- However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data.. - Results: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia.. - We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. - Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. - We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. - beijerinckii NCIMB 8052 in the latter phases of cultivation.. - Conclusions: We provided a complex analysis of the C. - We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - However, the knowledge of the genomic sequence itself does not provide any information regarding the gene regu- lation, which is crucial to improvements of the strains for industrial application. - In this paper, we present transcriptome dynamics during the cultivation of the promising butanol producer, C. - Until now, only the transcription of six selected genes involved in sporulation and solvent production was studied for this strain using RT-qPCR, yet the study supported the theory that solventogenesis is not regulated in the same way in all solventogenic clostridia [11]. - Here, we further investi- gate the specifics of the strain C. - The obtained transcriptome data includes the whole life cycle of the strain and therefore covers changes in metab- olism, i.e. - Moreover, the RNA-Seq technology has allowed us to study not only the temporal transcription of any gene but also to explore the accuracy of the current genome annotation. - Compared to the tran- scription profiling of the strain C. - To increase the robustness and validity of the experiment, each of the time-points was represented by three biological replicates rather, than verification using qPCR [13].. - metabolites formation with acid production in the first period followed by solvents formation (see Fig. - 1d) while a sharp decrease in pH occurred, so only acidogenic, non-sporulating and mostly motile cells were expected to be present in the sample. - The third sample set was with- drawn during the period of the most progressive rise in pH, suggesting a high rate of reutilization of the acids, to- gether with solvent formation. - However, there was no apparent increase in the production of acids in the fermentation data. - A relatively high amount of double-stained cells was present in the culture at all stages. - The staining pattern of the Clostridium culture at different time-points revealed dynamic changes in proportion of active cells within the first 13 h, with a detectable drop at the period with the lowest pH (the sixth hour), thus supporting the presumption that cells are highly-stressed by the presence of organic acids together with a low pH (when values slightly below pH 5 were reached). - The only noticeable changes in the OD measurements are the two slowdowns during the acidogenesis/solventogenesis transient states. - The FC data clearly shows that culture viability had already started to decline at around the 13th hour, which cor- responds to the apparent decrease in the number of regulated genes from that time.. - Surprisingly, after a decrease in the acid/solvent switch, the glu- cose consumption increased again and accompanied the T3–T4 transition state with the highest number of regulated genes.. - Although series A consisted of reads that were 50 bp long and series B and C consisted of reads that were 75 bp long, the whole series could be processed in the same way.. - The only following sequence-filtering step was the re- moval of the remaining residual rRNA contamination, even after the rRNA depletion. - (b) Flow cytometry – the distribution of cells within the population according to their fluorescence pattern for combined staining using PI and CFDA. - Values represent the mean of the biological replicates and error bars represent the standard deviations. - Nevertheless, in order to cover the expression of duplicated genes that were present in the C. - beijerinckii NRRL B-598 genome, the reads map- ping to multiple loci were also included in the gene ex- pression analysis (see Table 2). - However, the contribution of such reads was down-weighted in the expression ana- lysis depending on the number of times they mapped to the genome, so the sum of the total number of reads stayed intact.. - In the current RefSeq genome (NZ_CP out of the 5230 genes predicted by NCBI PGAP [14]. - Although none of the 198 pseudogenes overlapped with another pseudo- gene, 18 pseudogenes overlapped with genes directly and another 73 pseudogenes were at a distance from genes that could be covered by a single read. - those in the same operon, were covered by a single transcript. - Although genes in the third group were fully covered, this coverage consisted of two or more overlapping transcripts. - This group consisted of pseudo- genes that were transcribed and active genes that were possibly misidentified as pseudogenes due to errors in the genome assembly. - Only 11 genes were not transcribed at any of the six sam- pling points. - While the transcriptional profiles from the first three time-points (T1, T2, and T3) cor- respond to the transcription of the C. - Reproducibility of the experiment was verified using three biological replicates and by checking the expres- sion of six selected genes whose transcription profiles were observed during a previous study by Kolek et al.. - We explored differential expression of all genes and pseudogenes with detectable transcription among adja- cent time-points, in order to analyze changes in the transcription of particular genes over the whole fermen- tation process (see Fig. - The complete results of the differential expression ana- lysis, including log2fold changes and adjusted p-values, are available in Additional file 6.. - Similarly, 666 out of the 747 down-regulated genes were down-regulated uniquely between T3 and T4 (see Fig. - However, some of the uniquely up-regulated genes were down-regulated between another couple of time points and some of the uniquely down-regulated genes were up-regulated during another Table 3 Coverage of pseudogenes by transcripts. - 3 Analysis of the transcriptome reproducibility. - (b) 2D representation of the normalized expression data after dimensionality reduction by t-SNE to compare the samples collected at the six time-points (T1 – T6) coded by different colors. - other incomplete phage was more active with average RPKM = 86, but none of the genes were differentially expressed during the fermentation. - All genes were car- ried by a negative strand and 14 out of the 17 genes were covered by a single transcript, including one pseudo- gene (X276_RS17860) with a missing stop codon. - Cytometric data enabled the calculation of a specific rate of glucose consumption related to metabol- ically active cells in the population during different time periods of the cultivation, together with information about the overall culture condition.. - The majority of reads mapped to the genome without any mismatches and support an overall high quality of the genome assembly. - However, the transcription of the six selected genes under the same cultivation con- ditions was monitored