pkk223-3 vector

Multidrug-resistant Salmonella genomic island 1 (SGI1) is an integrated mobilizable element found in multiple enterobacterial pathogens. This chromosomal island requires excision of the conjugated IncA/C plasmid as a circular extrachromosomal form and mobilization of the conjugation in trans. Preliminary observations suggest that SGI1 is stably maintained in the host chromosome, but paradoxically there is also incompatibility between SGI1 and IncA/C plasmids. In the study here, using the Salmonella enterica Agona clonal bacterial population as a model, researchers demonstrate that the SGI1-encoded toxin-antitoxin (TA) system plays a critical role in its stable host maintenance when IncA/C plasmids are simultaneously present. This system was named sgiAT for Salmonella genomic island 1 antitoxin and toxin, respectively, and therefore appears to play a stabilizing role in situations where SGI1 is susceptible to loss by plasmid IncA/C-mediated excision. Furthermore, the incompatibility between SGI1 and IncA/C plasmids was experimentally confirmed for the first time.

In this study, the transformation efficiency of a plasmid vector expressing putative toxin S025 (plasmid pKH02) into E. coli strains carrying empty vector pKK223-3 or its pKH01 derivative expressing putative antitoxin S026 was first evaluated. As shown in Figure 2a, when expression was induced with arabinose at concentrations of 0.2% or 1%, respectively, the transformation efficiency of plasmid pKH02 expressing S025 was reduced by 100 to 1000 times relative to the empty plasmid vector pBAD33. On the other hand, under the same conditions, these reductions were not observed when plasmid pKH01 expressing the putative antitoxin S026 was present, thus indicating that S026 counteracts the toxic activity of S025. Serial dilutions of each E. coli strain from this experiment spotted on LB plates in the presence or absence of arabinose also showed these effects to the same extent as the transformation efficiency test (Fig. 2b). In the absence of S026, induction of S025 transcription rapidly displayed toxic activity against the E. coli host strain in less than 30 min, whereas when S026 was present, viability was not affected (Fig. 2c).

Figure 1. The SGI1 S026-S025 open reading frames encode a functional TA system. (Huguet, Kevin T., et al. 2016)