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 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. Concerning the first approach different modifications of acetate production and consumption pathways have been used to reduce acetate excretion in E. coli K growing in high concentration of glucose. In this study, several genes involved in the production-consumption of acetate were modified as a single or combined deletion in the low acetate producer E. coli BL21. The purpose of these genetic modifications was to see if it is possible to further reduce the acetate production in E. coli BL21, to examine if it is possible to convert E. coli BL21 to high acetate producer strain, and to suggest new approach for acetate reduction in E. coli K. Individual deletions of acs and aceA genes in E. coli BL21 did not show much difference in the metabolites accumulation pattern, but single deletion mutant ackA, and double deletion ackA-pta mutants showed gradually accumulated acetic acid to 3.1 and 1.7 g/L, respectively, with an extended lag time in the growth phase and high pyruvate formation. Single poxB knock-out in E. coli BL21 or additional poxB knock-out in the ackA-pta mutants did not change acetate accumulation pattern. It is suggested that poxB transcription lays a metabolic burden on the E. coli K strain, but does not affect the E. coli BL21 strain at standard growth conditions. But when the acetate production genes (ackA-pta-pta) were deleted in the E. coli BL21 strain, the bacteria was accumulated acetate. This may be an indication 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.
