Improving the genome annotation of the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5′-ends of primary transcripts

Schwientek, Patrick; Neshat, Armin; Kalinowski, Jörn; Klein, Andreas; Rückert, Christian; Schneiker-Bekel, Susanne; Wendler, Sergej; Stoye, Jens; Pühler, Alfred

doi:10.1016/j.jbiotec.2014.03.013

Cited by 16 publications

(12 citation statements)

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“…2 ). A similar 5′ UTR length distribution can be found in Actinoplanes sp., C. glutamicum and B. methanolicus [ 15 , 16 , 43 ]. However, leaderless transcripts are rare in P. riograndensis SBR5 as the present study only revealed 2 transcripts (P.riograndensis_final_2873 and P.riograndensis_final_5691) to be leaderless in this firmicute (Additional file 3 : Table S3).…”

Section: Discussionmentioning

confidence: 99%

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Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

et al. 2017

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BackgroundThe plant growth promoting rhizobacterium Paenibacillus riograndensis SBR5 is a promising candidate to serve as crop inoculant. Despite its potential in providing environmental and economic benefits, the species P. riograndensis is poorly characterized. Here, we performed for the first time a detailed transcriptome analysis of P. riograndensis SBR5 using RNA-seq technology.ResultsRNA was isolated from P. riograndensis SBR5 cultivated under 15 different growth conditions and combined together in order to analyze an RNA pool representing a large set of expressed genes. The resultant total RNA was used to generate 2 different libraries, one enriched in 5′-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5′-ends of the primary transcripts was used to identify 1082 transcription start sites (TSS) belonging to novel transcripts and allowed us to determine a promoter consensus sequence and regulatory sequences in 5′ untranslated regions including riboswitches. A putative thiamine pyrophosphate dependent riboswitch upstream of the thiamine biosynthesis gene thiC was characterized by translational fusion to a fluorescent reporter gene and shown to function in P. riograndensis SBR5.ConclusionsOur RNA-seq analysis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and will be a valuable basis for differential RNA-seq analysis of this bacterium.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4235-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

“…The scarcity of leaderless transcripts in the transcriptomes of the low-GC Gram-positives B. methanolicus [ 16 ] and P. riograndensis contrasts with the large proportion of leaderless transcripts present in the high-GC Gram-positive actinobacteria Actinoplanes sp. (20%) [ 43 ] and C. glutamicum (33%) [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

et al. 2017

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“…The distance distribution showed a maximum at 26–35 nucleotides (22%) before decreasing exponentially with increasing 5′-UTR lengths. This distance distribution is typical for bacteria that feature 5′-UTRs shorter than 100 bp and peak at a length of around 25–35 nt [17,49,50]. Similarly, the low number of leaderless transcripts in B. methanolicus MGA3 (6 with a 5′-UTR ≤ 10) is in accordance with in silico predictions for 953 bacterial genomes that revealed only about 25% of species belonging to the phylum Firmicutes possess leaderless transcripts and in these species 2.4% to 16.6% of all sequences are leaderless [51].…”

Section: Discussionmentioning

confidence: 99%

“…Actinoplanes sp. SE50/110 (20%) [49] . The exact translation mechanism of leaderless transcripts without a complete RBSs is still not fully elucidated, however, in those transcripts close proximity of the start codon to the 5' terminus is important for both ribosome binding and translation [53].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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BackgroundBacillus 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.ResultsAnalysis 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1239-4) contains supplementary material, which is available to authorized users.

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“…Recently, the complete genome sequence of Actinoplanes sp. SE50/110 was reported, which has a 9.24-Mb circular chromosome hosting 8270 putative protein coding sequences (CDSs) with a G+C content of 71.32% [18] , [19] . Comparative transcriptomics and proteomics were also applied to evaluate the different transcription and expression of acb cluster and other genes of SE50/110 cultivated in different media [20] , [21] , [22] , [23] .…”

Section: Introductionmentioning

confidence: 99%

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

Zhao

Xie

Peng

et al. 2017

Synthetic and Systems Biotechnology

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The α-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp. and used as a potent drug in the treatment of type-2 diabetes. In order to improve the yield of acarbose, an efficient genetic manipulation system for Actinoplanes sp. was established. The conjugation system between E. coli carrying ØC31-derived integrative plasmids and the mycelia of Actinoplanes sp. SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture (mycelia and E. coli), quantity of recipient cells, donor-to-recipient ratio and the concentration of MgCl2, which resulted in a high conjugation efficiency of 29.4%. Using this integrative system, a cloned acarbose biosynthetic gene cluster was introduced into SE50/110, resulting in a 35% increase of acarbose titer from 2.35 to 3.18 g/L. Alternatively, a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm) was constructed for gene inactivation, which has a conjugation frequency as high as 0.52%. Meanwhile, almost all 5-flucytosine-resistant colonies were sensitive to apramycin, among which 75% harbored the successful deletion of targeted genes. Using this replicating vector, the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated, and component C was eliminated as detected by LC-MS. Based on an efficient genetic manipulation system, improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

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Improving the genome annotation of the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5′-ends of primary transcripts

Cited by 16 publications

References 60 publications

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

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

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

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