Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 μM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.     

茶碱激活型RNA分子开关的构建及在枯草芽胞杆菌中调节外源基因表达的性能

枯草芽胞杆菌Bacillussubtilis在工业生物技术以及合成生物学领域作为一种重要的微生物可广泛用作代谢工程、重组蛋白表达以及新型基因电路的底盘。在B. subtilis中构建基于非编码RNA的高精准调节元件,能够实现不依赖蛋白质因子的基因表达调控,丰富B.subtilis基因表达通用工具。通过基因工程手段,设计了基于茶碱适体域的核糖开关E和适体核酶AZ调节元件,并与不同的B.subtilis内源组成型启动子适配,构建出茶碱激活型基因表达控制元件。测定这两种调节元件与6种组成型启动子组合匹配下报告基因GFP的荧光强度,鉴定并分析各调控元件的工作性能。并进一步以红色荧光蛋白mCherry和普鲁兰酶两种不同的异源蛋白验证核糖开关或适体核酶与启动子的最优组合。结果表明,同一种RNA调节元件与不同启动子组合呈现不同水平的调控效率。在核糖开关与启动子的组合中,启动子PsigW和核糖开关E组合(sigWE)对GFP表达的诱导率最高,达到16.8。在适体核酶与启动子的组合中,AZ与启动子P43、PrpoB组合(P43AZ和rpoBAZ)的诱导率最高,分别达到了6.1和6.2。进一步验证结果显示,sigWE调控mCherry的诱导率最高(9.2),而P43E调控普鲁兰酶的诱导率最高(32.8),产酶水平达到了81U/mL。核糖开关和适体核酶对GFP、mCherry、普鲁兰酶均能实现调控,但是不同元件组合的调控性能有所差异,对不同基因的调控效果也不尽相同。     

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.     

Expansion of the Tetracycline-Dependent Regulation Toolbox for Helicobacter pylori

In an effort to gain greater understanding of the biology and infection processes of Helicobacter pylori, we have expanded the functionality of the tetracycline-dependent gene regulation (tet) system to provide more improved and versatile genetic control and facilitate the generation of conditional mutants to study essential genes. Second-generation tetracycline-responsive H. pylori uPtetO5 promoters were based on the mutated core ureA promoter. Single point mutations at either the ribosomal binding site or the start codon were introduced to shift the regulatory range of three uPtetO5 derivatives. All promoters were tested for regulation by TetR and revTetR using dapD, a gene essential to peptidoglycan biosynthesis, as a reporter. All tet promoters were effectively regulated by both TetR and revTetR, and their regulation windows overlapped so as to cover a broad range of expression levels. tet promoters uPtetO5m1 and uPtetO5m2 could be sufficiently silenced by both TetR and revTetR so that the conditional mutants could not grow in the absence of diaminopimelic acid (DAP). Furthermore, through the use of these inducible promoters, we reveal that insufficient DAP biosynthesis results in viable cells with altered morphology. Overall, the development and optimization of tet regulation for H. pylori will not only permit the study of essential genes but also facilitate investigations into gene dosage effects on H. pylori physiology.     

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

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.     

The toxin-antitoxin system of the streptococcal plasmid pSM19035

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.