotic algae.

Aerobiosis

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.

Ammonia

Dark hexose metabolism by photoautotrophically and heterotrophically grown cells of the blue-green alga (Cyanobacterium) Nostoc sp. strain Mac.

Photoautotrophically grown cells of the blue-green alga (cyanobacterium) Nostoc sp. strain Mac assimilated and oxidized both glucose and fructose in the dark at different rates. The rate of fructose metabolism in these cells could be stimulated by casein hydrolysate, the effect being most pronounced at low sugar concentrations. This stimulation was not seen in cells grown heterotrophically in the dark, suggesting that it is a transitory phenomenon which disappears during the autotrophy-heterotrophy growth transition. The stimulation of fructose assimilation by casein hydrolysate was abolished by chloramphenicol or streptomycin, suggesting there are rate-limiting steps in protein biosynthesis in the dark that ultimately lead to inhibition of fructose uptake. Glucose metabolism did not show these phenomena, indicating there are differences in the metabolism of the two sugars.

Biological Transport

Regulation of uridylic acid biosynthesis in the cyanobacterium Anabaena variabilis.

The pathway of uridylic acid biosynthesis established by Leiberman, Kornberg, and Simms has been shown to be operative in the filamentous cyanobacterium Anabaena variabilis. The only enzyme of uridylic acid biosynthesis found to be lacking in two uracil-requiring strains of A. variabilis was aspartate transcarbamylase, the first enzyme in the pathway of de novo biosynthesis of uridvlic acid. Neither uracil-limited growth of a uracil-requiring mutant nor growth of the wild type in high concentrations of uracil resulted in substantial changes in the specific activities of enzymes of uridylic acid biosynthesis. It is therefore concluded that A. variabilis does not regulate all enzymes of this pathway by means of repression. However, control of the flow of intermediates through this pathway is possible by feedback inhibition of aspartate transcarbamylase by a variety of nucleotides.

Aspartate Carbamoyltransferase

Metagenome-assembled genomes for N2-fixing cyanobacterium Nostoc sp. TISTR 8405 and co-occurring microorganisms from a long-term laboratory culture.

We report here metagenome-assembled genomes from a long-term laboratory culture of the nitrogen-fixing cyanobacterium Nostoc sp. TISTR 8405, originally sourced from a Thai freshwater lake. The community consists of two additional co-occurring microorganisms, Erythrobacter sp. THAI-01 and Allorhizobium sp. THAI-01, and contains putative plasmids associated with Nostoc and Allorhizobium, respectively.

co-culture