RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms2C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms2C is only found in 2-thiocytidine (s2C) containing tRNAs, namely tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU at low abundances. ms2C is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s2C containing tRNA can be methylated to carry ms2C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms2C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach.

cCorynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum ⌬pgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum ⌬pgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized L-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmidencoded ptsG. L-Lysine yields and productivity with C. glutamicum ⌬pgi(pBB1-ptsG) were significantly higher than those with C. glutamicum ⌬pgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTSmediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast L-lysine production.T he Gram-positive, nonpathogenic bacterium Corynebacterium glutamicum is generally known for its employment in the large-scale industrial production of the amino acids L-glutamate and L-lysine (1). Furthermore, C. glutamicum strains for the efficient production of other amino acids such as L-valine have been developed (2, 3). The syntheses of these amino acids require reducing power in the form of NADPH. For example, synthesis of 1 mol L-lysine by either one of the two pathways present in C. glutamicum requires 4 mol of NADPH (4-6), and the synthesis of 1 mol of L-valine requires at least 2 mol of NADPH (7,8). Based on the stoichiometry of the metabolic pathways in C. glutamicum, elementary flux mode analyses indicated that efficient regeneration of the cofactor NADPH is indeed essential to obtain theoretical maximal yields for L-lysine (yield of product on substrate [Y P/S ], 0.82 mol Lys/mol Glc [9]) and L-valine (Y P/S , 0.86 mol Val/mol Glc [10]). C. glutamicum possesses four enzymes for the regeneration of NADPH from NADP: glucose 6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd) of the oxidative part of the pentose phosphate pathway (PPP) (11-13), isocitrate dehydrogenase of the tricarboxylic acid cycle (14,15), and the malic enzyme MalE (16, 17). The last enzyme was show...

The Gram-positive Corynebacterium glutamicum co-metabolizes most carbon sources such as the phosphotransferase system (PTS) sugar glucose and the non-PTS sugar maltose. Maltose is taken up via the ABC-transporter MusEFGK 2 I, and is further metabolized to glucose phosphate by amylomaltase MalQ, maltodextrin phosphorylase MalP, glucokinase Glk and phosophoglucomutase Pgm. Surprisingly, growth of C. glutamicum strains lacking the general PTS components EI or HPr was strongly impaired on the non-PTS sugar maltose. Complementation experiments showed that a functional PTS phosphorelay is required for optimal growth of C. glutamicum on maltose, implying its involvement in the control of maltose metabolism and/or uptake. To identify the target of this PTS-dependent control, transport measurements with 14 C-labelled maltose, Northern blot analyses and enzyme assays were performed. The activities of the maltose transporter and enzymes MalQ, Pgm and GlK were not decreased in PTS-deficient C. glutamicum strains, which was corroborated by comparable transcript amounts of musE, musK and musG, as well as of malQ, in C. glutamicum DptsH and WT. By contrast, MalP activity was significantly reduced and only residual amounts of malP transcripts were detected in C. glutamicum DptsH when compared to WT. Promoter activity assays with the malP promoter in C. glutamicum DptsH and WT confirmed that malP transcription is reduced in the PTS-deficient strain. Taken together, we show here for what is to the best of our knowledge the first time a regulatory function of the PTS in C. glutamicum and identify malP transcription as its target.

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. Working with bacterial mRNA is challenging because it lacks a poly(A)‐tail. However, methods for detecting RNA modifications, both sequencing and mass spectrometry, rely on efficient preparation of mRNA. Here, we compared size‐dependent separation by electrophoresis and rRNA depletion for enrichment of Escherichia coli mRNA. The purification success was monitored by qRT‐PCR and RNA sequencing. Neither method allowed complete removal of rRNA. Nevertheless, we were able to quantitatively analyze several modified nucleosides in the different RNA types. We found evidence for stress dependent RNA modification reprofiling in rRNA, but also several modified nucleosides in the mRNA enriched fractions showed significant changes.

Corynebacterium glutamicum is an environmental bacterium whose natural ability to produce and secrete large amounts of l-glutamate and l-lysine is exploited for the industrial production of these amino acids (