- It incompletely oxidizes a wide variety of carbohydrates regio- and stereoselectively in the periplasm using membrane-bound dehydrogenases with accumulation of the products in the medium. - As a consequence, only a small fraction of the carbon and energy source enters the cell, resulting in a low biomass yield. - RNAseq analysis revealed low expression levels for genes of the incomplete TCA cycle and also the mRNA half-lives of several of those were short and below the global mean. - Further analysis of the ribosome-associated rRNA revealed a 23S rRNA fragmentation pattern exhibiting new cleavage regions in 23S rRNAs which were previously not known.. - Conclusions: The very short mRNA half-lives of the H + -ATP synthase, which is likely responsible for the ATP-proton motive force interconversion in G. - Also, further studies are needed to unravel the multistep process of the 23S rRNA fragmentation in G. - 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. - L-sorbose, a precursor for vitamin C production, dihydroxyacetone, a chemical used for tanning lotions, or 6-amino-L-sorbose, a precursor of the antidiabetic drug miglitol [1–6]. - Genome sequencing revealed the absence of genes coding for enzymes of the central me- tabolism, such as 6-phosphofructokinase, succinyl-CoA synthetase, and succinate dehydrogenase [11]. - To overcome these hindrances, metabolic engineering was performed to complete the TCA cycle by introducing heterologous genes for succin- ate dehydrogenase and succinyl-CoA synthetase into the genome with simultaneous deletion of the genes for the membrane-bound and soluble glucose dehydrogenase, thus abolishing periplasmic and cytoplasmic glucose oxi- dation [12, 13]. - These steps led to an increase of the biomass yield on glucose by up to 60%, thereby reducing the costs for biomass formation. - In living cells, the abundance of mRNAs is a result of the balance between gene expression and degradation of mRNAs. - Generally, a correlation between the half-lives of transcripts and the cellular function of the encoded proteins was observed in these studies. - It is dependent on secondary structures of the 5′ and 3′ un- translated regions, posttranscriptional modifications such as polyadenylation, abundance of ribonucleases, presence of cleavage sites recognized by ribonucleases, interaction with small regulatory RNAs, as well as location of the mRNAs in the cell [20, 24].. - The mRNA decay analysis additionally revealed an apparent instability of the full-length 23S rRNA. - Enrichment of ribosomes and fur- ther analysis of the associated rRNAs uncovered that the 23S rRNA is fragmented in G. - Cells were grown in 500 mL shaking flasks with three baffles containing 50 mL of the mannitol medium (30 ° C, 140 rpm). - The DNA microarray analysis aimed at the determin- ation of the time-dependent mRNA level changes in G.. - For pairwise comparisons, the A mix and the B mix of the Agilent Spike-In Kit (Agilent Technologies) was used to spike t0 and tx RNA samples accordingly.. - This factor was calculated for each hybridization based on the log base 2 of the non-normalized ratio of median values of the Spike-In (+)E1A_r60_1 and (+)E1A_r60_a20 RNAs each having 32 array spots randomly scattered (Agilent. - Accordingly, the normalization factor was calculated that the log base 2 of the normalized ratio of median values of the 1:1 control RNAs is 0 on average. - A normalized ratio value was in- cluded in the calculation of the average of the triplicates for each time point if the following quality filter was ful- filled by the spot data (GenePix Pro 6.0): i) Flags ≥0 and ii) signal/noise ≥3 for Cy5 (F635Median / B635Median) or Cy3 (F532Median / B532Median). - The resulting data matrix with the average values of the four time points was used to calculate the mRNA half-lives and R 2 in Excel (Microsoft) by linear fit as described for E. - Sequencing reads were trimmed and strand-specifically mapped to the genome reference (CP000009) and the five plasmids pGOX1 to pGOX5 (CP000004 – CP000008) using the RNAseq analysis tool of the CLC Genomics Workbench (Qiagen Aarhus A/S) to determine absolute FPKM values [30]. - Therefore, 0.5 mL of the prepared cell lysate was injected into an ÄKTA pure FPLC system (GE Healthcare Life Sciences) equipped with two CIM®. - 5% of the average gene coverage and visualized with Origin (OriginLab).. - Global mRNA half-lives in G. - Ri- fampicin inhibits the activity of the RNA polymerase [32], thereby enabling the measurement of the time- dependent decrease of mRNA levels by turnover in the absence of mRNA de novo synthesis. - With 250 μg/mL and 200 μg/mL of rifampicin a notable drop of the cell density within 30 min was observed, suggest- ing cell damage and lysis to some extent (Fig. - With 150 μg/mL of rifampicin a growth inhibition within 30 min was observed without a drop of the cell density (Fig. - The 23S rRNA band was generally much weaker compared to the 