INTRODUCTION

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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 population. However, for many applications, intermediate expression levels are necessary to reduce metabolic burden or to achieve specific intracellular conditions. In these cases, one would like expression to be very low in the absence of inducer and vary directly with the level of inducer (6).

The ara operon is one of the most well-studied autocatalytic systems. In this system, genes encoding the arabinose transporters (araE and araFGH) and arabinose catabolic genes (araBAD) are under arabinose-inducible control through AraC; arabinose binds to the AraC protein, which positively regulates expression of transporters and negatively regulates its own expression (Fig. 1). Engineered plasmid vectors carrying the araC-P_BAD fragment from the ara operon have been used successfully in Escherichia coli and Salmonella enterica serovar Typhimurium as recombinant gene expression systems (3). This self-regulating system provides fine control of expression, tight repression in the absence of inducer, and induction over a 1,000-fold range in the presence of inducer (3). With the development of broad-host-range plasmids containing the araC-P_BAD repressor-promoter assemblage (13), this system is now also available for use in nonenteric, gram-negative bacteria.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Decoupled transporter-reporter system. AraC regulates expression from its own promoter (Pc) and from the araBAD promoter (PBAD). In the decoupled system, the araE genes, encoding the arabinose transporters, were placed under control of Ptac (pAK02).

Unfortunately, the response of the araC-P_BAD system in individual cells to arabinose concentration is not linear. In a recent study, Siegele and Hu (18) demonstrated all-or-none behavior of E. coli cells containing P_BAD::gfp constructs exposed to intermediate arabinose concentrations similar to that observed for the lac operon by Novick and Weiner (14). This phenomenon was attributed to the inducible L-arabinose transport systems (araE and araFGH).

Simulation experiments indicated that replacement of the native promoter for the gene encoding the transport protein with a constitutive or independently inducible promoter should effectively decouple the inducer transport-induction loop and eliminate autocatalytic responses (1). Using this strategy, it is possible to vary the average level of gene expression of the population by changing the level of gene expression in individual cells rather than the fraction of the population that is fully induced. In this article, we describe a transporter-reporter system used to examine the effect of independent expression of transport gene on reporter gene expression across the bacterial population.

	MATERIALS AND METHODS

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Reagents, strains, and plasmids. Chemical reagents were purchased from Fisher Scientific and Sigma (St. Louis, Mo.). Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from and used as recommended by Boehringer Mannheim (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). Oligonucleotide synthesis and sequencing were done by Genemed (South San Francisco, Calif.).

Induction experiments. Induction experiments were performed in C medium supplemented with 3.4% glycerol as a carbon source (4). For all constructs with the P_BAD promoter, E. coli strains were grown at 37°C under antibiotic selection with or without isopropyl--D-thiogalactopyranoside (IPTG) to an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.8. Cells were collected by centrifugation (15,000 × g), washed in C medium without a carbon source, resuspended in fresh C medium containing antibiotics, glycerol, IPTG, and/or arabinose (for the induction of gene expression) to an OD₆₀₀ of 0.1 to 0.2, and incubated for 6 h. Samples were taken routinely during the subsequent growth period for analysis.

L-Arabinose transport assay. The arabinose transport assay was performed using L-[¹⁴C]arabinose (American Radiolabeled Chemicals Inc., St. Louis, Mo.) and a Beckman LS 8000 scintillation counter. All cultures were grown on C medium with antibiotics and were preinduced with IPTG for 6 h to fully induce the transport genes and were not exposed to arabinose prior to addition of radioactive arabinose. Cells were harvested by centrifugation (12,000 × g), washed in C medium without arabinose, and resuspended in fresh medium to an OD₆₀₀ of 0.5. An aliquot of cells (0.5 ml) was added to 0.5 ml of medium at 37°C containing L-[¹⁴C]arabinose. The final arabinose concentration was 0.02% (1.33 mM) and had a specific activity of 55 µCi/mmol. Samples were placed at 37°C in a rotating incubator for 2 min. After incubation, the cells were filtered onto a 0.22-µm-pore-size Millipore membrane at room temperature (17) and washed with 10 ml of C medium containing 0.02% unlabeled arabinose. Filters were transferred into scintillation vials, filled with CytoScint scintillation liquid (ICN Biomedicals, Irvine, Calif.), shaken, and counted. A sample without cells was used to determine the background signal from nonspecific adsorption of labeled arabinose to the filters. Transport-deficient strains containing only control vectors (no transport genes) could not grow on minimal medium with arabinose as a carbon source and allowed no arabinose transport when induced with IPTG, arabinose, or both. Nonetheless, nonspecific adsorption of arabinose to cells was observed. It represented less than 10% of total radioactivity accumulated by catabolism-deficient and transport-deficient strains. All results presented here were corrected for such adsorption as well as for radioactivity nonspecifically bound to the filter.

