Heme-copper oxidases and their electron donors in cyanobacterial respiratory electron transport

Cyanobacteria are the paradigmatic organisms of oxygenic (plant-type) photosynthesis and aerobic respiration. Since there is still an amazing lack of knowledge on the role and mechanism of their respiratory electron transport, we have critically analyzed all fully or partially sequenced genomes for heme-copper oxidases and their (putative) electron donors cytochrome c(6), plastocyanin, and cytochrome c(M). Well-known structure-function relationships of the two branches of heme-copper oxidases, namely cytochrome c (aa(3)-type) oxidase (COX) and quinol (bo-type) oxidase (QOX), formed the base for a critical inspection of genes and ORFs found in cyanobacterial genomes. It is demonstrated that at least one operon encoding subunits I-III of COX is found in all cyanobacteria, whereas many non-N(2)-fixing species lack QOX. Sequence analysis suggests that both cyanobacterial terminal oxidases should be capable of both the four-electron reduction of dioxygen and proton pumping. All diazotrophic organisms have at least one operon that encodes QOX. In addition, the highly refined specialization in heterocyst forming Nostocales is reflected by the presence of two paralogs encoding COX. The majority of cyanobacterial genomes contain one gene or ORF for plastocyanin and cytochrome c(M), whereas 1-4 paralogs for cytochrome c(6) were found. These findings are discussed with respect to published data about the role of respiration in wild-type and mutated cyanobacterial strains in normal metabolism, stress adaptation, and nitrogen fixation. A model of the branched electron-transport pathways downstream of plastoquinol in cyanobacteria is presented.     

A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.     

Potent toxins in Arctic environments – Presence of saxitoxins and an unusual microcystin variant in Arctic freshwater ecosystems

Cyanobacteria are the predominant phototrophs in freshwater ecosystems of the polar regions where they commonly form extensive benthic mats. Despite their major biological role in these ecosystems, little attention has been paid to their physiology and biochemistry. An important feature of cyanobacteria from the temperate and tropical regions is the production of a large variety of toxic secondary metabolites. In Antarctica, and more recently in the Arctic, the cyanobacterial toxins microcystin and nodularin (Antarctic only) have been detected in freshwater microbial mats. To date other cyanobacterial toxins have not been reported from these locations. Five Arctic cyanobacterial communities were screened for saxitoxin, another common cyanobacterial toxin, and microcystins using immunological, spectroscopic and molecular methods. Saxitoxin was detected for the first time in cyanobacteria from the Arctic. In addition, an unusual microcystin variant was identified using liquid chromatography–mass spectrometry. Gene expression analyses confirmed the analytical findings, whereby parts of the sxt and mcy operon involved in saxitoxin and microcystin synthesis, were detected and sequenced in one and five of the Arctic cyanobacterial samples, respectively. The detection of these compounds in the cryosphere improves the understanding of the biogeography and distribution of toxic cyanobacteria globally. The sequences of sxt and mcy genes provided from this habitat for the first time may help to clarify the evolutionary origin of toxin production in cyanobacteria.     

Allosteric properties of cyanobacterial cytochrome c oxidase: inhibition of the coupled enzyme by ATP and stimulation by ADP

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.     

Exploration of a Possible Partnership among Orphan Two-Component System Proteins in Cyanobacterium Synechococcus elongatus PCC 7942

To understand the induction of the adaptive response under various stress conditions, it is important to determine the partnership between histidine kinase and response regulators in the bacterial two-component system (TCS). The genes encoding TCS partners are usually comprised of an operon in the genome, but many of them are orphans in the cyanobacterial genome. There is little information on their partnerships in Synechococcus elongatus PCC 7942. Our comprehensive analysis of protein-protein interactions among all 37 full-length proteins and the truncated domains of 24 orphans revealed a number of specific interactions. They involved evolutionarily well-conserved orphan proteins among cyanobacterial species such as Synpcc7942_0453/Ycf29, NblS/RpaB, NblS/SrrA, SasA/RpaA, and SasA/Synpcc7942_2466. Our investigation of the transphosphorylation of interaction partners indicates that orphan TCSs comprise a complex signaling network.     