using qRT-PCR in study of C.. - In the mentioned study by Kolek et al. - [11], an increase in ex- pression was observed in mid-cultivation for spoIIE and sigG and in the second part of cultivation for spoVD. - Moreover, the expression profiles of the remaining genes also showed the same pattern. - Butyrate kinase (buk, X276_RS1200) transcription was maximal at the begin- ning of the cultivation, decreased in time, and rose slightly at the end of cultivation. - The expression of ald and spo0A increased in the first third of cultivation and for ald also at the end of cultivation. - Moreover, the reproducibility of the experiment was supported by utilization of three bio- logical replicates and their high similarity in the sampling points visualized using tSNE in Fig. - samples in the original high-dimensional space, i.e.. - distances from the normalized expression profiles to the distances of the samples in the reduced space, i.e. - The position of the samples in the 2D space was then optimized until the samples with similar expres- sion profiles were placed close to each other and samples with very different expression profiles were at a further distance from each other. - Two main clusters, distinguish- ing samples from the first and the second half of the experiment, were present. - While the similarity of the rep- licates from the first cluster was supported mainly by the first coordinate tSNE1, the similarity in the other cluster was supported by the second coordinated tSNE2.. - beijerinckii NRRL B-598 (see Additional file 4) on the genome-wide scale were different, especially in the later phase of cultivation. - This could have been caused by structural reorganizations in the genomes of both strains or by differences in gene regulatory mechanisms. - beijerinckii NCIMB 8052 in the later phases could lie in the different phenotypic behavior of both strains at this stage. - This high activity was caused by genes transcribing into cell wall binding proteins, in the first region by the gene X276_RS24890 with average RPKM while in the second region by the gene X276_RS25120 with average RPKM 1.8∙10 4 . - The most noticeable change in the transcription on the genome wide scale was captured between T3 and T4 time-points when the highest number of differentially expressed genes was detected. - The massive change in the gene expression, which can be spotted in Fig. - Further transition between T4 and T5 could also show an entry to the irreversible phase of sporulation, in which two independent gene regulations were estab- lished in the mother cell and pre-spore and sporulation must be completed. - Overall culture attenuation after T4 is apparent from both a decrease of specific glucose con- sumption (Table 1) and from cytometric data that con- firmed the gradual increase in the proportion of inactive cells. - An increase in the specific rate of glucose consumption, corresponding to highly regulated genes coding for COG functional group C (energy production and conversion) (see Additional file 8), was detected together with an apparently improved viability.. - After the 13th hour COG D and COG L related to cell cycle control and replication respectively were not differentially expressed which was fully consistent with the decrease in cell growth and declining culture viability supporting a hypothesis of the switch of a highly proliferating cul- ture into a new strategy, securing genus preservation via ensuring a complete sporulation process. - Simultaneously COG F for nucleotide metabolism transport are up-regulated within the first two compared time sets and down-regulated in the latter two. - acetobutylicum down-regulated in the stationary phase.. - On the other hand, an increase in the latter stages is probably the result of culture phenotype desynchronization when all the cell types are again present, including motile cells. - Furthermore, some cells within the whole population might have undergone a massive change in energy me- tabolism and solvent production, which is associated with the switch of different genes in the period of transi- tion between T3 and T4 time-points. - The solvent forma- tion and acidogenesis/solventogenesis switch are usually explained as a stress response induced by accumulation of acids in the cultivation medium and pH decrease.. - However, the whole population situation was no longer critical at time-point T4 and the lower concentration of acids in the medium might have induced another metabolic change, this time associated with the direct formation of butanol/acetone from glucose. - However, a signifi- cant advantage of the reduced risk of low pH out- weighed this discomfort. - 1b) supports the hypothesis that not all cells in the population exhibit the same phenotype to cope with changing unfavorable living conditions. - Although they may represent large frac- tion of the strain-specific DNA sequences [29], the strain C. - beijerinckii NRRL B-598 is a promising butanol producer, we lack a precise descrip- tion of mechanisms within its fermentation metabolism, which prevent us from further modifications of the strain for industrial applications. - This allowed us to verify the reproducibility of the experiment and to gather the RNA-Seq dataset with the currently highest dynamic range available among solventogenic clostridia. - We analyzed the latest RefSeq annotation of the genome and confirmed its high accuracy. - Surprisingly, this change was not directly connected to the acidogenic/solventogenic change, nor the sporulation initiation but rather to a massive change in the energy metabolism and solvent production in a part of cell population as we discuss based on auxiliary HLPC and FC data.. - Multiforce 1 l bioreactors (Infors HT) with 630 ml TYA broth and agitation at 200 rpm were used for batch cultivation of the strain at 37 °C. - Quality and integrity of the samples were assessed using the Agilent RNA 6000 Nano Kit (Agilent) with the Agilent 2100 Bioanalyzer (Agilent). - The quality assessment after steps of the RNA-Seq reads processing was done using FastQC in combination with MultiQC to summarize the reports across all samples [32]. - Phage regions in the C. - A count table was reconstructed using the R/Bio- conductor featureCounts function included in the Rsubread package [45] and RPKM were computed using the R/Bioconductor edgeR package [46]. - Additional file 4: Circular plots showing average coverage of the genome by RNA-Seq reads in all six time points. - The RNA-Seq sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP033480.. - Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. - Complete genome sequence of the nitrogen-fixing and solvent-producing Clostridium pasteurianum DSM 525. - Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. - Pseudogenes in the ENCODE regions: Consensus annotation, analysis of transcription, and evolution. - Bacteriophage infections in the industrial acetone butanol (AB) fermentation process. - PHASTER: a better, faster version of the PHAST phage search tool
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