Abstract
BackgroundE. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR.ResultsThe 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).ConclusionsThe 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.
Highlights
E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration
We have investigated the effect of cra deletion on the central carbon metabolism in E. coli B (BL21) and E. coli K-12 (JM109) to verify the above assumption and to understand further the function of Cra in high cell density at high glucose, by following growth kinetics and gene expression
Cells growth parameters of the cra-positive and the cranegative strains Growth kinetics, glucose consumption and media conductivity profile of E. coli B (BL21) and E. coli K-12 (JM 109), without modification and with the cra gene deletion, are shown in Figure 1 and 2, and acetate and pyurvate production kinetics are shown in Figure 3. cra deletion in E. coli B (BL21) minimally affected growth, final cell yield, glucose consumption and maximal acetate and pyruvate accumulation
Summary
E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR. E. coli K-12 (JM109) accumulates acetate up to 11 g/L and its growth rate slows down; E. coli B (BL21) on the other hand, accumulates acetate to about 3 g/L and its growth rate is not affected Careful evaluation of these two strains revealed that E. coli B (BL21) has active glyoxylate shunt, gluconeogenesis, anaplerotic pathway, and TCA cycle compared with E. coli
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