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis.

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Molecular characterization of marine Cyanobacteria from the Indian subcontinent deduced from sequence analysis of the phycocyanin operon (cpcb-IGS-cpcA) and 16S-23S ITS region

Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17 % nucleotide variation, while cpcA exhibited 29 % variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteria. This study demonstrated that morphologically very similar strains might differ genotypically. Thus, molecular approaches comprising different gene regions in combination with morphological criteria may provide better taxonomical resolution of the order Oscillatoriales.     

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes

Summary A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elngation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12>EF-Tu>EF-G>S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.     

Non‐housekeeping,non‐essential GroEL (chaperonin) has acquired novel structure and function beneficial under stress in cyanobacteria

GroELs which are prokaryotic members of the chaperonin (Cpn)/Hsp60 family are molecular chaperones of which Escherichia coli GroEL is a model for subsequent research. The majority of bacterial species including E. coli and Bacillus subtilis have only one essential groEL gene that forms an operon with the co‐chaperone groES gene. In contrast to these model bacteria, two or three groEL genes exist in cyanobacterial genomes. One of them, groEL2, does not form an operon with the groES gene, whereas the other(s) does. In the case of cyanobacteria containing two GroEL homologs, one of the GroELs, GroEL1, substitutes for the native GroEL in an E. coli cell, but GroEL2 does not. Unlike the E. coli GroEL, GroEL2 is not essential, but it plays an important role which is not substitutable by GroEL1 under stress. Regulation of expression and biochemical properties of GroEL2 are different/diversified from GroEL1 and E. coli GroEL in many aspects. We postulate that the groEL2 gene has acquired a novel, beneficial function especially under stresses and become preserved by natural selection, with the groEL1 gene retaining the original, house‐keeping function. In this review, we will focus on difference between the two GroELs in cyanobacteria, and divergence of GroEL2 from the E. coli GroEL. We will also compare cyanobacterial GroELs with the chloroplast Cpns (60α and 60β) which are thought to be evolved from the cyanobacterial GroEL1. Chloroplast Cpns appear to follow the different path from cyanobacterial GroELs in the evolution after gene duplication of the corresponding ancestral groEL gene.     

Molecular confirmation of Planktothrix rubescens as the cause of intense,microcystin—Synthesizing cyanobacterial bloom in Lake Ziros,Greece

Cyanobacterial blooms occur increasingly often and raise ecological concerns worldwide. In Mediterranean freshwater ecosystems algal blooms are commonly attributed to Microcystis, Anabaena, and Aphanizomenon genera while Planktothrix is the most common bloom forming cyanobacterium in deep Northern and prealpine European oligotrophic to mesotrophic lakes. In the framework of an undertaken study of cyanobacterial species in lakes of Northwestern Greece we investigated the cyanobacterial diversity in Lake Ziros throughout a 15-month period (January 2006–March 2007) by using molecular methods. Surprisingly, a severe cyanobacterial bloom occurred during the study period, which upon microscopic examination and detailed molecular characterization found to be caused by Planktothrix rubescens species. The appearance of P. rubescens from November 2006 coincided with poor cyanobacterial diversity and resulted in a thick epilimnetic bloom in March 2007 (3.1 × 10⁸ cells/l and microcystin concentration 199 μg/l). Genotype composition of the total cyanobacterial community of the lake was analyzed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rnn operon (rRNA-ITS). A P. rubescens strain closely related to Kpr strain from Lake Klinckenberg, The Netherlands, was found to dominate. The importance of this observation is expanded by the fact that microcystin concentrations recorded in Lake Ziros were the highest measured ever in Greek aquatic ecosystems examined so far and also found amongst the highest recorded worldwide.     

Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix-dominated freshwater lakes

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.