A comparative genomics approach to understanding the biosynthesis of the sunscreen scytonemin in cyanobacteria

BACKGROUND: The extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis ofscytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria. RESULTS: Six putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly of scytonemin from central metabolites, based on genetic, biochemical, and sequence similarity evidence. Also in this cluster are redundant copies of genes encoding for aromatic amino acid biosynthetic enzymes. These can theoretically lead to tryptophan and the tyrosine precursor, p-hydroxyphenylpyruvate, (expected biosynthetic precursors of scytonemin) from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found there as well. We identified four other cyanobacterial strains containing orthologues of all of these genes, three of them by database searches (Lyngbya PCC 8106, Anabaena PCC 7120, and Nodularia CCY 9414) and one by targeted sequencing (Chlorogloeopsis sp. strain Cgs-089; CCMEE 5094). Genomic comparisons revealed that mostscytonemin-related genes were highly conserved among strains and that two additional conserved clusters, NpF5232 to NpF5236 and a putative two-component regulatory system (NpF1278 and NpF1277), are likely involved in scytonemin biosynthesis and regulation, respectively, on the basis of conservation and location. Since many of the protein product sequences for the newly described genes, including ScyD, ScyE, and ScyF, have export signal domains, while others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell. Basic structural monomer synthesis and initial condensation are most likely cytoplasmic, while later reactions are predicted to be periplasmic. CONCLUSION: We show that scytonemin biosynthetic genes are highly conserved among evolutionarily diverse strains, likely include more genes than previously determined, and are predicted to involve compartmentalization of the biosynthetic pathway in the cell, an unusual trait for prokaryotes

Characterization of the juvenile green turtle (Chelonia mydas) microbiome throughout an ontogenetic shift from pelagic to neritic habitats

The gut microbiome of herbivorous animals consists of organisms that efficiently digest the structural carbohydrates of ingested plant material. Green turtles (Chelonia mydas) provide an interesting model of change in these microbial communities because they undergo a pronounced shift from a surface-pelagic distribution and omnivorous diet to a neritic distribution and herbivorous diet. As an alternative to direct sampling of the gut, we investigated the cloacal microbiomes of juvenile green turtles before and after recruitment to neritic waters to observe any changes in their microbial community structure. Cloacal swabs were taken from individual turtles for analysis of the 16S rRNA gene sequences using Illumina sequencing. One fecal sample was also obtained, allowing for a preliminary comparison with the bacterial community of the cloaca. We found significant variation in the juvenile green turtle bacterial communities between pelagic and neritic habitats, suggesting that environmental and dietary factors support different bacterial communities in green turtles from these habitats. This is the first study to characterize the cloacal microbiome of green turtles in the context of their ontogenetic shifts, which could provide valuable insight into the origins of their gut bacteria and how the microbial community supports their shift to herbivory

Expression of Scytonemin Biosynthesis Genes under Alternative Stress Conditions in the Cyanobacterium Nostoc punctiforme

The indole-alkaloid scytonemin is a sunscreen pigment that is widely produced among cyanobacteria as an ultraviolet radiation (UVR) survival strategy. Scytonemin biosynthesis is encoded by two gene clusters that are known to be induced by long-wavelength radiation (UVA). Previous studies have characterized the transcriptome of cyanobacteria in response to a wide range of conditions, but the effect on the expression of scytonemin biosynthesis genes has not been specifically targeted. Therefore, the aim of this study is to determine the variable response of scytonemin biosynthesis genes to a variety of environmental conditions. Cells were acclimated to white light before supplementation with UVA, UVB, high light, or osmotic stress for 48 h. The presence of scytonemin was determined by absorbance spectroscopy and gene expression of representative scytonemin biosynthesis genes was measured using quantitative PCR. Scytonemin genes were up-regulated in UVA, UVB, and high light, although the scytonemin pigment was not detected under high light. There was no scytonemin or upregulation of these genes under osmotic stress. The lack of pigment production under high light, despite increased gene expression, suggests a time-dependent delay for pigment production or additional mechanisms or genes that may be involved in scytonemin production beyond those currently known

Gene Expression Patterns Associated with the Biosynthesis of the Sunscreen Scytonemin in Nostoc punctiforme ATCC 29133 in Response to UVA Radiation▿

Under exposure to UV radiation, some cyanobacteria synthesize sunscreen compounds. Scytonemin is a heterocyclic indole-alkaloid sunscreen, the synthesis of which is induced upon exposure to UVA (long-wavelength UV) radiation. We previously identified and characterized an 18-gene cluster associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133; we now report on the expression response of these genes to a step-up shift in UVA exposure. Using quantitative PCR on cDNAs from the N. punctiforme transcriptome and primers targeting each of the 18 genes in the cluster, we followed their differential expression in parallel subcultures incubated with and without UVA. All 18 genes are induced by UVA irradiation, with relative transcription levels that generally peak after 48 h of continuous UVA exposure. A five-gene cluster implicated in the process of scytonemin biosynthesis solely on the basis of comparative genomics was also upregulated. Furthermore, we demonstrate that all of the genes in the18-gene region are cotranscribed as part of a single transcriptional unit

The Response Regulator Npun_F1278 is Essential for Scytonemin Biosynthesis in the Cyanobacterium Nostoc Punctiforme ATCC 29133

Following exposure to long-wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole-alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 co-transcribed genes. A two-component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator Npun_F1278 was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain D1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, D1278 was incapable of producing scytonemin. A phenotypic characterization of D1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid-soluble pigment complement of D1278 compared to the wild type. The mutant, however, had a slower specific growth rate under white light and produced ~2.5-fold more phycocyanin per cell under UVA than the wild type. Since D1278 does not produce scytonemin, this study demonstrates that the response regulator gene Npun_F1278 is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production

Molecular Genetics and Genomic Analysis of Scytonemin Biosynthesis in Nostoc punctiforme ATCC 29133▿

The indole-alkaloid scytonemin is the most common and widespread sunscreen among cyanobacteria. Previous research has focused on its nature, distribution, ecology, physiology, and biochemistry, but its molecular genetics have not been explored. In this study, a scytonemin-deficient mutant of the cyanobacterium Nostoc punctiforme ATCC 29133 was obtained by random transposon insertion into open reading frame NpR1273. The absence of scytonemin under conditions of induction by UV irradiation was the single phenotypic difference detected in a comparative analysis of the wild type and the mutant. A cause-effect relationship between the phenotype and the mutation in NpR1273 was demonstrated by constructing a second scytoneminless mutant through directed mutagenesis of that gene. The genomic region flanking the mutation revealed an 18-gene cluster (NpR1276 to NpR1259). Four putative genes in the cluster, NpR1274 to NpR1271, with no previously known functions, are likely to be involved in the assembly of scytonemin. Also in this cluster, there is a redundant set of genes coding for shikimic acid and aromatic amino acid biosynthesis enzymes, leading to the production of tryptophan and tyrosine, which are likely to be biosynthetic precursors of the sunscreen