Construction and characterization of the first Arcobacter butzleri - Escherichia coli shuttle vector.

This study addresses a key limitation in the genetic characterisation of Arcobacter butzleri, an emerging food- and waterborne pathogen associated with gastrointestinal and systemic infections in humans and animals. Despite the identification of numerous putative virulence genes, functional validation has been hindered by the absence of suitable complementation tools. Here, we report the development and characterisation of the first shuttle vector capable of replication in both Escherichia coli and A. butzleri. A total of 117 A. butzleri isolates were screened for plasmids, leading to the identification and sequencing of a novel cryptic plasmid, pABRW13 (3,402 bp), from a waterborne strain. This plasmid revealed four open reading frames, including a putative replication-associated gene. These elements were incorporated into a novel shuttle vector, pIMM24 (7,425 bp), together with a tetracycline resistance cassette (tetO), using the pBluescript KS derivative pMW2 vector as backbone. The resulting construct successfully replicated in both hosts, exhibited stable maintenance in E. coli and moderate stability in A. butzleri, and conferred increased tetracycline resistance. Functional validation was achieved by complementation of a fliS knockout mutant, restoring motility to near wild-type levels. Overall, pIMM24 represents a significant advancement in the genetic toolkit available for A. butzleri, enabling gene complementation studies and facilitating deeper insights into its physiology and pathogenic mechanisms.