In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.
To examine the role of chronic (in)activity on muscle carnosine (MCarn) and how chronic (in)activity affects MCarn responses to β-alanine supplementation in spinal-cord injured athletes, sixteen male athletes with paraplegia were randomized (2:1 ratio) to receive β-alanine (n=11) or placebo (PL, n=5). They consumed 6.4 g‧d-1 of β-alanine or PL for 28 days. Muscle biopsies of the active deltoid and the inactive vastus lateralis (VL) were taken before and after supplementation. MCarn in the VL was also compared with the VL of a group of individuals without paraplegia (n=15). MCarn was quantified in whole muscle and in pools of individual fibers by High-performance Liquid Chromatography. MCarn was higher in chronically inactive VL vs. well-trained deltoid (32.0±12.0 vs. 20.5±6.1 mmol‧kg-1 DM; p=0.018). MCarn was higher in inactive vs. active VL (32.0±12.0 vs. 21.2±7.5 mmol‧kg-1 DM; p=0.011). In type-I fibers, MCarn was significantly higher in the inactive VL than in the active deltoid (38.3±4.7 vs. 27.3±11.8 mmol‧kg-1 DM, p=0.014). MCarn increased similarly between inactive VL and active deltoid in the β-alanine group (VL: 68.9±55.1%, p=0.0002; deltoid: 90.5±51.4%, p<0.0001), with no changes in the PL group. MCarn content was higher in the inactive VL than in the active deltoid and the active VL, but this is probably a consequence of fiber type shift (type I to type II) that occurs with chronic inactivity. Chronically inactive muscle showed an increase in MCarn after BA supplementation equally to the active muscle, suggesting that carnosine accretion following β-alanine supplementation is not influenced by muscle inactivity.
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