Convergent evolution of [D-Leucine(1)] microcystin-LR in taxonomically disparate cyanobacteria

BACKGROUND: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. RESULTS: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu(1)] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA(2) adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA(2) adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu(1)] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met(1)] microcystin-LR. CONCLUSIONS: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu(1)] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.Peer reviewe

Genome Sequence of Dickeya solani, a New soft Rot Pathogen of Potato, Suggests its Emergence May Be Related to a Novel Combination of Non-Ribosomal Peptide/Polyketide Synthetase Clusters

Peer reviewe

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.</p

Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella

Microcystins (MCs) are serine/threonine phosphatase inhibitors synthesized by several members of the phylum Cyanobacteria. Mining the draft genome sequence of the nostocalean MC-producing Fischerella sp. strain CENA161 led to the identification of three contigs containing mcy genes. Subsequent PCR and Sanger sequencing allowed the assembling of its complete biosynthetic mcy gene cluster with 55,016 bases in length. The cluster encoding ten genes (mcyA-J) with a central bidirectional promoter was organized in a similar manner as found in other genera of nostocalean cyanobacteria. However, the nucleotide sequence of the mcy gene cluster of Fischerella sp. CENA161 showed significant differences from all the other MC-producing cyanobacterial genera, sharing only 85.2 to 74.1% identities. Potential MC variants produced by Fischerella sp. CENA161 were predicted by the analysis of the adenylation domain binding pockets and further investigated by LC-MS/MS analysis. To our knowledge, this study presents the first complete mcy cluster characterization from a strain of the genus Fischerella, providing new insight into the distribution and evolution of MCs in the phylum Cyanobacteria. (C) 2017 Elsevier Ltd. All rights reserved.Peer reviewe

Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S–23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains

Genome derived insights into the biology of the hepatotoxic bloom forming cyanobacterium Anabaena sp. strain 90

Background Cyanobacteria can form massive toxic blooms in fresh and brackish bodies of water and are frequently responsible for the poisoning of animals and pose a health risk for humans. Anabaena is a genus of filamentous diazotrophic cyanobacteria commonly implicated as a toxin producer in blooms in aquatic ecosystems throughout the world. The biology of bloom-forming cyanobacteria is poorly understood at the genome level. Results Here, we report the complete sequence and comprehensive annotation of the bloom-forming Anabaena sp. strain 90 genome. It comprises two circular chromosomes and three plasmids with a total size of 5.3 Mb, encoding a total of 4,738 genes. The genome is replete with mobile genetic elements. Detailed manual annotation demonstrated that almost 5% of the gene repertoire consists of pseudogenes. A further 5% of the genome is dedicated to the synthesis of small peptides that are the products of both ribosomal and nonribosomal biosynthetic pathways. Inactivation of the hassallidin (an antifungal cyclic peptide) biosynthetic gene cluster through a deletion event and a natural mutation of the buoyancy-permitting gvpG gas vesicle gene were documented. The genome contains a large number of genes encoding restriction-modification systems. Two novel excision elements were found in the nifH gene that is required for nitrogen fixation. Conclusions Genome analysis demonstrated that this strain invests heavily in the production of bioactive compounds and restriction-modification systems. This well-annotated genome provides a platform for future studies on the ecology and biology of these important bloom-forming cyanobacteria.Peer reviewe

A High-Sensitivity Method for Detection and Measurement of HMGB1 Protein Concentration by High-Affinity Binding to DNA Hemicatenanes

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies

Can dissonance engineering improve risk analysis of human–machine systems?

The paper discusses dissonance engineering and its application to risk analysis of human–machine systems. Dissonance engineering relates to sciences and technologies relevant to dissonances, defined as conflicts between knowledge. The richness of the concept of dissonance is illustrated by a taxonomy that covers a variety of cognitive and organisational dissonances based on different conflict modes and baselines of their analysis. Knowledge control is discussed and related to strategies for accepting or rejecting dissonances. This acceptability process can be justified by a risk analysis of dissonances which takes into account their positive and negative impacts and several assessment criteria. A risk analysis method is presented and discussed along with practical examples of application. The paper then provides key points to motivate the development of risk analysis methods dedicated to dissonances in order to identify the balance between the positive and negative impacts and to improve the design and use of future human–machine system by reinforcing knowledge

Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins

Correlating corneal arcus with atherosclerosis in familial hypercholesterolemia

Abstract Background A relationship between corneal arcus and atherosclerosis has long been suspected but is controversial. The homozygous familial hypercholesterolemia patients in this study present a unique opportunity to assess this issue. They have both advanced atherosclerosis and corneal arcus. Methods This is a cross-sectional study of 17 patients homozygous for familial hypercholesterolemia presenting to the Clinical Center of the National Institutes of Health. Plasma lipoproteins, circumferential extent of arcus, thoracic aorta and coronary calcific atherosclerosis score, and Achilles tendon width were measured at the National Institutes of Health. Results Patients with corneal arcus had higher scores for calcific atherosclerosis (mean 2865 compared to 412), cholesterol-year score (mean 11830 mg-yr/dl compared to 5707 mg-yr/dl), and Achilles tendon width (mean 2.54 cm compared to 1.41 cm) than those without. Corneal arcus and Achilles tendon width were strongly correlated and predictive of each other. Although corneal arcus was correlated with calcific atherosclerosis (r = 0.67; p = 0.004), it was not as highly correlated as was the Achilles tendon width (r = 0.855; p Conclusion Corneal arcus reflects widespread tissue lipid deposition and is correlated with both calcific atherosclerosis and xanthomatosis in these patients. Patients with more severe arcus tend to have more severe calcific atherosclerosis. Corneal arcus is not as good an indicator of calcific atherosclerosis as Achilles tendon thickness, but its presence suggests increased atherosclerosis in these hypercholesterolemic patients.</p