The Sequence of the Ribosomal Binding Site Controls the Gene Expression in Brucella
Abstract BackgroundBrucella is an important pathogen causing Brucellosis. Vaccine strains obtained by a single knockout cannot combine low virulence and immunogenicity. Our study modified the SD sequence and spacer sequence of the RBS of Brucella to affect its protein expression. We altered the RBS of LPS-associated genes to reduce LPS-associated protein expression while retaining LPS integrity.ResultsWe first established an evaluation system based on the reporter gene red fluorescent protein mCherry. The mCherry expression could be changed by altering the Shine Dalgarno sequence and spacer sequence of RBS. After optimizing the Shine Dalgarno sequence, mCherry expression was increased 4-fold in E. coli and decreased by 1/4 in Brucella. The mCherry expression was increased 1.5-fold in E. coli and decreased to 1/2 in Brucella when the length of the spacer sequence was 0. When the spacer sequence was NA (N = 4, 8, 12nt) or NG (N = 4, 8, 12nt), mCherry expression was reduced in both E. coli and Brucella. Accordingly, two mutant strains were constructed in an attempt to decrease the expression of LptA and LpxO, Brucella LPS-related genes, by 1/4. Silver staining experiments of LPS SDS-PAGE revealed an alteration in the composition of LPS in the two mutant strains. Polymyxin B experiments revealed that both mutant strains were more sensitive to Polymyxin B resistance.Conclusion: In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and efficacy of vaccines.