An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.
The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria. (+info)
Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.
Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes. (+info)
Extinction coefficients and midpoint potentials of cytochrome c(6) from the cyanobacteria Arthrospira maxima, Microcystis aeruginosa, and Synechocystis 6803.
Cytochrome c(6) is a soluble heme protein that serves as a photosynthetic electron transport component in cyanobacteria and algae, carrying electrons from the cytochrome bf complex to photosystem I. The rapid accumulation of cytochrome c(6) sequence data from a wide range of species, combined with significant advances in determining high resolution three-dimensional structures, provides a powerful database for investigating the relationship between structure and function. The fact that the gene encoding cytochrome c(6) can be readily modified in a number of species adds to the usefulness of cytochrome c(6) as a tool for comparative analysis. Efforts to relate cytochrome c(6) sequence information to structure, and structural information to function depend on knowledge of the physical and thermodynamic properties of the cytochrome from different species. To this end we have determined the optical extinction coefficient, the oxidation/reduction midpoint potential, and the pH dependence of the midpoint potential of cytochrome c(6) isolated from three cyanobacteria, Arthrospira maxima, Microcystis aeruginosa, and Synechocystis 6803. (+info)
Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat.
The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C(18) cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C(18) reverse-phase column. The specific growth rate (mu) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing mu. The MC-LR and MC-RR contents on a dry weight basis were highest at mu of 0.1/day at 339 and 774 microg g(-1), respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower mu, whereas the MC-producing rate was linearly proportional to mu. The C fixation rate at an ambient irradiance (160 microeinsteins m(-2) s(-1)) increased with mu. The ratios of the MC-producing rate to the C fixation rate were higher at a lower mu. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions. (+info)
DNA-DNA reassociation among a bloom-forming cyanobacterial genus, Microcystis.
DNA base composition and DNA-DNA hybridization among the cyanobacterial genus Microcystis were determined using nine axenic Microcystis strains, including the three 'morphological' species of Microcystis aeruginosa, Microcystis viridis and Microcystis wesenbergii. These Microcystis species showed a similar DNA base composition (42.1-42.8 mol% G + C) and demonstrated more than 70% DNA relatedness, confirming their synonymy based on bacterial criteria. (+info)
Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system.
BACKGROUND: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids. (+info)
Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate.
Cell quotas of microcystin (Q(MCYST); femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q(MCYST) and specific growth rate (mu), from which we propose a generalized model that enables Q(MCYST) at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q(MCYST) from mu, mu(max) (maximum specific growth rate), Q(MCYSTmax) (maximum cell quota), and Q(MCYSTmin) (minimum cell quota). Under the conditions examined in this study, we predict a Q(MCYSTmax) of 0.129 fmol cell(-1) at mu(max) and a Q(MCYSTmin) of 0.050 fmol cell(-1) at mu = 0. Net MCYST production rate (R(MCYST)) asymptotes to zero at mu = 0 and reaches a maximum of 0.155 fmol cell(-1) day(-1) at mu(max). MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with mu, whereas the MCYST/protein ratio reached a maximum at intermediate mu. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with mu, leading to an increase in intracellular MCYST concentration at high mu. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components. (+info)
Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (Phycocyanin Intergenic Spacer) phylogenies.
The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for the N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37 Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMT-specific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance from mcyC. All nontoxic strains also contained uma1, which is not cotranscribed with mcyABC. The consistent linkage of mcyC to uma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the various Microcystis clades. (+info)