Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Speciation in red algae: members of the Ceramiales as model organisms

Red algae (Rhodophyta) are an ancient group with unusual morphological, biochemical, and life-history features including a complete absence of flagella. Although the red algae present many opportunities for studying speciation, this has rarely been explicitly addressed. Here, we examine an aspect of paternal gene flow by determining fertilization success of female Neosiphonia harveyi (Ceramiales), which retains a morphological record of all successful and unsuccessful female gametes. High fertilization rates were observed except when there were no males at all within the tidepool, or in a submerged marina environment. Small numbers of reproductive males were able to saturate fertilization rates, suggesting that limited availability of sperm may be less significant in red algae than previously thought. In another member of the Ceramiales, Antithamnion, relatively large chromosomes permit karyological identification of polyploids. The Western Pacific species Antithamnion sparsum is closely related to the diploid species Antithamnion defectum, known only from the Eastern Pacific, and appears to have evolved from it. Molecular evidence suggests that A. sparsum is an autopolyploid, and that the European species known as Antithamnion densum is divergent from the A. sparsum/defectum complex.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena . We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.     

Novel morphology in Enteromorpha (Ulvophyceae) forming green tides

"Green tides" are vast accumulations of unattached green macroalgae associated with eutrophicated marine environments. They have major ecological and economic impacts globally, so an understanding of their origin and persistence is required in order to make management decisions. Blooms predominantly consist of two common fouling genera of the Ulvales, Ulva (distromatic sheets) and Enteromorpha (monostromatic tubes). In the Baltic Sea and elsewhere green tides have increased over the last few decades. On the west coast of Finland, summer blooms consist of monostromatic sheets resembling Monostroma (Codiolales). We identified these as Enteromorpha intestinalis by comparative analyses of rDNA internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 sequences, the first time monostromatic sheets have been found in the genus Enteromorpha. Ordinary attached E. intestinalis sporulated freely in culture, but the sheets reproduced only by cell regeneration into typical tubular thalli. The ITS sequences were identical to those of attached E. intestinalis populations in southwestern Finland, but differed by two substitutions from other Baltic sequences. We infer that this bloom originated from local attached populations and now reproduces clonally by fragmentation. This study provides further evidence of radical changes in gross morphology of green algae under eutrophicated conditions and the need for molecular identification.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.