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.
Highlights
Haemophilus parasuis (H. parasuis, HPS) is a Gram-negative, non-spore-forming, pleomorphic bacterium which is the etiological agent of Glässer’s disease
H. parasuis was routinely cultured in Tryptic Soy Broth (TSB; Difco, Franklin Lakes, NJ, USA) or on Tryptic Soy agar (TSA; Difco, Franklin Lakes, NJ, USA) supplemented with 5% inactivated bovine serum (Solarbio, Beijing, China) and 0.1% (w/v) nicotinamide adenine dinucleotide
The results showed that minimal inhibitory concentration of SM (MIC-S) for SC1401 to be 16 μg ml−1; 24 μg ml−1 of SM was bactericidal (Table 1)
Summary
Haemophilus parasuis (H. parasuis, HPS) is a Gram-negative, non-spore-forming, pleomorphic bacterium which is the etiological agent of Glässer’s disease. How to cite this article Dai et al (2018), A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation. The resulting illness is characterized by polyarthritis, fibrinous polyserositis and meningitis in pigs, producing significant mortality and morbidity in pig farms and leading to serious economic losses in the pork industry throughout the world (Oliveira & Pijoan, 2004). Numerous gaps in our knowledge of molecular mechanisms of invading internal organs causing local and disseminated infection still remain at the present time (Zhou et al, 2016)
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