Tight Regulation, Modulation, and High-Level Expression by Vectors Containing the Arabinose PBAD Promoter

We have constructed a series of plasmid vectors (pBAD vectors) that contain the PBAD promoter of the Arab and operon (arabinose) and the gene that encodes the positive and negative regulator of this promoter, araC. Using the phone gene and phoA fusions to monitor expression in these vectors, we show that the induction/repression ratio can be 1200-fold, compared to 50-fold for PTAC-based vectors.

The expression of phoA can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Furthermore, the induction and repression kinetics is very fast and is significantly affected by the ara allele in the host strain.

Thus, the use of this system that can be switched on and off quickly and efficiently allows the study of important aspects of bacterial physiology in a very simple way and without temperature changes. We have taken advantage of the strict regulation of the PBAD promoter to study the phenotypes of essential genes null mutations and explored the use of pBAD vectors as an expression system.

In bacterial physiology studies, it is often useful to express a cloned gene from an inducible promoter and assess the effect of the expression or depletion of the gene product in mutants might lack the chromosomal gene. In these situations, it is very It is desirable to use a system that can be shut down efficiently.

This is particularly the case when the phenotype caused by the absence of a protein can be obscured by leaks of a repressed promoter or when even low levels of a protein are harmful to the cell. Furthermore, it would be desirable to modulate the expression system to achieve levels of synthesis similar to those of the wild-type gene.

However, the repertoire available of Escherichia coli expression systems tend to produce high levels of the corresponding cloned gene product (4, 13, 18, 45, 48) and in many cases still produce substantial levels of synthesis under uninduced or repressed conditions (4, 13, 15, 16, 48, 49).

These systems include controllable expression vectors based on the strong inducible promoters PLAC (48), PTAC (13), PTRC (4), PL and PR (18), and PT7 (45). Some are better repressed than others, but induction of expression requires temperature changes (18, 45) and produces very high levels of protein, resulting in conditions detrimental to cell growth and viability (17, 45). We have been studying the function of several essential elements. genes encoding membrane proteins involved in cell division of E. coli (7a, 22).

To analyze their role, we have sought to deplete cells of proteins and then examine phenotype of cells so depleted under conditions that would minimize alterations in cell physiology. For these purposes, we wish to use a plasmid that satisfies the following two conditions: (i) protein synthesis must be interrupted rapidly and efficiently without temperature changes, and (ii) expression Before depletion, very high levels of protein should not be produced, which by itself can give a phenotype or influence the depletion phenotype.

To achieve these conditions, we construct a set of vectors (pBAD vectors) containing the PBAD promoter of the arabinose operon and its regulatory gene, araC (28, 41). The AraC protein is both a positive and a negative regulator (6, 30). In the presence of arabinose, the transcription of the PBAD promoter is activated; in its absence, transcription occurs in very low levels (28, 29). Non-induced levels can be reduced even also by growth in the presence of glucose.

Glucose reduces levels of cyclic 39.59-AMP, which reduces the expression of the catabolite-repressed PBAD promoter (32). The properties of the mechanism of expression and repression of PBAD by AraC have been studied extensively, and their interactions have been dissected at the molecular level (reviewed in reference 41).

Furthermore, the plasmids described above in which transcriptional or translational fusions to araB were used to establish genes under the control of PBAD and AraC suggested that the expression of this system was strictly regulated (12, 26, 38, 40).

Here, we describe the construction of vectors pBAD and the characteristics of repression, induction and modulation of the expression of genes cloned in them. In addition, we show the application of the strict regulation of vectors to the study of null mutations of essential genes and discuss the relevance of this and the expression properties of the vector for bacteria physiology.

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