A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system

Abstract

The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal Streptomyces avidinii streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group, D-biotin. The entire construct was designed to allow the soil bacterium Pseudomonas putida to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.

Figure 2

Construction of plasmids pCC-s05 and pRO-ilp (A), and physical maps of their killing and regulatory elements (B). stv or s, Streptavidin gene; T7 RNA pol or pol, T7 RNA polymerase gene (T7 gene 1); lys, T7 lysozyme gene (T7 gene 3.5); kan, kanamycin-resistance gene; luc^ΔC, a truncated luc gene encoding an N-terminal fragment of luciferase; MCS, a fragment of pUC18 containing part of the multiple cloning site. B, BamHI; Bg, BglI; D, DraI; Ea, EagI; EI, EcoRI; EV, EcoRV; H, HindIII; Hp, HpaI; K, KpnI; N, NheI; Nr, NruI; P, PstI; Sa, ScaI; Sc, SacI; Sl, SalI; Sm, SmaI. Unique cleavage sites are marked with asterisks.

Figure 1

Scheme of a tightly regulated biological containment system to control survival of bacteria by availability of 3MB or other hydrocarbon effectors of the XylS protein. (A) Survival. (B) Induction of a lethal phenotype.

Figure 3

Kinetics of the induced suicide of P. putida cultures. Bacteria were grown to a mid-exponential growth phase in LB medium containing 3MB, ampicillin, and kanamycin. After washing with LB, cells were diluted 100-fold with LB supplemented with antibiotics with or without 3MB (A), or resuspended in the same medium without 3MB (B), and further incubated. Broken lines with open symbols refer to the additional presence of IPTG in LB. Plasmid combinations: s04, pCC-s04/pRO-ilp; s05, pCC-s05/pRO-ilp. CFU, colony forming unit. Each data point is the mean of two or three independent experiments.