Controlling Bdellovibrio Bacteriovorus Gene Expression and Predation Using Synthetic Riboswitches

Bdellovibrio bacteriovorus is a predatory bacterium that feeds on Gram-negative bacteria including a very wide range of pathogens and thus has a high potential as a biocontrol agent against multi-drug resistant infections¹. Although the potential applications of B. bacteriovorus can greatly benefit from its genetic engineering, genetic manipulation of B. bacteriovorus is not always straightforward due to its special life cycle which depends on a prey bacterium for survival. In the current study, we investigated the possibility of controlling gene expression in B. bacteriovorus using synthetic riboswitches. Two previously reported riboswitches that respond to thiamine pyrophosphate (TPP)² and six theophylline-responsive riboswitches³ were examined for their efficacy in B. bacteriovorus using mCherry as a reporter gene. The TPP riboswitches were not effective in B. bacteriovorus exhibiting high background expression in the absence of externally added thiamine. On the other hand, some of the theophylline riboswitches tested were significantly more effective. The best theophylline riboswitch (Riboswitch F) showed an ON/OFF ratio of 14 with a low background expression in the absence of theophylline. This riboswitch was subsequently tested for its ability to control native gene expression in B. bacteriovorus. For that purpose, two native genes were targeted; fliC3 which codes for flagellin that is essential for flagellar assembly and hence predation, and the flagellar sigma factor fliA (sigma 28)⁴. Chromosomal knock-in of riboswitch F upstream of either of those two genes led to delayed plaque formation on agar plates prepared using Escherichia coli MG1655 as a prey in the absence of theophylline and almost complete disappearance of plaques for the double knock-in mutant which had riboswitch F upstream of both genes. The plaque formation, however, was restored when theophylline was added at 1 mM final concentration. Predation of the knock-in mutants in liquid cultures using the same prey also showed some dependence on theophylline, though, to a lesser extent compared to the agar plates.

In conclusion, we showed that chemical regulation of gene expression in B. bacteriovorus and its behavior are possible using synthetic riboswitches, specifically, the theophylline responsive riboswitches. Our strategy not on only facilitates genetic manipulation to study the biology of B. bacteriovorus, but also brings us closer to the construction of an engineered bacterium with programmable predatory behavior.

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