Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter

Abstract

We have constructed a series of plasmid vectors (pBAD vectors) containing the P BAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/ repression can be 1,200-fold, compared with 50-fold for P TAC -based vectors. phoA expression 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. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the P BAD promoter to study the phenotypes of null mutations of essential genes 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 lacking the chromosomal gene. In these situations, it is highly desirable to use a system that can be efficiently shut off. This is particularly the case when the phenotype caused by the absence of a protein can be obscured by leakiness from a partially repressed promoter or when even low levels of a protein are detrimental to the cell. Also, it would be desirable to modulate the expression system to achieve synthesis levels similar to those of the wild-type gene. However, the available repertoire of Escherichia coli expression systems usually produce high levels of the corresponding cloned gene product (4,13,18,45,48) and in many cases still produce substantial levels of synthesis in uninduced or repressed conditions (4,13,15,16,48,49). These systems include controllable expression vectors based on the strong inducible promoters P LAC (48), P TAC (13), P TRC (4), P L and P R (18), and P T7 (45). Some are better repressed than others, but induction of expression requires changes of temperature (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 genes that encode membrane proteins involved in cell division of E. coli (7a, 22). To analyze their role, we have sought to deplete cells of the proteins and then examine the phenotype of cells so depleted under conditions that would minimize alterations in cell physiology. For these purposes, we wished to use a plasmid that would satisfy the following two conditions: (i) the synthesis of the proteins should be shut off rapidly and efficiently without changes of temperature, and (ii) expression before depletion should not produce very high levels of protein, which itself may give a phe...

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli

“…Plasmids included pZAQ and pAQ (Yi et al, 1985) provided by J. Lutkenhaus, pBAD18Cm (Guzman et al, 1995) and pLMG161 (pBAD18ftsQ ) (Guzman et al, 1997) provided by L. M. Guzman and pGC401 . Plasmid pBAD18CmftsA was constructed by amplifying the ftsA gene from chromosomal DNA by PCR, surrounded by restriction sites for NheI (5Ј) and SphI (3Ј); the primers used were 5Ј-GGGGCTAGCGGAGCGGAGTAG-GCTGGGCG and 5Ј-GGGGCATGCACCTTCAATGCGCTCG-CGCA; the amplified fragment was cloned in NheI-and SphIdigested pBAD18Cm.…”

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

“…44). To prevent overexpression of Hfq, the mutants were expressed under control of the inducible araBAD promoter 45 . In agreement with previous work 7 , in the absence of arabinose or with vector alone, very little Hfq accumulated (Fig.…”

A tightly regulated inducible expression system utilizing the fim inversion recombination switch

“…Thus, numerous inducible expression systems have been developed to express a desired gene in a switch-like fashion, such as the IPTG-induced trc promoter (Ptrc) (Amann et al, 1988) and the arabinose-induced araBAD promoter (P BAD ) systems (Guzman et al, 1995). However, their non-induced basal expression can be fairly significant, limiting their use in complementation studies or in propagation of toxic genes.…”

“…However, their non-induced basal expression can be fairly significant, limiting their use in complementation studies or in propagation of toxic genes. Leaky expression can be mitigated by altering the ribosome binding site of the target gene (Guzman et al, 1995) but this often results in a reduced induced expression level as well. Podhajska et al (1985) developed an inversion-based expression system using the phage l Int and the attP/attB recombination.…”