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...

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.

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.

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 (

Running title: Coordination of PTS sugar uptake and central metabolism in C. glutamicum 22 Content category: Biochemistry and Physiology 23Abbreviations: cdm -cell dry mass; WT, wild-type. 24 ABSTRACT 28Corynebacterium glutamicum co-metabolizes most carbon sources, such as glucose and 29 sucrose. Uptake of those sugars by the PTS involves a glucose-and a sucrose-specific 30 permease EII Glc (ptsG) and EII Suc (ptsS), respectively. Block of glycolysis by deletion of pgi 31 (encodes phosphoglucoisomerase) redirects glucose-driven carbon flux towards pentose 32 phosphate pathway. C. glutamicum Δpgi grows poorly with glucose but has unaffected, good 33 growth with sucrose. However, addition of glucose to sucrose-cultivated C. glutamicum Δpgi 34 immediately arrested growth via inhibition of the EII Suc -mediated sucrose uptake and 35 reduction of ptsS-mRNA amounts. Kinetic analyses revealed that sucrose uptake inhibition in 36 C. glutamicum Δpgi took place within 15 s after glucose addition. We show that inhibition of 37 PTS-mediated sucrose uptake occurs as direct response to glucose-6-P accumulation. 38Moreover, addition of non-PTS substrates, which are metabolized to glucose-6-P such as 39 maltose or glucose-6-P itself (uptake was enabled by heterologously produced UhpT), led to 40 similar growth and sucrose uptake inhibition as glucose addition. Despite EII Glc not being 41 involved in uptake of these substrates, negative effects on sucrose uptake after addition of 42 maltose and glucose-6-P were absent in the EII Glc -deficient strain C. glutamicum ΔpgiΔptsG. 43These results show that the ptsG-encoded EII Glc is part of a novel mechanism for perception of 44 intracellular glucose-6-P accumulation and instantaneous inhibition of EII Suc -mediated 45 sucrose uptake in C. glutamicum. This novel mode of control of PTS activity by an early 46 glycolytic metabolite probably allows efficient adaptation of sugar uptake to the capacity of 47 IMPORTANCE 50Coordination of substrate uptake and metabolism are a prerequisite for efficient co-utilization 51 of substrates, a trait typical for the Gram-positive C. glutamicum. Sucrose uptake via the PTS 52 permease EII Suc in this organism immediately was inhibited in response to intracellular 53 accumulation of the glycolysis intermediate glucose-6-phosphate. This inhibition depends 54 exclusively on the presence but not activity of the PTS permease EII Gluc . Thus, C. glutamicum 55 possesses a novel, immediate, and PTS-dependent way to control and coordinate both uptake 56 and metabolization of multiple substrates by monitoring of their metabolic levels in the cell. 57This offers new insights and interesting concepts for a further rational engineering of this 58 industrially important production organism and exemplifies a putative general strategy of 59 bacteria for the coordination of sugar uptake and central metabolism. 60 61 sucrose metabolism. As characterized here, sucrose in this bacterium is exclusively taken up 126 6 via the sucrose-specific, ptsS-encoded EII Suc with a ...

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.