OBJETIVOS DEL CUESTIONARIO COMO PROCEDIMIENTO DE MUESTREO

2.1. Expression levels of fructose operon in different conditions

Expression of the fructose operon in P. putida KT2440 was measured with a fruB′-′lacZ translational fusion in plasmid pMCH1 using the classical β-galactosidase protocol in different conditions (fructose, glucose and succinate, Fig. 30). As expected the highest levels of expression were obtained on fructose as carbon source. Surprisingly, on glucose the activity level albert low was 10 times higher than on succinate (23 ± 8 vs 2 ± 1 U Miller). This interesting observation will be studied in depth in the next section. It should be noted that in vivo metabolic measurements of FBP and F1P (Fig. 31) were consistent with our gene expression data since the highest levels of both metabolites were obtained on fructose as carbon source (FructoseF1PFBP via fruBKA). As was mentioned in Chapter 1 the presence of significant FBP levels on glucose and succinate can be explained by the presence of the discussed cyclic ED pathway. On the other hand, presence of low amounts of F1P on glucose and succinate (see Fig. 31) is intriguing since to the best of our knowledge, there are not known enzymes for producing F1P during growth on other carbon source than fructose.

Figure 30. fruBKA promoter activity in P. putida as measured by translational fusions to lacZ gene in plasmid pMCH1. As expected the highest PTS^Fru activity levels were measured on fructose as carbon source (545 ± 56 U Miller). On the other hand, PTS^Fru system expression was very low on glucose (23 ± 8 U Miller) and succinate (2 ± 1 U Miller). A schematic diagram is shown of the fruB′^-′lacZ gene fusion fragment in plasmid pMCH1. The negative control corresponds to P. putida KT2440 electroporated with pMCH3 (empty vector, red).

Figure 31. In vivo concentrations of F1P and FBP in P. putida. F1P and FBP were measured in P. putida by HPLC-MS on succinate (black), glucose (orange) and fructose (purple). Highest levels of F1P and FBP were detected on fructose where PTS^Fru is active.

Fructose intake is performed by FruAB to produce F1P and then this compound is transformed to FBP by FruK.

But, for the most part, the results of gene expression and metabolic data allow us to conclude that the PTS^Fru system is expressed only on fructose where the extracellular sugar is phosphorylated to F1P and subsequently phosphorylated again to generate FBP.

2.2. Activity of PTS^Fru system on glucose as carbon source is due to inherent fructose contamination on glucose preparations

From β-galactosidase activity measurements a higher activity of the fructose operon promoter on glucose (23 ± 8 U Miller) was noticeable with respect to succinate (2 ± 1 U miller). This observation was reproducible not only with the Miller protocol (Fig. 30) but also with the protocol based on chemiobioluminescence (Fig. 32b). This higher activity of fructose operon could explain previous observations made in our laboratory suggesting that FruB is active during growth on glucose and is able to phosphorylate PtsN in this condition (for details see section 4 in this Chapter). The notion that FruB can phosphorylate to PtsN on glucose is quite surprising and not trivial. We hypothesize two possible explanations for this phenomenon: [i] that glucose preparations could be contaminated with small amounts of fructose that can activate the system or [ii] that that the glycolitic activity in P. putida may be enough to derepress fruB expression, i.e., that FBP or other glycolytic metabolite could be transformed to F1P by an unknown enzyme. To adress these possibilities, first we constructed a fruB mutant, which is unable to transport fructose as carbon source (Fig. 32a). If fructose is present in the medium as a contaminant the sugar is unable to enter the cell in this mutant. We measured the expression of the operon in fruB strain as well in wild type strain with the β-Galacto-Light Plus^TM system (Fig. 32b) which is more reliable for measuring low amounts of β-galactosidase.

As can be observed in Figure 32b, promoter activity decreased in the ΔfruB strain to same levels on glucose as the wild strain on succinate, suggesting that the higher activity of the fruBKA operon on glucose with respect to succinate is due to the contamination of this sugar with fructose. However, it is still surprising that small amounts of fructose in presence of a preferential carbon source such as glucose may increase ten times the promoter activity in respect to the baseline. To clarify this observation we measured the promoter activity on glucose (10 mM, 99.5% purity, SIGMA) with increasing concentrations of fructose in the culture medium (0, 10 μM and 100 μM). These very low concentrations of fructose are equivalent to mixtures of glucose:fructose of 99.9:0.1 and 99:1 respectively.

Figure 32. Growth and fructose operon promoter activity of P. putida fruB mutant. (a) Growth of P. putida KT2440 (purple) and ΔfruB mutante (orange) in minimum medium with fructose as the only carbon source. (b) Expression of fructose operon in wild type strain and ΔfruB mutant on succinate (green) and glucose (purple) as carbon sources. Experiments were performed with β-Galacto-Light Plus^TM system. A schematic diagram is shown of the fruB′^-′lacZ gene fusion fragment in plasmid pMCH1.

The negative control corresponds to P. putida KT2440 electroporated with pMCH3 plasmid (empty vector).

Results (Fig. 33) showed clearly that very low concentrations of fructose in the medium are enough to activate the expression of the fructose operon. Therefore, the small quantities of F1P that are produced due to fructose contamination are recognized by Cra, leading to slight derepression of the system. This observation is consistent with our in vitro studies of Chapter 2 in which we demonstrated the high affinity of the regulator by F1P. In contrast, the activity of the fructose operon promoter in fruB strain remains unchanged, due to the inability of the strain to internalize fructose and increase the intracellular concentrations of F1P.

Figure 33. Activity of fructose operon promoter in P. putida KT2440 (purple) and fruB mutant (green) on glucose 10 mM (0.2%) with increasing concentrations of fructose. Experiments were performed with the β-Galacto-Light Plus^TM system. Note that the activity of the fructose operon in ΔfruB strain is not significantly altered by the addition of fructose in the medium. A schematic diagram is shown of the fruB′^-′lacZ gene fusion fragment in plasmid pMCH1. The negative control corresponds to P. putida KT2440 electroporated with pMCH3 plasmid (empty vector, red).