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Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

Irla M, Neshat A, Brautaset T, Rückert C, Kalinowski J, Wendisch VF - BMC Genomics (2015)

Bottom Line: Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements.The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons.Several of the genes related to methylotrophy had highly abundant transcripts.

View Article: PubMed Central - PubMed

Affiliation: Genetics of Prokaryotes, Faculty of Biology & Center for Biotechnology, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany. mairla@cebitec.uni-bielefeld.de.

ABSTRACT

Background: Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends.

Results: Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.

Conclusion: The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

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Related in: MedlinePlus

Absolute number of identified transcription start sites in correlation to the length of their 5′-untranslated regions (5′-UTRs). The 1,642 TSSs located upstream or in coding direction of known CDSs were used to determine the length of the 5′-UTRs for each CDS. The 5′-UTR length was calculated as the distance between an identified TSS to the next TLS. The absolute number of TSSs is grouped in 5 bp intervals of 5′-UTR lengths (1–5, 6–10, 11–15 etc.), whereas the most distant right bar represents all 5′-UTRs longer than 500 bases.
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Fig2: Absolute number of identified transcription start sites in correlation to the length of their 5′-untranslated regions (5′-UTRs). The 1,642 TSSs located upstream or in coding direction of known CDSs were used to determine the length of the 5′-UTRs for each CDS. The 5′-UTR length was calculated as the distance between an identified TSS to the next TLS. The absolute number of TSSs is grouped in 5 bp intervals of 5′-UTR lengths (1–5, 6–10, 11–15 etc.), whereas the most distant right bar represents all 5′-UTRs longer than 500 bases.

Mentions: The distances of 1,642 TSSs belonging to the leader regions of protein-coding genes to the next TLSs revealed a median 5′-UTR length of 51 nt (Figure 2). More than 99% of the transcripts analyzed in this study have 5′-UTR leader sequences of ≥ 10 nt. 22% of transcripts showed a 5′-UTR length of 26–35 nt and 25% of transcripts contain leader sequences longer than 100 nt, which suggests that they might contain regulatory RNA structures [27]. Only six transcripts (<0.5%) possess a 5′-UTR shorter than 10 nt, which implies that they do not retain a (complete) RBS. However, no leaderless transcript was found for B. methanolicus MGA3 since all transcripts had 5′-UTRs ≥ 2 nt. Only two transcripts showed leaders shorter than 5 nt. The corresponding genes are BMMGA3_05465 encoding the CtaG protein which participates in the formation of active cytochrome caa3 and sigF encoding the sporulation-specific sigma factor σF [28].Figure 2


Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

Irla M, Neshat A, Brautaset T, Rückert C, Kalinowski J, Wendisch VF - BMC Genomics (2015)

Absolute number of identified transcription start sites in correlation to the length of their 5′-untranslated regions (5′-UTRs). The 1,642 TSSs located upstream or in coding direction of known CDSs were used to determine the length of the 5′-UTRs for each CDS. The 5′-UTR length was calculated as the distance between an identified TSS to the next TLS. The absolute number of TSSs is grouped in 5 bp intervals of 5′-UTR lengths (1–5, 6–10, 11–15 etc.), whereas the most distant right bar represents all 5′-UTRs longer than 500 bases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4342826&req=5

Fig2: Absolute number of identified transcription start sites in correlation to the length of their 5′-untranslated regions (5′-UTRs). The 1,642 TSSs located upstream or in coding direction of known CDSs were used to determine the length of the 5′-UTRs for each CDS. The 5′-UTR length was calculated as the distance between an identified TSS to the next TLS. The absolute number of TSSs is grouped in 5 bp intervals of 5′-UTR lengths (1–5, 6–10, 11–15 etc.), whereas the most distant right bar represents all 5′-UTRs longer than 500 bases.
Mentions: The distances of 1,642 TSSs belonging to the leader regions of protein-coding genes to the next TLSs revealed a median 5′-UTR length of 51 nt (Figure 2). More than 99% of the transcripts analyzed in this study have 5′-UTR leader sequences of ≥ 10 nt. 22% of transcripts showed a 5′-UTR length of 26–35 nt and 25% of transcripts contain leader sequences longer than 100 nt, which suggests that they might contain regulatory RNA structures [27]. Only six transcripts (<0.5%) possess a 5′-UTR shorter than 10 nt, which implies that they do not retain a (complete) RBS. However, no leaderless transcript was found for B. methanolicus MGA3 since all transcripts had 5′-UTRs ≥ 2 nt. Only two transcripts showed leaders shorter than 5 nt. The corresponding genes are BMMGA3_05465 encoding the CtaG protein which participates in the formation of active cytochrome caa3 and sigF encoding the sporulation-specific sigma factor σF [28].Figure 2

Bottom Line: Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements.The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons.Several of the genes related to methylotrophy had highly abundant transcripts.

View Article: PubMed Central - PubMed

Affiliation: Genetics of Prokaryotes, Faculty of Biology & Center for Biotechnology, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany. mairla@cebitec.uni-bielefeld.de.

ABSTRACT

Background: Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends.

Results: Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.

Conclusion: The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Show MeSH
Related in: MedlinePlus