Abstract
Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism.
Highlights
Expression in minimal medium that depends on the leucine-responsive regulatory protein, Lrp, which is lost upon carbon starvation; transcription of stpA is silenced by H-NS14,19
The hns and stpA genes differ in their genomic locations: hns is in the Ter macrodomain of the chromosome close to the point where chromosome replication ends, making hns among the last genes on the chromosome to be duplicated at completion of the cell cycle[2,20]
The results showed that 5′ -stpA[untranslated region (UTR)]-hns[open reading frames (ORFs)]-3′ was less stable than native hns mRNA (Fig. 4a,b) despite generating elevated levels of H-NS in SL1344RX (Fig. 3)
Summary
Expression in minimal medium that depends on the leucine-responsive regulatory protein, Lrp, which is lost upon carbon starvation; transcription of stpA is silenced by H-NS14,19. Moving the entire hns gene together with its negatively-auto-regulated promoter and upstream regulatory region to different locations on the chromosome did not alter cell physiology in E. coli[26]. Since the exchanges began at the translation initiation codons and ended at the translation stop codons, all other components of the participating genes – the transcription regulation, initiation and termination signals – remained in place and unchanged. This allowed us to investigate the effects of ORF exchange while minimizing the number of other variables
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