Limits...
Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation.

Flaherty BL, Van Nieuwerburgh F, Head SR, Golden JW - BMC Genomics (2011)

Bottom Line: Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation.We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0116, USA.

ABSTRACT

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.

Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.

Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation. We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development. These data provide detailed information on the Anabaena transcriptome as filaments undergo heterocyst development and begin nitrogen fixation.

Show MeSH

Related in: MedlinePlus

Analysis of the nifB-fdxN-nifS-nifU operon structure, 5' end, and expression levels in response to nitrogen deprivation. RNA-seq read coverage is shown on a log scale across the nifB-fdxN-nifS-nifU operon in heterocyst chromosomes for 0, 6, 12, and 21 hours after the removal of combined nitrogen. The log scale is required to allow depiction of both the low numbers of reads at earlier time points and the large increase in reads at 21 h. The 59,428-bp fdxN element (denoted by the gray triangle) is excised from the chromosome in heterocysts [6], and the break in nucleotide numbering for the vegetative-cell chromosome is marked with «». The position of the previously mapped mRNA 5' end for the operon is at 282 bases upstream of the nifB start codon (marked by the black downward arrow in the 21 h graph) [30].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3141674&req=5

Figure 3: Analysis of the nifB-fdxN-nifS-nifU operon structure, 5' end, and expression levels in response to nitrogen deprivation. RNA-seq read coverage is shown on a log scale across the nifB-fdxN-nifS-nifU operon in heterocyst chromosomes for 0, 6, 12, and 21 hours after the removal of combined nitrogen. The log scale is required to allow depiction of both the low numbers of reads at earlier time points and the large increase in reads at 21 h. The 59,428-bp fdxN element (denoted by the gray triangle) is excised from the chromosome in heterocysts [6], and the break in nucleotide numbering for the vegetative-cell chromosome is marked with «». The position of the previously mapped mRNA 5' end for the operon is at 282 bases upstream of the nifB start codon (marked by the black downward arrow in the 21 h graph) [30].

Mentions: Unlike previous whole-transcriptome analyses in Anabaena, deep sequencing provides information on all transcripts and can help identify 5' and 3' ends and characterize operon structure; however, transcriptional start sites versus processing sites cannot be differentiated with these methods. We used RNA-seq to identify distinct 5' ends for many transcripts, characterized by a set of reads with a common 5' end and the absence of upstream reads (GEO accession #GSE26633). These 5' ends often corresponded with published transcriptional start and/or processing sites; for example psbB, petF, nrrA, psbAI, and nifB, all of which were previously analyzed via primer extension [30-35] (Figure 2 and 3).


Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation.

Flaherty BL, Van Nieuwerburgh F, Head SR, Golden JW - BMC Genomics (2011)

Analysis of the nifB-fdxN-nifS-nifU operon structure, 5' end, and expression levels in response to nitrogen deprivation. RNA-seq read coverage is shown on a log scale across the nifB-fdxN-nifS-nifU operon in heterocyst chromosomes for 0, 6, 12, and 21 hours after the removal of combined nitrogen. The log scale is required to allow depiction of both the low numbers of reads at earlier time points and the large increase in reads at 21 h. The 59,428-bp fdxN element (denoted by the gray triangle) is excised from the chromosome in heterocysts [6], and the break in nucleotide numbering for the vegetative-cell chromosome is marked with «». The position of the previously mapped mRNA 5' end for the operon is at 282 bases upstream of the nifB start codon (marked by the black downward arrow in the 21 h graph) [30].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141674&req=5

Figure 3: Analysis of the nifB-fdxN-nifS-nifU operon structure, 5' end, and expression levels in response to nitrogen deprivation. RNA-seq read coverage is shown on a log scale across the nifB-fdxN-nifS-nifU operon in heterocyst chromosomes for 0, 6, 12, and 21 hours after the removal of combined nitrogen. The log scale is required to allow depiction of both the low numbers of reads at earlier time points and the large increase in reads at 21 h. The 59,428-bp fdxN element (denoted by the gray triangle) is excised from the chromosome in heterocysts [6], and the break in nucleotide numbering for the vegetative-cell chromosome is marked with «». The position of the previously mapped mRNA 5' end for the operon is at 282 bases upstream of the nifB start codon (marked by the black downward arrow in the 21 h graph) [30].
Mentions: Unlike previous whole-transcriptome analyses in Anabaena, deep sequencing provides information on all transcripts and can help identify 5' and 3' ends and characterize operon structure; however, transcriptional start sites versus processing sites cannot be differentiated with these methods. We used RNA-seq to identify distinct 5' ends for many transcripts, characterized by a set of reads with a common 5' end and the absence of upstream reads (GEO accession #GSE26633). These 5' ends often corresponded with published transcriptional start and/or processing sites; for example psbB, petF, nrrA, psbAI, and nifB, all of which were previously analyzed via primer extension [30-35] (Figure 2 and 3).

Bottom Line: Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation.We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0116, USA.

ABSTRACT

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.

Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.

Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation. We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development. These data provide detailed information on the Anabaena transcriptome as filaments undergo heterocyst development and begin nitrogen fixation.

Show MeSH
Related in: MedlinePlus