The role of Cra in regulating acetate excretion and osmotic tolerance in E. coliK-12 and E. coliB at high density growth

Son Young-Jin, Phue Je-Nie, Trinh, Lee, Shiloach, Shiloach Joseph

Description

E. 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. Results 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.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 530
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Sonet al.Microbial Cell Factories2011,10:52 http://www.microbialcellfactories.com/content/10/1/52

R E S E A R C H

The role of Cra in regulating acetate and osmotic tolerance inE. coliK12 at high density growth 1†1†1 2 1* YoungJin Son , JeNie Phue , Loc B Trinh , Sang Jun Lee and Joseph Shiloach

Open Access

excretion andE. coli

B

Abstract Background:E. coliB (BL21), unlikeE.coliK12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate thanE. coliK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 inE. coliB 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 negativeE. coliB (BL21) andE. coliK12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and realtime PCR. Results:The deletion of thecragene inE. coliB (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene inE.coliK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) andpoxB(the acetate producing gene) were downregulated in both strains, whileacs(acetate uptake gene) was downregulated only inE.coliB (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth ofE. coliK12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producingbetoperon (betABT). Conclusions:The transcriptional changes caused by the deletion ofcragene 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 thatcra deletion caused transcription inhibition of thebetoperon inE. coliK12 (JM109) but did not affect this operon transcription inE. coliB (BL21). This property, together with the insensitivity to high glucose concentrations, makes this theE. coliB (BL21) strain more resistant to environmental changes.

Background Acetate accumulation is one of the main concerns during high cell density growth ofE. coli[1,2]. It was established that acetate concentrations above 40 mM (2.4 g/L) nega tively affect cellular growth and recombinant protein pro duction [35]. Acetate accumulation is dependent on the bacterial strain [6] and is affected by high growth rate and low oxygen concentration [4,7]. Methods have been developed to reduce acetate accumulation, including dif ferent glucose feeding strategies, usage of lower acetate

* Correspondence: [e-mail] †Contributed equally Full list of author information is available at the end of the article

producing carbon sources, and the development of mutant strains with altered acetic acid metabolic flux [810]. The acetic acid production pattern ofE. coli B(BL21) is different from that ofE. coliK12 (JM109) especially when the bacteria grow to high densities at high glucose concentrations [11].E. coliK12 (JM109) accumulates acetate up to 11 g/L and its growth rate slows down; E. coliB (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 thatE. coliB (BL21) has active glyoxylate shunt, gluconeogenesis, ana plerotic pathway, and TCA cycle compared withE. coli

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