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. 2009 Jul;191(14):4639-46.
doi: 10.1128/JB.00134-09. Epub 2009 May 8.

Gene Expression Patterns Associated With the Biosynthesis of the Sunscreen Scytonemin in Nostoc Punctiforme ATCC 29133 in Response to UVA Radiation

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Gene Expression Patterns Associated With the Biosynthesis of the Sunscreen Scytonemin in Nostoc Punctiforme ATCC 29133 in Response to UVA Radiation

Tanya Soule et al. J Bacteriol. .
Free PMC article

Abstract

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 the 18-gene region are cotranscribed as part of a single transcriptional unit.

Figures

FIG. 1.
FIG. 1.
(A) N. punctiforme genomic region associated with scytonemin biosynthesis (not drawn to scale). The arrows represent ORFs and indicate the transcriptional direction, while the double lines indicate separation of the gene clusters in the genome. The hatched ORFs are scytonemin core genes, the stippled ORFs are genes associated with aromatic amino acid biosynthesis, the white ORFs are miscellaneous genes that do not fit any particular functional category, the gray ORFs are putative regulators, and the black ORFs are genes in the scytonemin five-gene satellite cluster. (B) Scytonemin-associated genomic region in Nodularia spumigena CCY 9414. Homologous ORFs are labeled as in panel A (clarified where appropriate) and are annotated to match the name given for the N. punctiforme homolog.
FIG. 2.
FIG. 2.
Cotranscription assay for pairs of contiguous genes. Shown is an agarose gel of PCR products from reverse-transcribed RNA of UVA-induced cells, which targets several genes in the 18-gene cluster. N. punctiforme genomic DNA (+) was run on the gel next to the corresponding PCR products of reverse-transcribed RNA (cDNA, in duplicate reactions) to verify the correct product size. The lane at the left is the molecular size standard (in bp). The presence of a product of appropriate size was taken as preliminary evidence of cotranscription. The bands within the rectangles were excised for sequencing confirmation of the product identity. Products are labeled by the names of the adjacent genes that were targeted. Note that a PCR product linking aroB-trpE (primer set a is shown in this image) was not detected on this gel; hence, evidence of cotranscription was not detected in the first two RT-PCR attempts.
FIG. 3.
FIG. 3.
Differential induction of scytonemin biosynthesis in cultures used for gene expression analyses. Solid line, mean specific content in the control series (white light only); dashed line, mean specific content in cultures under white light supplemented with UVA. The error bars are standard deviations for three independent determinations.
FIG. 4.
FIG. 4.
Gene expression dynamics of scytonemin-associated genes in response to UVA radiation, based on qPCR of reverse-transcribed RNA extracts. The data are presented as the change in gene expression of the UVA-exposed cultures with respect to the white-light-alone control cultures over time. Time zero indicates initial exposure to UVA. (A) The scytonemin assembly core genes. (B) Genes in the scytonemin gene cluster involved in tryptophan and tyrosine biosynthesis. (C) Miscellaneous genes in the main scytonemin gene cluster. (D) The genes in the five-gene satellite cluster. (E) Expression time course of the shikimic acid genes. (F) Housekeeping copies (*) of tyrA and trpA (solid lines) compared to their homologs in the scytonemin gene cluster (dashed lines).
FIG. 5.
FIG. 5.
Cotranscription of the 18-gene cluster associated with scytonemin biosynthesis in N. punctiforme. Genes in the leftmost column are represented by arrows drawn to ORF length scale indicating the transcriptional direction. In the second column from the left, the brackets (drawn to scale) represent genomic regions targeted in adjacent-gene RT-PCR. Multiple brackets represent multiple primer pairs targeting adjacent genes. Up to three different primer pairs were used to confirm a negative result between contiguous genes. The third column shows gene pairs whose cotranscription was demonstrated by RT-PCR and confirmed by sequencing. The last column depicts the overall transcriptional unit deduced from the previous data.
FIG. 6.
FIG. 6.
Multiple primer pairs used for repeated RT-PCR between aroB (NpR1267) and trpE (NpR1266) to determine cotranscription between the two genes. Primer pairs are represented by vertical blocks with similar shading, and complementary forward and reverse primers (sets a, b, and c) are joined by brackets, with the product size in parentheses.

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