Hypersymmetry in a transcriptional terminator of Escherichia coli confers increased efficiency as well as bidirectionality.

Wright, Joy; Kumar, Ashok; Hayward, Richard S.

doi:10.1002/j.1460-2075.1992.tb05249.x

Cited by 25 publications

(23 citation statements)

References 24 publications

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“…The NcoI-digested PCR product was inserted into the unique NcoI site of the broad-host-range vector pBBR122 (MoBiTec) creating plasmid pCW3. The unique BamHI and ScaI sites of pCW3 were treated with DNA polymerase I Klenow fragment to create blunt ends and transcriptional terminators, T L "( (Wright et al, 1992), were then cloned into these sites to generate P1pBHR-T. To facilitate detection of phagemid transduction in P. aeruginosa, the ampicillin-resistance gene including its putative promoter was amplified by PCR from pBluescript II SK using primers 5h-CGCTTACAATTTAGG-TGGCAC-3h and 5h-AACTTGGTCTGACAGTTACC-3h and cloned into the DraI sites of P1pBHR-T, thereby creating plasmid P1pBHR-bla.…”

Section: Methodsmentioning

confidence: 99%

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Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

et al. 2002

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The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-hostrange origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii ) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

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Section: Methodsmentioning

confidence: 99%

“…P1pBHR-T was recovered from transduced cells by the alkaline lysis method (QIAprep miniprep kit, Qiagen). terminator is a natural element derived from the E. coli α-operon (Wright et al, 1992). Only relevant restriction sites are indicated.…”

Section: Methodsmentioning

confidence: 99%

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

et al. 2002

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“…In contrast, the artificial promoter (Pro2) contains a consensus C1 operator site flanked by consensus Ϫ10 and Ϫ35 promoter elements. To prevent read-through from cryptic promoters and runaway transcription, the ribosomal terminators rrnB T1 T2 (5) and TL 17 (29) were placed at the 5Ј and 3Ј ends of the expression cassette, respectively (Fig. 1B).…”

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confidence: 99%

“…The vector was modified to contain two antibiotic-resistant markers to facilitate selection. The expression cassette is flanked by terminators at the 5Ј (5) and 3Ј (29) ends. The sequence is available on request.…”

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confidence: 99%

Controlled Expression in Klebsiella pneumoniae and Shigella flexneri Using a Bacteriophage P1-Derived C1-Regulated Promoter System

et al. 2001

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The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in Shigella flexneri and Klebsiella pneumoniae. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in S. flexneri and up to 50-fold in K. pneumoniae. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.Many regulated promoter systems have been described for use in Escherichia coli. These systems include promoters regulated by LacI (2), AraC (9), and TetR (18) or combinations that can provide both low basal and high induced expression. Each system has shown utility with varying success in other bacteria, such as Pseudomonas aeruginosa (6), Corynebacterium glutamicum (4), Agrobacterium tumefaciens (22), and Xanthomonas campestris (27). However, few or no data exist for a regulated promoter system in the medically important species Klebsiella pneumoniae (14) and Shigella flexneri. Klebsiella species cause approximately 8% of nosocomial infections in the United States and are commonly found in both humans and the environment (23). In contrast, Shigella species, found mainly in humans, cause shigellosis, which is characterized by cramps, fever, and dysentery (1).The temperate bacteriophage P1 can infect and lysogenize several enterobacterial species, including K. pneumoniae and Shigella dysenteriae (21,30). Stable lysogeny is maintained by the action of the components of the tripartite immunity system (for a review, see reference 10). The C1 repressor protein acts as a central regulator by binding to and negatively regulating promoter elements for a variety of genes (7,8,11,12,16,17,28). The C1 asymmetric operator sites (consensus sequence ATTGCTCTAATAAATTT) are widely dispersed over the P1 genome and are numbered according to their positions on the P1 genetic map (10,30).In this report, we describe a temperature-sensitive C1-regulated promoter system in a broad-host-range plasmid for controlled gene expression in both K. pneumoniae and S. flexneri.Characteristics and construction of the expression vectors. The lacZ reporter gene vectors were constructed in the broadhost-range gram-negative plasmid pBBR122 (Mobitec). The lacZ gene was placed under the transcriptional control of two C1-regulated promoters (Fig. 1A). The Pro1 promoter is based on the promoter responsible for driving ban gene expression in bacteriophage P1 and has been shown to be effectively repressed in E. coli in the presence of C1 (11, 26). It consists of two overlapping C1 operator sites but lacks consensus E. coli Ϫ10 and Ϫ35 promoter elements. In contrast, the artificial promoter (Pro2) contains a consensus C1 operator site flanked by consensus Ϫ10 and Ϫ35 promoter elements. To prevent read-through from cryptic promoters and runaway transcription, the ribosomal terminators rrnB T1 T2 (5) and TL 17 (29) we...

