Teoria del funcional de la densitat DFT

At the first time point (1 h), differentially expressed genes were 869 of the total of 2860 genes. More than half of these differentially expressed genes, 55.12%, were positively regulated and 44.8% were negatively regulated. From the up-regulated genes, some of them were involved in amino acid biosynthesis process and genes encoding products that have the ability to degrade host tissue or digest extracellular proteins were observed with 2 fold changes Table 12. Induction of these genes during the stress response may be attributed to the deactivation of CodY repressive activity as a result of decreased level of the CodY ligand, GTP, during (p)ppGpp synthesis processes (Figure 50) (Geiger et al., 2012, Pohl et al., 2009).

Up-regulation of amino acid biosynthesis and protease genes is in line with previous work that used mupirocin to trigger the stringent response (Reiß et al., 2011, Geiger et al., 2010). Positive induction of these genes might indicate how treated cells cope with amino acid limitation, mimicked by mupirocin treatment, by increasing free cellular amino acids levels through increasing their biosynthetic processes as well as providing them from the surrounding environment.

Moreover, groups of stress adaption and regulation genes were up- regulated, such as spxA (4.5 fold), which plays a role in transcription

stress proteins family which enhances cell survival rate when it exposed to prolonged stress conditions. This has been reported by Drumm to help to establish chronic persistent infection in M. tuberculosis (Drumm et al., 2009).Toxin antitoxin system genes, SAOUHSC 02692(8 fold) and SAOUHSC 02757 (16 fold) were also up-regulated, which is also a classical feature of stress response (Reiß et al., 2011). Furthermore, Hfq gene, which encodes host factor 1 protein, that is involved in stress response and virulence factors in S.aureus was increased 3.6 fold. Most of these genes have been shown to be up-regulated during different stress response conditions, such as oxidative stress, nutrient deprivation, acidic and alkaline shocks, and their activation mechanism is not yet known (Anderson et al., 2006, Reiß et al., 2011). However, recent work has revealed that, during amino acid starvation, the universal stress protein gene SAOUHSC 01819 seems to be up-regulated independently from CodY in S. aureus (Geiger et al., 2012). Increasing transcription of these genes can obviously indicate the huge alteration in genes regulation that is needed to adjust the cell physiology and its molecular functions in order to allow the cell to survive in this condition.

Nevertheless, the highest proportions of the positively expressed genes encoded hypothetical proteins (63.8%), which reflect the lack of knowledge of the stress response process. Rieb and his colleagues have suggested that hypothetical proteins might play a role in adapting cell physiology to stress response (Reiß et al., 2011). Another possibility is that they might contribute in regulating gene transcription, translation and other biological

processes that need to be tuned with the surrounding environmental conditions.

On the other hand, there were 390 significantly differently down-regulated genes at the first time point. Among these genes 63.5% were known to be involved in essential biological activity, such as replication, transcription, translation machineries, RNA and DNA pathway precursors, metabolic pathways and transport systems, which is in agreement with previous authors (Geiger et al., 2012, Geiger et al., 2010a, Reiß et al., 2011) who have reported down-regulation of replication, transcription, translation machinery genes after mupirocin treatment. In B.subtilis most of rRNA gene transcription starts with guanosine nucleotides, which can subsequently be influenced via GTP intracellular levels (Krásný and Gourse, 2004).

Furthermore, the transcriptional start of two rRNA operons in S.aureus have been mapped and have confirmed GTP’s role in initiating their primary promoters, which can explain the GTP effect on their expression (Geiger et al., 2012, Krásný et al., 2008). Down-regulation of these genes is highly conserved in Gram positive bacteria during the stringent response which can justify the stalled cell growth at this time point,1 h , for the treated cells. Furthermore, functions of 36.4% of the genes differentially down- regulated are unknown yet. One can speculate that these genes may either contribute in the molecular functions mentioned above, or may be involved in repressive activity on stress response genes during normal conditions. It is worth mentioning that Anderson and his colleagues claimed that inhibitory concentrations of mupirocin did not increase relA transcript titre appreciably (Anderson et al., 2006). In this work transcription of the gene

that is responsible for ppGpp synthesis, RSH was increased 1.2 relatively to control. Although this was not a significant increase according to the

criteria used here, the hallmark of stringent response ppGpp was detected via direct detection using HPLC after 1 h of sublethal concentration of mupirocin treatment this low level of expression might be related to the level of the stringent response that caused by the sublethal mupirocin. Furthermore, up-regulation of genes that are involved in amino acid biosynthesis, transcription regulation, stress response, virulence factors and the down-regulation of genes that contribute to transcription,

translation and replication are in agreement with previous observations using bactericidal concentrations of mupirocin (Anderson et al., 2006, Geiger et al., 2012, Reiß et al., 2011), which strongly supports the fact that the sublethal concentration of mupirocin used here was capable of

triggering the stringent response during at least 1 h of treatment which has not been reported in previous studies and supports the HPLC results in chapter 4.