Global mRNA decay and 23S rRNA fragmentation in Gluconobacter oxydans 621H

Abstract

BackgroundGluconobacter oxydans is a strictly aerobic Gram-negative acetic acid bacterium used industrially for oxidative biotransformations due to its exceptional type of catabolism. 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. Additionally, central carbon metabolism is characterized by the absence of a functional glycolysis and absence of a functional tricarboxylic acid (TCA) cycle. Due to these features, G. oxydans is a highly interesting model organism. Here we analyzed global mRNA decay in G. oxydans to describe its characteristic features and to identify short-lived mRNAs representing potential bottlenecks in the metabolism for further growth improvement by metabolic engineering.ResultsUsing DNA microarrays we estimated the mRNA half-lives in G. oxydans. Overall, the mRNA half-lives ranged mainly from 3 min to 25 min with a global mean of 5.7 min. The transcripts encoding GroES and GroEL required for proper protein folding ranked at the top among transcripts exhibiting both long half-lives and high abundance. The F-type H+-ATP synthase transcripts involved in energy metabolism ranked among the transcripts with the shortest mRNA half-lives. 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. The mRNA decay analysis also revealed an apparent instability of full-length 23S rRNA. 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.ConclusionsThe very short mRNA half-lives of the H+-ATP synthase, which is likely responsible for the ATP-proton motive force interconversion in G. oxydans under many or most conditions, is notably in contrast to mRNA decay data from other bacteria. Together with the short mRNA half-lives and low expression of some other central metabolic genes it could limit intended improvements of G. oxydans’ biomass yield by metabolic engineering. Also, further studies are needed to unravel the multistep process of the 23S rRNA fragmentation in G. oxydans.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5111-1) contains supplementary material, which is available to authorized users.

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“…The resulting cell pellets were directly frozen in liquid nitrogen and stored at − 80 °C until RNA preparation. The preparation of RNA, cDNA synthesis, hybridization using Agilent’s 4-plex DNA microarray platform, and data analysis were carried out as described (Kranz et al 2018 ).…”

Library Selection with a Randomized Repertoire of (βα)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression

“…Total RNA was isolated using the RNeasy system (Qiagen) and quality-checked as described previously. 28 The synthesis of Cy3-or Cy5-labeled cDNA and two-color hybridization to DNA microarrays was carried out, as described. 28 Custom-made 4 × 44K DNA microarrays were obtained from Agilent Technologies and were designed using Agilent's eArray platform (https://earray.chem.agilent.com/ earray).…”

“…28 The synthesis of Cy3-or Cy5-labeled cDNA and two-color hybridization to DNA microarrays was carried out, as described. 28 Custom-made 4 × 44K DNA microarrays were obtained from Agilent Technologies and were designed using Agilent's eArray platform (https://earray.chem.agilent.com/ earray). The array design comprises oligonucleotides for the annotated genes of E. coli MG1655 and other bacterial genomes, as well as Agilent's control spots.…”

“…The pelleted cells were shock-frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated using the RNeasy system (Qiagen) and quality-checked as described previously . The synthesis of Cy3- or Cy5-labeled cDNA and two-color hybridization to DNA microarrays was carried out, as described .…”

The industrial versatility of Gluconobacter oxydans: current applications and future perspectives

“…Recent developments rely on comprehension of metabolic features and genetic manipulation of G. oxydans. Research has been undertaken (Mientus et al 2017;Kranz et al 2018;Zhou et al 2019a;Qin et al 2021) to elucidate G. oxydans metabolism, which could pave the way for the improved performance of strains. For example, the genome of a strain with high oxidative power and low growth yield (G. oxydans ATCC 621H) was compared with that of a strain with good growth yield (G. oxydans DSM 3504) in order to understand how to boost growth and increase productivity (Kostner et al 2015).…”