In our previous work, by using system biology approaches, we concentrated on the regulation of the central carbon metabolism in E. coli K and B strains. We expanded this work by investigating the effect of Cra on the growth, acetate production and gene expression in these two strains. We showed that the deletion of the Cra gene in E. coli B minimally affected the growth and acetate accumulation, while the deletion of the same gene in E.coli K caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. 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). The conclusions were that Cra deletion caused transcription inhibition of the bet operon in E. coli K but did not affect this operon transcription in E. coli B. This property, together with the insensitivity to high glucose concentrations, makes E. coli B strain more resistant to environmental changes and better equipped for high density growth and recombinant protein production. We expanded our research towards understanding the effect of various stress condition on E. coli growth and especially the role of small regulatory RNAs that believed to be expressed when E. coli is exposed to stress conditions. Our assumption is that by manipulating the expression of small RNAs it will be possible to minimize the environmental effect on the bacterial growth and recombinant protein production. We showed that In E. coli K, which is sensitive to high glucose concentration;the small RNA SgrS was not expressed. By over-expressing this molecule it was possible to reduce the stress effect caused by the high glucose concentration and to allow this E. coli strain to grow as well as E. coli B. This observation opens new approach towards controlling bacterial metabolism utilizing non-coding RNA. We continued this line of work to identify small RNA that can increase the bacteria resistance to acid conditions, but so far we were unable to affect the bacteria resistance to low pH by manipulating level of FnrS And Gady that were expressed differentially at this growth conditions . Another possible stress factor is oxygen. The use of oxygen-enriched air is a common strategy that supports high density growth of E. coli. However high dissolved oxygen concentrations may also promote oxidative stress in the cells through the formation of reactive oxygen species (ROS). To determine the effect of elevated oxygen concentrations on the growth characteristics, specific genes expression and enzyme activities in two E. coli strains, parental and SOD-deficient strain, were evaluated when the dissolved oxygen level was increased from 30% to 300%. No significant differences in the growth parameters were observed in the parental strain except for a temporary decrease of the respiration and acetate accumulation profile. By performing transcriptional analysis, it was determined that the parental strain responded to the oxidative stress by activating the SoxRS regulon. However, following the dissolved oxygen switch, the SOD-deficient strain activated both SoxRS and OxyR regulons but was unable to resume its initial growth rate. The transcriptional analysis and enzyme activities results indicated that when E. coli is exposed to dissolved oxygen shift, the superoxide stress regulator SoxRS is activated and causes the stimulation of the superoxide dismutase system. This enables the E. coli to protect itself from the poisoning effects of oxygen. In addition, since the OxyR protecting system was not activated it showed that H2O2 did not increase to stressing levels.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$214,450
Indirect Cost
City
State
Country
Zip Code
Baez, Antonino; Majdalani, Nadim; Shiloach, Joseph (2014) Production of recombinant protein by a novel oxygen-induced system in Escherichia coli. Microb Cell Fact 13:50
Baez, Antonino; Shiloach, Joseph (2013) Escherichia coli avoids high dissolved oxygen stress by activation of SoxRS and manganese-superoxide dismutase. Microb Cell Fact 12:23