A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation

Abstract

The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.

hsdS, Belonging to the Type I Restriction-Modification System, Contributes to the Streptococcus suis Serotype 2 Survival Ability in Phagocytes

“…An insertion mutation of SS2 ZY05719, obtained by a TnYLB-1 insertion from pMar4s, demonstrated that the capacity of anti-phagocytosis (This term means phagocytosis resistance, which comes from ref Bergman et al, 2009 ; Yamaguchi et al, 2009 ; Hsieh et al, 2016 ) compared with the wild-type strain was significantly reduced in our earlier research (Liu et al, 2016 ). The TnYLB-1 insertion site was between the T 1384 and A 1385 bases in the open reading frame (ORF) of the host specificity determinant specificity subunit ( hsdS ) gene (locus_tag ZY05719_RS06855) (Figure 1A ).…”

The serine/threonine protein kinase ofStreptococcus suisserotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier

“…suis mutants have been successfully identified, and Tn323 mutant has the poorest invasion ability. TnYLB‐1 insertion site of the Tn323 mutant was sequenced as previously described (Liu et al, ), and the interrupted gene was stk . The stk gene was maybe related to S .…”

Screening for phagocytosis resistance-related genes via a transposon mutant library of Streptococcus suis serotype 2

“…However, mechanism of SS2 resistance to non-opsonophagocytosis are unknown and remain to be elucidated. Therefore, we screened for more pathogenesis-associated genes via a SS2 mutant library constructed previously in this lab [ 3 ].…”

“…Phagocytosis is a crucial step of host immunity, and hence, the phagocytosis resistance ability of SS2 has attracted our attention. A transposon mutant library of ZY05719 was previously constructed in our laboratory [ 3 ]. This mutant library contained 2400 strains of SS2 mutants in total.…”

“…This mutant library contained 2400 strains of SS2 mutants in total. The mariner Himar1 transposon was randomly inserted into the genome, and the transposon distribution within the SS2 genome was shown to be random without any hot spots [ 3 ]. Transposon mutants have been used to screen for virulence-associated genes by generating altered phenotypes compared to wild-type strains [ 4 – 7 ].…”