	RESULTS

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Arabinose transport. To determine if the cloned and overexpressed araE gene was functional, an arabinose transport assay was performed. Since in any inducible system inducer must be transported into the cell, knowledge of its transport is important for understanding the effect it may play on the promoter modulation. Under normal conditions, arabinose is transported into E. coli via arabinose permeases. (AraE and AraFGH) and any changes in transport efficiency will influence intracellular sugar pools and affect P_BAD induction.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   L-[14C]arabinose accumulation by E. coli CW2587 with various transporter-reporter systems. Cells harbored arabinose transport genes on pAK01 (PtaclacUV5::araE), pAK02 (Ptac::araE), or pKKATEB (Ptac::araFGH) and the reporter gene on pCSAK50 (araC-PBAD::gfpuv). Data for cultures preinduced with 32 µg of IPTG per ml for 6 h prior to addition of L-[14C]arabinose (black bars) and uninduced cells (open bars) are shown. Assays were performed 2 min after radioactive arabinose was added. All numbers are the averages of triplicate experiments.

Induction experiments. Flow cytometry experiments were conducted to examine the homogeneity of gene expression in several strains with mutations in none, one, or both of the arabinose transport systems and containing the IPTG-inducible arabinose transport gene on pAK02 (P_tac::araE) and the gfp gene under control of P_BAD promoter pCSAK50 (P_BAD::gfp). Flow cytometry analysis showed that the fluorescence of individual cells could be reliably detected (Fig. 3 and 4). After only 2 h of induction with arabinose, the cultures produced enough green fluorescent protein (GFP) to give a quantifiable fluorescence signal. Prior to arabinose addition (time zero), both cultures displayed background fluorescence intensities. Two hours after induction, cultures of CW2513 had two distinct subpopulations (Fig. 4A). With time, the mean fluorescence of the subpopulation that was fully induced increased, and the fraction of the population that was uninduced decreased. Six hours after induction, only 10 to 15% of the cells remained uninduced. In contrast to the results obtained with CW2513, cultures of CW2587 had a homogeneous population at all times after induction (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Population homogeneity of various E. coli strains harboring the transporter-reporter plasmid system. All cultures harbored the transporter gene on pAK02 (Ptac::araE) and reporter gene on pCSAK50 (araC-PBAD::gfpuv). Cells were grown in the presence of 32 µg of IPTG per ml. Flow cytometry analysis was performed 4 h after addition of arabinose. In control experiments, all strains harbored pCSAK50 and pMMB207. Experiments were performed in triplicate. Results from a single experiment are presented.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Time course of fluorescence intensity in E. coli CW2513 (A) and CW2587 (B) harboring pAK02 (Ptac::araE) and pCSAK50 (araC-PBAD::gfpuv). All cells were preinduced with 32 µg of IPTG per ml for 6 h prior to arabinose induction. Arabinose (0.02%) and IPTG (32 µg/ml) were added to the culture at time zero.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   GFP expression as function of the arabinose concentration in cultures of E. coli CW2513 (open squares) and CW2587 (filled squares) harboring pAK02 (Ptac::araE) and pCSAK50 (araC-PBAD::gfpuv). Cells were preinduced with 32 µg of IPTG per ml. Six hours after addition of arabinose, the percentage of the population that was induced (A) and the population-averaged fluorescence (Fluorescence/OD ratio) (B) were determined.

	DISCUSSION

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Inducible promoter systems have been used extensively to control and probe cellular processes and to express heterologous genes for high-level protein production. For many years, it has been known that the lacZYA operon suffers from an all-or-none expression response (11, 14). Recently, it was shown that the ara operon has similar characteristics (18). These carbohydrate-responsive systems have evolved this all-or-none method of gene expression to allow low-level expression in the absence of the substrate and rapid, high-level response when the substrate is present. These characteristics are ideal for overexpression of a heterologous gene; in the absence of inducer, the gene is not expressed, and in the presence of inducer, the gene is overexpressed. Unfortunately, these characteristics are not suitable for the subtle and regulatable control of gene expression that may be essential for probing cellular processes and for manipulating cellular metabolism. These reasons have motivated the work described above.

The all-or-none or autocatalytic phenomenon arises because the transporter for the inducer is under control of the inducer itself. Upon exposure to an inducer, the inducer is transported into the cell by some minimal number of transporters in the membrane. If a threshold level of inducer accumulates inside the cell, the transporter gene and any other associated genes are induced. The production of more transport protein and the subsequent import of more inducer rapidly cascades to maximal gene expression.

