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

Abstract

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.

Impact of CRISPR interference on strain development in biotechnology

“…As ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12 and S. thermophilus have been employed for genome editing at elevated temperatures [65,68], future construction and application of catalytically dead Thermo‐dCas9 variants may widen the application of CRISPRi screen to more thermophilic or hyperthermophilic bacteria and archaea. In the case of B. methanolicus , three genes differing in expression strength [69] were repressed. Reduced spore formation, but increased biofilm formation, was observed when targeting stage zero sporulation protein A gene spo0A , perturbed growth with mannitol occurred upon targeting mannitol‐1‐phosphate 5‐dehydrogenase gene mtlD , and hydrogen peroxide dismutation was impaired as a consequence of catalase gene repression supporting functional annotation of these genes [30].…”

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle

“…Since purified protein corresponding to the annotated Ta of B. methanolicus MGA3 (Ta MGA3 ) showed no activity when expressed in E. coli, however, crude extracts of MGA3 did show Ta activity, the available transcriptome data from RNA-sequencing [26] were used to search for a possible transcription start site (TSS) upstream of the start codon of our proposed Ta coding sequence ta MGA3put (Fig. 3).…”

“…Indeed, a putative TSS preceded by a − 10 region (TTTCAA(T)) and a − 35 region (TTGAAA) were found. The − 35 region represents the consensus sequence [26], while the − 10 region shares only a low similarity with the B. methanolicus consensus sequence (TATAAT) [26].…”

Methanol as carbon substrate in the bio‐economy: Metabolic engineering of aerobic methylotrophic bacteria for production of value‐added chemicals

“…Interestingly, this bacterium has three genes encoding active Mdhs; one of them encoded on the natural plasmid pBM19 and two encoded on the chromosome. All three genes encode active Mdhs with broad substrate specificities for alcohols, and combined transcriptome and proteome analyses indicated that the plasmid‐encoded variant is upregulated and most abundant in the cells during methylotrophic growth . The alternative B. methanolicus strain PB1 has a different organization and regulation of its mdh genes, and this was accompanied by a significantly different growth profile during fed‐batch methanol fermentation .…”