DERECHO DE TRANSMISIÓN DERECHO DE REPRESENTACIÓN

With an aim to study what happens within cells when the two known thiol substrates of CydDC are exogenously added to cells lacking CydDC (as in the cytochrome bd-I restoration experiments), transcriptional analysis of cydDC cells were performed with the addition of GSH and cysteine to the growth medium. In this situation, cytochrome bd-I restoration is expected, and a large number of changes to gene expression are likely to be due to a gain of function of the oxidase. For this reason, transcriptional changes in a cydDC cydAB mutant with the addition of GSH and cysteine were also studied.

As a large number of genes were identified, the results of the two microarray experiments were processed to produce a more focused subset of genes. This was achieved first by using TFinfer to deduce which transcription factors have activities that are significantly affected by the thiols. This permitted an understanding of the environmental stimuli imposed on cells by thiol addition. A major aim of this study was to examine the effects of thiol addition beyond the restoration of cytochrome bd-I, so transcription factors were grouped; TFs affected in both cydDC and cydDC cydAB cells (considered to be independent of cytochrome bd-I restoration), and TFs that were only significantly affected in cydDC cells but not cydDC cydAB cells (considered to be cytochrome bd-I restoration dependent). Lists of genes that are controlled by these two groups of TFs were listed in Tables 4-1 and 4-2, only genes that were shown to be significantly up- or down- regulated were included. These two tables were then used to create a metabolic map (Figure 6-2).

Changes in gene expression that were independent of cytochrome bd-I restoration included a repression of genes involved in motility, the TCA cycle, dihydroxyacetone degradation, as well as amino acid biosynthesis and transport. An induction of genes related to respiration was observed including appBC that encodes cytochrome bd-II and hyaABCDF genes that encode hydrogenase-I. Placing these changes in gene expression on a metabolic map (Figure 6-2), showed that the repression of genes encoding enzymes of the TCA cycle are accompanied by a repression of genes encoding enzymes that feed into the TCA cycle too.

Addition of thiols repressed expression of TCA cycle genes via the ArcA transcription factor. The same genes were up-regulated in the cydD vs. wild type

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microarray, the authors of the latter study proposed that misfolded proteins and fatty acids are broken down to feed into the TCA cycle in order to restore the balance of metabolic flux (Holyoake et al., 2016). NMR metabolomic data showed that in the absence of cydD, levels of succinate were elevated while those of fumarate were simultaneously depleted, which suggested an increase in succinate dehydrogenase activity. Succinate dehydrogenase catalyses the oxidation of succinate to fumarate and simultaneously reduces ubiquinone to ubiquinol (Figure 3-12). Taken together this indicates that loss of CydDC-exported thiols affects the redox status of the quinone pool and that TCA cycle genes are up-regulated to aid in re-balancing of the redox status. This is interesting as ArcB senses respiratory growth conditions via the redox state of the quinone pool using a thiol-based redox switch (Iuchi and Lin, 1988; Malpica et al., 2004; Alvarez et al., 2013) and ArcA, the corresponding response regulator controls expression of cydABX genes encoding cytochrome bd-I, it suggests that in wild type cells, CydDC-mediated thiol export affects the transcription of the genes encoding cytochrome bd-I, adding a new level to the relationship between the two protein complexes. Previously it had been shown that the regulation of cydDC and cydABX operons are not coordinated (Section 1.2), these results however suggest some form of relationship between the level of activity of CydDC and transcription of cydABX. The repression of motility genes suggests that swarming motility is reduced by the addition of thiols. Which is surprising considering that Pittman et al. 2002 showed that exogenous cysteine restores motility defects of a cydD mutant, (most likely by permitting the correct disulphide folding of FlgI). It had been expected that addition of both cysteine and GSH would restore assembly of the flagella suprastructure by balancing the redox environment of the periplasm. It is therefore possible that contrary to a re-balancing of the periplasmic redox environment, the amount of thiols added is surplus to requirement and instead the periplasm is becoming more reduced than is observed in wild type cells. It would be of interest to observe the motility of cydDC cells in the presence of exogenous thiols to determine the actual effect of both thiols. A larger repression of motility genes was observed after the addition of thiols to cydDC cydAB cells when compared to cydDC cells. Looking at the previously performed cydB vs. wild type microarray data, flagella genes were repressed and accompanied by a reduced swarming motility (Shepherd et al., 2010). The authors suggest that repression

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of motility genes is an energy conserving mechanism in the face of reduced energetic efficiency of cytochrome bd-II. It is thus likely that the difference between repression of motility genes is due to the lack of cytochrome bd-I restoration in the cydDC cydAB double mutant and the larger reliance upon cytochrome bd-II as shown by the increased regulation of appBC in the cydDC cydAB background.

Changes in gene expression that are considered dependent upon the restoration of cytochrome bd-I include a repression of genes of the 2-methylcitrate cycle, this cycle overlaps with the TCA cycle and utilises propionate to produces pyruvate. Northern- blot analysis has shown than unlike acnB, prpD is exclusively transcribed during growth on propionate (Brock et al. 2002). This suggests that in the absence of exogenous glutathione and cysteine, cydDC cells utilise propionate as a carbon source.

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Figure 6-2. Model for the processes influenced by CydDC substrates in the periplasm: environmental signals and downstream effects

The gene transcript levels and transcription factor activities that are up- and down- regulated in the cydDC GSH/cysteine vs. cydDC and cydDC cydAB GSH/cysteine vs. cydDC cydAB are shown in red and blue, respectively. The gene transcript levels and transcription factor activities that are up- and down-regulated only in the cydDC GSH/cysteine vs. cydDC microarray are shown in orange and purple, respectively. The green and purple lines denote steps in the 2-methylcitrate cycle and -oxidative of fatty acids. Dotted lines show potential environmental stimuli responded to. Abbreviations: Ac-CoA, Acetyl CoA; Dha, dihydroxyacetone; GSH, reduced glutathione; TF, transcription factor.

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