A streptomycin resistance marker inH. parasuisbased on site-directed mutations inrpsLgene to perform unmarked in-frame mutations and to verify natural transformation

Abstract

Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R) or at 88th (AAA → AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively.

The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis

“…Comparable variation has also been found in Glaesserella parasuis (G. parasuis), a naturally transformable, gram-negative, pleomorphic bacterium of the Pasteurellaceae group (Bigas et al, 2005;Dai et al, 2021). Although a comparison of the entire genome sequence of G. parasuis isolates published in NCBI showed that all strains contained the identified homologous genes required for natural transformation, such as pilA, comEA, comM, etc., many G. parasuis isolates are non-transformable (Dai et al, 2016(Dai et al, , 2018b. The molecular mechanism of such diversity is unclear, however, it may due to significant differences in the regulatory systems of the different strains.…”