Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

Abstract

The arabinose-inducible promoter P BAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-␤-D-thiogalactopyranoside-inducible P tac and P taclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P BAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.In 1957, Novick and Weiner (14) studied expression of the lac operon in the presence of inducer concentrations less than that needed for maximal induction (subsaturating concentrations). This early study demonstrated that a fraction of cells in the population was fully induced while the remainder was uninduced and that the number of fully induced cells varied directly with the concentration of inducer. They referred to this mechanism as "all-or-none" or autocatalytic gene expression (14). Autocatalytic gene expression systems contain the genes encoding the transporter under the control of the transported molecule (the inducer). More recently, autocatalytic behavior was also reported for the ara operon (18).Although the all-or-none phenomenon associated with autocatalytic expression systems was demonstrated more than 40 years ago, many of the expression systems currently available continue to be based on similar frameworks and used without regard for this phenomenon. For systems in which population heterogeneity is not important and high-level gene expression is desired, autocatalytic systems remain an ideal choice; expression can be induced to a maximal level in all cells of the populat...

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

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Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter

“…KHLEBNIKOV and OTHERS than varying the level of gene expression in individual cells of the culture, the concentration of arabinose in the medium changes the fraction of cells that are fully induced. Recently, we showed that expression of araE from an arabinose-independent (IPTG-inducible) promoter allows regulatable gene expression control from P BAD in individual cells (Khlebnikov et al, 2000). In this paper we show that expression of araE from constitutive promoters of various strengths on medium-copy plasmids or on the chromosome allows homogeneous expression from P BAD and that the level of araE expression affects the level of expression from P BAD at a given inducer concentration.…”

Continuous Control in Bacterial Regulatory Circuits

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