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“…To increase the number of cloning sites, the oligonucleotides also contained a SpeI site at the 5Ј end. To reduce readthrough from cryptic promoters into the 5Ј end of the expression cassette, the TL 17 transcriptional terminators (54) were cloned into the SpeI site. To prevent "runaway" transcription, the terminators were also cloned at the 3Ј end of the expression cassette (EcoRV site).…”

Section: Methodsmentioning

confidence: 99%

Development of a Thermally Regulated Broad-Spectrum Promoter System for Use in Pathogenic Gram-Positive Species

Schofield

Westwater

Hoel

et al. 2003

Appl Environ Microbiol

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Selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria. In this report, we describe the evaluation of a series of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Using the lacZ gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying C1 operator sites. The promoters exhibited extremely low basal expression and, under inducing conditions, gave high levels of expression (100-to 1,000-fold induction). We demonstrate that the promoter system could be modulated by temperature and showed rapid induction and that the mechanism of regulation occurred at the level of transcription. Controlled expression with the same constructs was also demonstrated in the gram-negative bacterium Escherichia coli. However, low basal expression and the ability to achieve derepression were dependent on both the number of mismatches in the C1 operator sites and the promoter driving c1 expression. Since the promoters were designed to contain conserved promoter elements from gram-positive species and were constructed in a broad-host-range plasmid, this system will provide a new opportunity for controlled gene expression in a variety of gram-positive bacteria.The overuse of antibiotics has contributed to the emergence and increasing prevalence of antibiotic-resistant bacteria. Gram-positive cocci such as Staphylococcus aureus and enterococci are the leading cause of hospital-acquired infections (39). S. aureus causes a variety of infections ranging from localized skin suppuration to life-threatening septicemia. Alarmingly, S. aureus isolates resistant to vancomycin, the last effective antibiotic, are emerging worldwide (22). Enterococcus species are a leading cause of urinary tract infection, nosocomial infection, and surgical-wound infection (39). Enterococcus faecalis is responsible for the majority of enterococcal infections (26,41) and, for the time being, usually remains sensitive to at least one antibiotic. In contrast Enterococcus faecium, which causes fewer infections, is more likely to be resistant to all antibiotics.The recent emergence of antibiotic-resistant gram-positive bacteria has highlighted the need for genetic studies addressing the mechanism of bacterial pathogenesis. Regulated promoters are essential for the functional analysis of genes through expression studies (1) and reverse genetics. However, only a few regulated promoters are available for use in enterococci and streptococci. The tetracycline-regulated promoter system has been shown to function in Streptococcus pneumoniae (47), Bacillus subtilis (11), and S. aureus (1, 25). In addition, the xylose-inducible promoter system has been used for B. subtilis and staphylococci (27,52,56). However, the levels of regulation achieved with these systems are below those obtained for gram-negative bacteria (33), and tight basal e...

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Hypersymmetry in a transcriptional terminator of Escherichia coli confers increased efficiency as well as bidirectionality.

Cited by 25 publications

References 24 publications

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

Controlled Expression in Klebsiella pneumoniae and Shigella flexneri Using a Bacteriophage P1-Derived C1-Regulated Promoter System

Development of a Thermally Regulated Broad-Spectrum Promoter System for Use in Pathogenic Gram-Positive Species

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