Several strategies can be used to decouple autocatalytic systems. One strategy is to place the gene for the transport system under control of a promoter that is not regulated by the inducer that the transport system transports. This can be accomplished by transforming a transport-deficient strain or a strain carrying a low-capacity transport system with a plasmid that carries an independently regulated transporter. The second strategy is to synthesize an inducer analog that can diffuse across the cell membrane without a transport system (e.g., IPTG). In the latter case, one may need to use a transport-deficient strain, as an inducer analog may be transported by the same transport proteins that act on the natural inducer and suffer the same all-or-none regulation. Hence, we chose the former strategy.

The high-capacity arabinose transporter system has been studied extensively (7-10, 17). AraE is a proton symporter, in contrast to AraFGH, which is binding protein dependent. In this work, the expression of araE was engineered to be dependent on the IPTG concentration in the growth medium (Fig. 1). The vectors carrying araE contained either a strong P_tac promoter or a moderate P_taclacUV5 promoter and the gene encoding the lac repressor (lacl^q) for tight control over expression. In addition, expression of araE from the low-copy-number RSF1010-based plasmids (approximately 12 copies per cell) minimized any possible toxic effects of overexpression of membrane-associated proteins.

Our results showed that plasmid encoded L-arabinose transport in the transport-deficient strains was not completely dependent on IPTG. Even in the absence of IPTG, P_BAD was induced above basal expression levels in the presence of arabinose. However, the more tightly regulated P_taclacUV5 promoter system had a lower basal expression level and consequently less arabinose accumulated inside the cells. There are two potential reasons. (i) The P_tac promoter was leaky. (ii) Arabinose was transported via other transporters. In the early experiments with the P_tac::araFGH system (pKKATEB), Horazdovsky and Hogg (5) found that even in the absence of the inducer, a significant level of L-arabinose accumulated in a similar strain. Thus, the basal expression level of the transport gene from the plasmid might be high enough to allow transport of arabinose in amounts sufficient for induction of arabinose-dependent promoters. In any case, the gene encoding the transport protein would be controlled independently of arabinose and should not suffer from all-or-none regulation.

As demonstrated above, IPTG preinduction led to a significant increase in arabinose transport at intermediate arabinose levels. In this case, the cultures were exposed to arabinose for a very short time, so there was minimal effect of arabinose on the expression of genes encoding the transport proteins. However, when both IPTG and arabinose were present in the culture broth for a long period of time, there was a significant inhibition of radioactively labeled arabinose accumulation (data not shown). Most likely, the unlabeled arabinose that accumulated inside the cells inhibited further accumulation of labeled arabinose.

Introduction of an independently regulated transporter in CW2513 had no effect on population homogeneity. In contrast, expression of the independently regulated transporter in the araE mutant strains CW2549, CW2553, and CW2587 resulted in a single population in cultures preinduced with IPTG. In the case of CW2587, a homogeneous population was achieved even at the lowest arabinose level tested (0.0002%), at least 100-fold-less inducer than was needed to induce all of the other strains to comparable levels. Through the entire range of arabinose concentrations, the transport-deficient cultures demonstrated higher specific and overall GFP production compared to that of wild-type CW2513.

It should be noted that the recA strain CW2587 had a more uniform population than the recA⁺ strains CW2549 and CW2553. As recA is known to affect plasmid stability, this result is not surprising. Nevertheless, the recA⁺ strains have homogeneous populations at all arabinose concentrations.

Finally, at saturating arabinose concentrations (2%), a decline in the specific fluorescence was observed. This phenomenon might be ascribed to arabinose catabolism, resulting in a lower internal arabinose concentration and lower GFP production per cell.

The presence of a functional araE gene on the chromosome would appear to be primarily responsible for the all-or-none response. However, it is not entirely clear why the plasmid-encoded transport system did not alleviate the all-or-none response in the araE⁺ CW2513. It is possible that once arabinose was transported into CW2513, expression from the chromosomal copy of the arabinose transport gene was higher than the low-level, IPTG-induced expression from the plasmid.

In summary, the results presented here show that replacing the native inducer transport system with one under independent control can eliminate the all-or-none response characteristic of many inducible systems. Furthermore, by placing the transport system under independent control, the resulting inducible promoter is regulatable with significantly lower inducer concentrations. This design should prove useful in metabolic redesign of cells and should serve as a prototype for redesign of other inducible promoter systems suffering from the all-or-none phenomenon.