This title appears in the Scientific Report : 2014 

The 6 RNA of $\textit{Corynebacterium glutamicum}$
Pahlke, Jennifer (Corresponding Author)
Biotechnologie; IBG-1
2014
Jülich Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag 2014
II, 144 S.
Heinrich-Heine-Universität Düsseldorf, Diss., 2014
978-3-95806-003-6
Dissertation / PhD Thesis
ohne Topic
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health 76
The 6C RNA family is a class of small RNAs (sRNAs) with as yet unknown function present and conserved within the phylum Actinobacteria, including the Gram-positive soil bacterium $\textit{Corynebacterium glutamicum}$. In this work studies were conducted to characterize properties of the 6C RNA from $\textit{C. glutamicum}$ ATCC 13032 and to shed light on possible physiological roles in this species. The following results were obtained: (i) The 6C RNA was found to be present in $\textit{C. glutamicum}$ throughout growth in CGXII medium with glucose and exhibited RNA level changes not greater than two-fold. RNA stability tests using rifampicin revealed a 6C RNA half-life of >120 min, showing that it is a very stable sRNA. Quantitation of the 6C RNA transcripts yielded about 325 and about 260 molecules per cell in the exponential and in the stationary growth phase, respectively, which is much more compared to numbers of individual mRNAs. (ii) 6C RNA pull-down experiments using crude protein extracts of $\textit{C. glutamicum}$ revealed 3-phosphoglycerate kinase (PGK) as a candidate protein interaction partner. However, an interaction of 6C RNA with purified PGK could not be confirmed yet by electrophoretic mobility shift assays, possibly indicating that a further factor is required. (iii) The 6C RNA is not essential for $\textit{C. glutamicum}$ and the growth of a 6C RNA deletion mutant was very similar to the wild type under various cultivation conditions tested, except when treated with the SOS response-inducing antibiotic mitomycin C (MMC), which caused a growth defect of the Δ6C RNA mutant. Reporter fusion constructs with promoters of the LexA-regulated genes $\textit{recA, recN, cglIM}$ and $\textit{dnaE2}$ indicated that the SOS response level in the Δ6C RNA mutant is lower in the stationary growth phase compared to the wild type. This leads to the suggestion that the 6C RNA might be involved in the SOS response or plays a more general role in the stress defense of $\textit{C. glutamicum}$. Complementation studies of the Δ6C RNA mutant by plasmid-born expression of the 6C RNA from its native promoter revealed an unexpected and exceptional growth phenotype in the presence of MMC. Quantitation of 6C RNA revealed an about five-fold increased level in the complemented strain. (iv) The increased 6C RNA level resulted in an elongated cell shape under standard conditions and a dramatically branched cell shape in the presence of MMC, indicating that the 6C RNA might be involved in the regulation of cell division in $\textit{C. glutamicum}$. (v) 6C RNA deletion as well as 6C RNA overexpression led to changes in the global mRNA expression profile. Notably, the WhiB-like transcriptional regulator WhcD had a decreased mRNA level in the 6C RNA overproducer. WhcD is homologous to the septation and cell division-related regulator WhmD in $\textit{Mycobacterium smegmatis}$. A Δ$\textit{whcD}$ mutant showed a reduced growth and a strongly altered cell morphology resembling that of the 6C RNA overproducer in the presence of MMC. Thus, the homologous proteins indeed seem to be functionally equivalent in both actinobacteria. These results suggest that the 6C RNA may have an impact on the expression of $\textit{whcD}$ or its mRNA stability, thereby playing a role in some aspect of septation and cell division in $\textit{C. glutamicum}$.