Ólafur S. Andrésson

Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

Significance Remarkable chemical families are being recognized by studying diverse symbioses. We identified, through metagenomics, the first cyanobacterial trans-AT polyketide biosynthetic pathway in the Nostoc symbiont of the lichen Peltigera membranacea and showed its expression in natural thalli. An isotope-based technique designed for characterizing minute amounts of material confirmed predictions that its product, nosperin, is a distinct member of the pederin family of compounds that was previously thought exclusive to animal–bacteria associations. The unexpected discovery of nosperin in lichen expands the structural range and known distribution of this family of natural products and suggests a role associated with symbiosis. Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.

Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

Heat-labile bacterial alkaline phosphatase from a marine Vibrio sp.

Psychrophilic organisms have successfully adapted to various low-temperature environments such as cold ocean waters. Catalysts with increased catalytic efficiencies are produced, generally at the expense of thermal stability due to fewer non-covalent stabilizing interactions. A marine bacterial strain producing a particularly heat-labile alkaline phosphatase was selected from a total of 232 strains isolated from North-Atlantic coastal waters. From partial 16S rRNA sequences the strain was characterized as a Vibrio sp. An alkaline phosphatase was purified 151-fold with 54% yield from the culture medium using a single step affinity chromatography procedure on agarose-linked L-histidyldiazobenzylphosphonic acid. The active enzyme was a 55 +/- 6 kDa monomer. The enzyme had optimal activity at pH 10 and was strikingly heat-labile with a half-life of 6 min at 40 degrees C and 30 min at 32 degrees C. This enzyme from Vibrio sp. had a higher turnover number (k(cat)) and higher apparent Michaelis-Menten factor (K(m)) than the enzyme from Escherichia coli, a clear-indication of cold-adaptation. Inorganic phosphate was a competitive inhibitor with a relatively high K(i) value of 1.7 mM. Low affinity for phosphate may contribute to higher turnover rates due to more facile release of product.

The long terminal repeat is a determinant of cell tropism of maedi-visna virus.

Maedi-visna virus (MVV) is a lentivirus of sheep, mainly affecting the lungs and the central nervous system. Long terminal repeat (LTR) sequence variability is common in tissue culture-derived isolates of MVV as well as those of other lentiviruses. The role of this sequence variation in MVV replication has not been explored. PCR amplification of the LTRs of an MVV isolate revealed two product sizes, the larger containing a 53 bp duplication. PCR products containing the two size variants of the LTRs were cloned into an infectious molecular clone of MVV and the resulting chimeric viruses were tested for growth in various cell types. The chimeric virus containing only one copy of the 53 bp sequence was found to grow more slowly in sheep choroid plexus cells, sheep fibroblasts and sheep synovial cells than the virus with the 53 bp duplication. Both viruses grew equally well in macrophages. These results indicate that the LTRs determined the extended cell tropism of MVV.

Duplicated Sequence Motif in the Long Terminal Repeat of Maedi-Visna Virus Extends Cell Tropism and Is Associated with Neurovirulence

ABSTRACT Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.

Biological and Genetic Differences Between Lung- and Brain-Derived Isolates of Maedi-Visna Virus

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.

Cloning and heterologous transcription of a polyketide synthase gene from the lichen Solorina crocea.

Lichens and most ascomycete fungi produce polyketide secondary metabolites often with valuable biological activities. Their biosynthesis is primarily governed by large iterative multifunctional type I polyketide synthases. Although there has been good progress studying filamentous non-lichenized fungi, there is limited information on polyketide biosynthesis in lichens and their mycobionts, due to their slow growth, difficulties in establishing pure cultures, and the absence of methods for direct genetic manipulation. However, heterologous expression in a surrogate host offers an alternative approach for exploring lichen polyketide biosynthesis. Here, we report cloning of a type I polyketide synthase gene from the foliose lichen Solorina crocea and its heterologous transcription in the filamentous fungus Aspergillus oryzae, including processing of the transcript. No new polyketide product was detected. The lichen polyketide synthase showed greatest homology with uncharacterized genes from filamentous fungi and lower homology with proteins catalysing biosynthesis of the decaketide alternapyrone and the tetraketide side-chain of squalestatin. The technology platform utilized here presents a useful tool for functional characterization of fungal biosynthetic genes and provides a means for novel production of valuable compounds.

Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea: Features and phylogeny

Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.

Cloning and heterologous expression of Solorina crocea pyrG.

Abstract A pyrG gene, encoding orotidine 5′-monophosphate decarboxylase, was cloned from a phage library derived from the lichen Solorina crocea. Phylogenetic analysis and a survey of geographically well-separated specimens were used to verify that the gene represented the fungal component of the lichen. Both coding and upstream sequences of S. crocea pyrG exhibited features typical of fungal genes. A 132-bp intron interrupting the coding region between nucleotides 157 and 288 was confirmed by RT-PCR and sequencing. Transformation of Aspergillus nidulans with S. crocea pyrG, controlled by either its native promoter or the A. nidulans trpC promoter, resulted in uridine-independent strains that exhibited appreciable growth only at 24 °C. Southern analysis indicated multiple integrations of S. crocea pyrG. These results demonstrate that heterologous expression may be used to investigate genes from lichens.

Effects of abiotic stressors on lutein production in the green microalga Dunaliella salina

BackgroundRecent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution.ResultsNitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at.ConclusionsThis study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.