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

Abstract

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...

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

“…Moreover, the results suggested that differences in C1 expression correlated with the ability to achieve derepression. Partial derepression when the repressor is in excess has been demonstrated previously for regulated promoter systems in gram-negative bacteria (33,45). Indeed, ProA might be expected to be more active than ProB, resulting in higher levels of C1 expressed, since it contains more conserved nucleotides.…”

“…To ensure efficient translation, the primers amplifying lacZ incorporated a contrived RBS derived from gram-positive species (6). This resulted in a 200-fold increase in ␤-Gal activity in E. faecalis (data not shown) compared to that with the lacZ RBS (GGA GG[N] 6 ATG) used for reporter gene studies of gram-negative bacteria (45); consequently, the former RBS was also incorporated into the forward primers amplifying c1 and bof. At the permissive temperature, C1 binds to its operator sites and prevents transcription from the reporter gene, while at the nonpermissive temperature, C1 is thermally unstable, thereby allowing transcription to proceed.…”

“…Controlled expression using a temperature-sensitive C1-regulated promoter system has been demonstrated previously for gram-negative bacteria (44,45) by using a promoter responsible for driving ban gene expression in bacteriophage P1 (18). The ban promoter consisted of two overlapping C1 operator sites, and, although it did not contain consensus Ϫ35 and Ϫ10 hexamers, it was highly active in enteric gram-negative bacteria (43,45). However, reporter gene analysis indicated that this promoter was inactive in the gram-positive species E. faecalis and S. aureus (data not shown).…”

“…The amount of repressor produced is crucial to the effectiveness of a promoter system; small amounts of repressor can result in partial repression, while too much repressor results in the inability to achieve derepression (45,46). Therefore, the c1 gene was placed under the transcriptional control of two promoters (ProA and ProB; Fig.…”

“…The C1 repressor protein acts as a central regulator by controlling the expression of a variety of genes (3,7,18,19) by binding to C1 asymmetric operator (7) sites (consensus sequence, ATTGCTCTAATAAATTT). A bacteriophage P1-derived promoter in conjunction with the temperature-sensitive C1 repressor (40) has been used to regulate gene expression in gram-negative bacteria (45). In this report we demonstrate that the P1 temperature-sensitive C1 repressor can be used to control gene expression by using synthetic promoters in the pathogenic gram-positive species E. faecium, E. faecalis, and S. aureus.…”