PERFIL LOGISTICO DE COLOMBIA HACIA CANADA

In this Thesis, we analyzed the metabolic fluxes in the central carbon metabolism of isogenic PTS mutant strains of P. putida (Chapter 1) growing on a PTS sugar (fructose) or on a non-PTS sugar (glucose). Two main conclusions regarding the influence of PTS proteins could be drawn from those data: [i] that the PTS mutants did not affect the carbon flux distribution of the pathways implicated in the upper metabolism (EDP, EMP and PPP) with the exception of fruB mutants which are unable to transport

fructose in the cell, and [ii] that PtsN is involved in the regulation of the pyruvate shunt in the central metabolism, irrespective of the carbon source used and of its phosphorylation state.

The regulation of PtsN on pyruvate shunt was observed on first instance through flux analysis (see Figs. 13 and 14), and then confirmed with enzymatic assays (Fig. 16). In both approaches, a higher activity of the malic enzyme and pyruvate carboxylase were observed in the ptsN mutant, as well as in the ptsN/ptsO mutant with respect to the wild type strain suggesting a repressor role of PtsN on the pyruvate shunt. Moreover, our results suggest that the regulatory influence is not dependent of the phosphorylation status of the protein, as:

[i] No effects on the metabolic fluxes were observed in a ptsP/fruB mutant (see Fig. 12).

This strain lacks both EI components of the PTS and therefore does not support phosphorylation of PtsN, thus EIIA^Ntr would not be phosphorylated under any condition (Pfluger and de Lorenzo, 2008), and

[ii] No changes in malic enzyme activity were observed in ptsNHA and ptsNHE mutants (see Fig. 17). These strains contain a ptsN allele with changes in the triplet corresponding to the phosphorylable position H68 of PtsN (ptsNHA HisxAla and ptsNHE HisxGlu).

These genetic modifications generate that ptsNHA strain has in any time the PtsN protein in dephosphorylated form whereas ptsNHE strain has this protein in a state that resembles to the phosphorylated species. The fact that no changes were observed in malic enzyme activity in a scenario where the PtsN is fully phosphorylated or dephosphorylated suggest that the observed effect of PtsN can be traced down to the sole presence/absence of the protein irrespective of its phosphorylation state.

While most studies suggest that the dephosphorylated form of PtsN is responsible for its regulatory functions (Begley and Jacobson, 1994; Lee et al, 2005; Luttmann et al, 2009;

Pfluger-Grau et al, 2011; Segura and Espin, 1998), the fact that the PtsN protein regulates the activity of an enzyme independently of their state of phosphorylation is not new. Recently Choi et al. (2010) reported in Salmonella a direct protein-protein interaction between EIIA^Ntr and the response regulator SsrB, a component required for Salmonella virulence. This work showed with in vivo and in vitro experiments that EIIA^Ntr interact with SsrB and it performs its regulatory function in a fashion entirely

independent of the phosphorylation state. On the other hand, other studies have reported regulatory functions of PtsN that depends on the phosphorylated form (Cases et al, 1999; Hayden and Ades, 2008). With these premises, the simplest interpretation is that the state of phosphorylation is one of the features that PtsN uses to carry out its multiple regulatory functions. Therefore, we believe that the various phenotypes observed in PtsN mutants may be due to any of the following: [i] the phosphorylation state, [ii] the amount of protein expressed and, [iii] the ability of PtsN to interact with other macromolecules to form protein complexes.

Perhaps, the two forms of EIIA^Ntr (phosphorylated and nonphosphorylated) have specialized in controling different sets of proteins, so the regulatory functions are carried out through several/different mechanisms. According to the literature (Lee et al, 2007;

Choi et al, 2010; Pfluger-Grau et al, 2011) it seems clear that one of these mechanisms is through direct protein-protein interaction suggesting that PtsN may have a role in scaffolding multiprotein complexes.

Our experiments do not allow to deduce whether this regulation is due to direct protein-protein interaction of PtsN with the pyruvate shunt enzymes or by a secondary effect due to altered intracellular metabolite composition. For instance, it is known that in Bacillus subtilis acetyl-CoA has an inducing effect on the activity of the pyruvate carboxylase (Diesterhaft et al, 1973). However, in other bacteria, as e.g. in Pseudomonas citronellosis, Methanobacterium thermautotrophicum, and Clostridium glutamicum, no dependence on the presence of acetyl-CoA has been observed on Pyruvate carboxylase (Attwood, 1995; Jitrapakdee and Wallace, 1999). Recently, it was shown that the non-phosphorylated form of PtsN binds to the AceE component of PDH in vitro, downregulating its activity (Pfluger-Grau et al, 2011). Therefore, a ptsN mutant should have higher fluxes towards acetyl-CoA, if not even higher amounts of this metabolite.

Analyzing the metabolic net fluxes only a slight increase is predicted for the reaction catalyzed by the PDH complex in the ptsN mutants (see Figs. 13, 14). However, measurements of the PDH activity in cell free extracts of P. putida wild type and ptsN mutant showed ~2-fold higher activity of the enzyme complex in the absence of PtsN (Pfluger-Grau et al, 2011). Thus, we cannot exclude that the higher activity of the pyruvate carboxylase is at least to some extend an effect of its induction by acetyl-CoA.

Even when we cannot provide a detailed functional mechanism, we have evidence that

the pyruvate shunt is induced in the absence of PtsN. As mentioned above, the pyruvate shunt has been related with the production of additional amounts of OAA for gluconeogenesis, amino acids biosynthesis (aspartate, isoleucine, threonine), and energy production via the citric acid cycle (Diesterhaft et al, 1973). The fact that PtsN repress the pyruvate shunt (and therefore modulates the pool of oxaloaceate) indicates that PtsN would indirectly regulate the biosynthesis of amino acids.

Although the observed differences in ptsN⁺/ptsN^- strains in the fluxes and enzymatic assays are somewhat low, small changes in central metabolic reactions can lead to significant changes in other regulatory processes. Therefore, considering the robustness of the central metabolism it is very remarkable that PtsN can regulate its activity. We are just at the beginning of understanding some of the signals and regulatory devices orchestrating the metabolic flux distribution through the pyruvate shunt node and thereby guiding the carbon flow to the different metabolic modules.

In summary, we produced evidence that PtsN is one player in the complex machinery that is responsible for carbon flux distribution in the central metabolism of P. putida.

Our data suggest a role of PtsN in the fine-tuning of the central carbon metabolism helping to adjust the metabolic fluxes to satisfy the anabolic and energetic demands of overall cell physiology.

4. F1P is the one and only effector of the transcriptional regulator Cra