In our previous work, we established that the glyoxylate shunt, the TCA cycle and acetate uptake by acetyl-CoA synthetase are more active in E.coli B than in E.coli K. By implementing the system biology approach, we showed that in addition to what we knew, other metabolic pathways are active differently in the two strains. These are glucoenogensis, sfcA shunt, ppc shunt, glycogen biosynthesis and fatty acid degradation. It was found that in E.coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconegensis enzyme phosphoenolpyruvate synthase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF), cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations, these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis genes (fbp, pckA, and ppsA) lead to glycogen accumulation and are constitutively active in E. coli BL21 regardless of glucose feeding strategy, our assumption was that the Cra protein (catabolite repressor/activator, formally called FruR) is responsible for this effect. To further understand this phenomenon we decided to check the effect of the Cra on the growth and acetate production by E. coli B and K, and evaluate the gene transcription pattern between the Cra positive and the Cra negative strains by using microarrays amd by following the behavior of the mutant organism. The deletion of the cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT). Conclusions: The transcriptional changes caused by the deletion of cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone;rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion caused transcription inhibition of the bet operon in E. coli K-12 (JM109) but did not affect this operon transcription in E. coli B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the E. coli B (BL21) strain more resistant to environmental changes.The results indicated that in E. coli B there is no considerable difference between the Cra positive strain and the Cra negative strain, acetate production is a bit lower in the Cra positive strain but the growth kinetics and the glucose consumption are similar. In E. coli K, however, there is a significant difference, the Cra negative strain is stop growing at a concentration about one third of the final cell concentration of the Cra positive strain, as for the acetate production, there is no difference between the two strains. There is also an indication that the E. coli K cra negative lost it ability to grow in the presence of higher concentration of salt. We applied two additional approaches for the possible understanding of the differences between the two strains. One approach was to follow mutated BL21 strains and the other one to look at the role of small RNA, a regulatory RNA. The results of our first approach were included in last year report, they indicated that perhaps acetate is not only a by-product of carbon metabolism;it is possible that it also plays a role in cellular metabolism. It is likely that there are alternative acetate production pathways in the cells. Concerning the small RNA approach we decided to concentrate on the Sgrs a small RNA which affects the expression of the glucose transporter IICBGLU by inactivating the corresponding mRNA ptsG. The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encoding the glucose transporter IICBGlc), was studied in both E. coli K-12 (MG1655 and JM109) and E. coli B (BL21). It is known that the transcription level of sgrS increases when E. coli K-12 (MG1655 and JM109) is exposed to the non-metabolized glucose alpha methyl glucoside (MG) or when the bacteria with a defective glycolysis pathway is grown on glucose. The increased level of sRNA SgrS downregulates ptsG by inhibiting and destabilizing ptsG mRNA and consequently reduces the level of the glucose transporter IICBGlc. The suggested trigger for this action is the accumulation of the corresponding glucose-phosphate. In the course of the described work, it was found that E. coli B (BL21) and E. coli K-12 (JM109) responded similarly to MG: both strains increased SgrS transcription and reduced ptsG transcription. However, the two strains responded differently to high glucose concentration (40 g/L). E. coli B (BL21) responded by increasing sgrS transcription and reducing ptsG transcription and E. coli K-12 (JM109) did not respond to the high glucose concentration, and therefore transcription of sgrS was not detected and ptsG mRNA level was not affected. The results suggest that E. coli B (BL21) tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization. Based on this information, it was assumed that over expression of SgrS enables E. coli B (BL21) to reduce its acetate excretion by slowing the glucose transport. When SgrS was over-expressed in both E. coli K-12 strains by inducing expression from an external plasmid, it was possible to reduce their acetate excretion levels to those seen in E. coli B. This observation opens a new approach towards controlling bacterial metabolism through the utilizing of non-coding RNA.
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