16S rRNA and almost disappeared after 15 min, suggesting a decay or further processing of the expected mature full-length 23S rRNA transcripts with and 2711 nt in G.. - normalization, as it was done in other mRNA decay stud- ies where the rRNAs of the host were considered as stable enough to be used for data normalization [18]. - c Schema of the experimental design. - Subsequently, the mRNA half-lives can be estimated via linear regression of the log ratios over time. - Based on the four time points of the analysis and filtering for R 2 >. - 0.7 of the linear fit [18], we obtained half-life values for 1193 transcripts. - For example, genes with shorter half-lives may exhibit the decrease in the mRNA level at earlier sampling times, while at later sampling times there is no further decrease for several reasons including inherent limits of the DNA microarray technology used. - Therefore, many genes exhibiting this type of mRNA decay kinetics will likely not fulfill a well-intentioned R 2 criterion for fil- tering results of the linear regression analysis when later time points are included. - Depending on the range of the absolute sampling times it is legitimate to check the out- come of half-life estimations by linear regression with a focus on earlier time points. - Together, this resulted in mRNA half-life estimations for of the protein-cod- ing genes of G. - Overall, the calculated mRNA half-lives of the 2500 genes mainly ranged from approximately 2 min to 25 min. - In previous studies, correlations between the stability of mRNAs and the functional group of the gene products were observed . - Independent of the RNA abundance reflected by the FPKM values, on the individual transcript level the shortest mRNA half-lives were found for GOX1807 (2.2 min) annotated as GTP pyrophosphokinase involved in (p)ppGpp metabolism, for GOX0730, GOX1845, and GOX1426 (2.3 min) encoding hypothetical proteins, for GOX0666 and GOX2394 (2.4 min) involved in ferric iron uptake and purine synthesis, and for GOX1113 (2.4 min) encoding subunit A of an F 1 F o -type ATP syn- thase (Additional file 1: Table S1). - As reported previously, this ATP synthase is an ortholog of the H + -translocating ATP synthases of Acetobacter pasteurianus, Gluconacetobacter diazotro- phicus and other α-proteobacteria [34]. - 2 Distribution of mRNA half-lives. - Transcripts of the molecular chaperones GroES (GOX1901) and GroEL (GOX1901) exhibited high expression values as well as long half-lives. - The operons of the F 1 F o -type ATP synthase encoded by atpBEFF ’ (GOX1110 – 13) and atpHAGDC (GOX1310 – 14. - belong to the operons/genes with the shortest mRNA half-lives in G. - In comparison, the transcripts of the second F 1 F o -type ATP synthase encoded by GOX2167 – 75. - Among the genes of the incomplete TCA cycle. - operon (GOX2167–75) which is an ortholog of the Na + -translocating F 1 F o -type ATP synthases present al- ways in addition to the H + -ATP synthase in the archaea Methanosarcina barkeri and M. - a ) For the F 1 F o ATP synthase encoded by two operons the data of the operon GOX1310–14, which was close to the top 20, were also included. - Their transcript half-lives close to 4 min follow the trend of the inverse relationship between FPKM and half-live. - The transcripts of the PQQ-dependent mDHs 1 (GOX1857), 3 (GOX1441), and 4 (GOX0516), sorbitol dehydrogenase (GOX2094–. - oxydans has an unusual central metabolism, we were also interested in the stability of transcripts en- coding enzymes of the central carbon metabolism in- cluding the pentose phosphate pathway (PPP), the Entner-Doudoroff pathway (EDP), and the pyruvate me- tabolism. - of the most-stable transcripts in G. - The tran- scripts of the PPP genes exhibited half-lives below the global mean ranging from 3.8 min to 5.1 min. - Genes of the incomplete TCA cycle ex- hibited the lowest apparent expression in the central metabolism based on the FPKM values, with the aconitase transcript (GOX1335) as the most stable (9.3 min) above the global mean, and with the transcripts of odhB (GOX1073, 3.4 min) encoding dihydrolipoamide succinyl transferase (E2) of the 2-oxoglutarate dehydrogenase, mqo (GOX2070, 4 min) encoding malate:quinone oxidoreduc- tase, and lpd (GOX2292, 4 min) encoding dihydrolipoa- mide dehydrogenase exhibiting the shortest half-lives (Fig. - oxydans RNA sam- ples always raised questions on the quality of the prepared RNA, for example in DNA microarray studies. - 4 mRNA half-lives and FPKM expression values for genes of the central carbon metabolism. - The sum of the protein content in the elution fractions of the four peaks typically comprised 85% to 95% of the total protein applied to the column (Additional file 1: Table S4). - To narrow down the regions of fragmentation in the 23S RNA transcripts, the ribosome-associated RNA was sequenced via next-generation sequencing and the paired-end reads were mapped to the sequences of the 16S and 23S rRNA gene loci