pBAD18

Salmonella enterica serovars Typhi can express several flagellar antigens. Indonesian Salmonella typhi isolates express the novel antigen H: z66 (z66), encoded by the linear plasmid pBSSB1. Thirty-three open reading frames (ORFs) have been identified, of which ORF031 encodes the flagellar antigen H: z66. Previously, the protein Fis has been shown to affect the stability of pBSSB1. However, the specific regions involved in proliferation and stable survival of linear plasmids have not yet been identified. Here, researchers identified open reading frame (ORF) 009 as a toxin-encoding gene in a plasmid by introducing a translation stop codon in the ORF. Ectopic expression of ORF009 in the pBAD18 vector demonstrated that ORF009 encodes a toxin. This fragment stabilizes plasmid pUC18, which was previously destabilized by mutations in the pcnB (plasmid copy number control) gene encoding poly(A) polymerase. According to phase contrast microscopy, most ORF009-expressing cells were not viable.

In this study, the expression vector pBAD18 contains the arabinose promoter PBAD, which is repressed by glucose and induced arabinose in the growth medium. ORF009 was cloned downstream of PBAD using EcoR I and Pst I restriction sites, and the resulting construct was named pBAD18-ORF009. Similar growth patterns (OD₆₀₀) were observed in the presence of glucose and arabinose in the presence of pBAD18 alone. Cells containing pBAD18-ORF009 showed reduced growth when arabinose was present but grew normally in the presence of glucose. Cells containing pBAD18 or pBAD18-ORF009 showed different viable bacterial counts in the presence of glucose and arabinose. In the presence of arabinose, viable counts of cells containing pBAD18-ORF009 decreased 10⁴-fold after 30 min. Thus, expression of ORF009 resulted in 99.99% cell death, although not lysis, compared to cells containing functional ORF009.

Figure 1. Growth of cultures containing pBAD18 or pBAD18-ORF009 in presence of glucose (0.2%) or arabinose (0.2%) measured in terms of OD₆₀₀ (a) and viable counts (b). (Ahsan S, Summers D., 2018)