from G. - To identify possible frag- mentation positions based on the mapping coverage we searched for regions with less than 5% coverage of the average coverage for the entire gene locus (Fig. - For the other three 23S rRNA genes, the positions of two of the three regions differed by maximal two nucleotides:. - 5 Graphical representation of the 23S rRNA (a, b) and 16S rRNA (c) mapping coverage. - a Mapping coverage of the 23S rRNA locus GOX1319 (2711 nt). - b Mapping coverage of the 23S rRNA locus GOX1159 (2709 nt) which indicates an additional fragmentation site. - This shows the presence of the T and suggests that GOX1159 was very likely transcribed into RNA. - For the 16S rRNA genes we did not find such low coverage regions indicating the presence of the mature full-length transcripts in the ribosomes (Fig. - Only for the second and fourth fragmentation position an extension of the low coverage regions by 55 nt and by 31 nt was observed.. - In this study, we estimated the mRNA half-lives in G.. - oxydans 621H on a global scale and detected and ana- lyzed fragmentation of the 23S rRNA transcripts. - With a mean half-life of 3 min the mRNAs of the H + -ATP synthase were among the transcripts with the shortest half-lives.. - Comparative DNA microarray analysis of the effects of. - oxydans revealed an approxi- mately 2-fold decreased expression of the H + -ATP syn- thase and 2- to 3-fold increased expression of the Na + -ATP synthase [34]. - Actually, one would expect that genes of the energy metabolism including ATP synthases are among the medium or most stable transcripts due to their classification as housekeeping genes [50]. - In α-proteobacteria, for example Rhizobia- ceae, Bradyrhizobiaceae, and Rhodobacteraceae, pro- cessing of an IVS located close to the 5′-end of the 23S rRNA likely also involves RNase III in an early step. - The multistep process generates a fragment of approximately 130 nt from the 5′-end, which is sepa- rated by the IVS from the main segment of the 23S. - One of the three is found at nt 1139–1160 within a region without conservation, where also one IVS is present in the 23S rRNAs of S.. - Since these var- iations are located within the fragmentation region and not at their ends or in the flanking regions, the results suggest further rRNA processing of the frag- ments after the cleavage. - A more general role for rRNA fragmentation was speculated recently in a study of the mammal naked mole-rat in the context of translational fidelity [55]. - In the. - It was speculated that rRNA fragmentation may change the folding or dynamics of the large ribosomal sub- unit, altering the rate of GTP hydrolysis and/or interaction of the large subunit with tRNA during accommodation, thus finally affecting the fidelity of protein synthesis. - Rather, a quick mRNA turnover for fast adaptation of the cell to environmental changes is made possible by the shorter half-lives of highly expressed genes. - The very short mRNA half-lives of the H + -ATP synthase, which is likely responsible for the ATP-proton motive force inter- conversion in G. - Together with the short mRNA half-lives and the relatively low expression of some genes of the incomplete TCA cycle, which exhibited the lowest expression values in the central carbon metabol- ism, these could be bottlenecks in G. - Our Illumina sequencing and mapping analysis of the RNA fragments isolated from enriched ribosomes re- vealed three potential fragmentation regions which were previously not known from 23S rRNA fragmentation stud- ies in other bacteria. - FPKM expression values and mRNA half-lives.. - mRNA half-lives of operons and monocistronic transcripts. - The authors thank Ilka Maria Axmann for helpful discussion and Alexander Vogel for deposition of the RNAseq data in the ENA archive and at the Gluconobacter portal www.gluconobacterfactory.de.. - The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. - The funding organization did not influence the design of the study, the collection, analysis, and interpretation of data, and the writing of the manuscript.. - The source of the wild type Gluconobacter oxydans 621H strain (DSM 2343) used in this study was the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).. - Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans. - Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. - DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate.. - Influence of oxygen limitation, absence of the cytochrome bc(1) complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology. - Characterization of the N-ATPase, a distinct, laterally transferred Na + -translocating form of the bacterial F-type membrane ATPase. - Mutational analysis of the pentose phosphate and Entner-Doudoroff pathways in Gluconobacter oxydans reveals improved growth of a Dedd Deda mutant on mannitol. - Fragmentations of the large- subunit rRNA in the family Rhizobiaceae
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