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. 2012 Nov 30;7(11):e50987. doi: 10.1371/journal.pone.0050987

Figure 1. Construction and confirmation of Δhp0197 and a complementary strain, cΔhp0197.

Figure 1

(A) Construction strategy for Δhp0197, which was derived from the SS2 strain 05ZY. Approximately 600 bp of sequence flanking hp0197 was constructed into the temperature-sensitive S. suis-E. coli shuttle vector pSET4s, hp0197 was replaced with egfp. (B) The egfp gene could not be amplified from the WT strain (lane 1 and 2) but was amplified from the Δhp0197 (lane 5 and 6) and cΔhp0197 strains (lane 3 and 4). (C) A PCR targeted to the 176 bp and 252 bp from the upstream and downstream sequence of hp0197 sequence respectively with primer pairs of HP0197-P1/HP0197-P2 was performed to confirm the successful construction. And a 2.1-kb band could be amplified from DNA of the WT strain (lanes 1 and 2), and a 1.2-kb DNA fragment could be amplified from the Δhp0197 mutant (lanes 5 and 6). Two bands were amplified from the cΔhp0197 strain (lanes 3 and 4). Lane 7 in A and B were PCR negative controls. (D) HP0197 antibodies did not bind proteins extracted from the Δhp0197 mutant strain (lane 2) but was able to bind proteins extracted from the WT strain (lane 1) and the cΔhp0197 strain (lane 3) in the immunoblot assay. (E) The growth rates of S. suis strains in THB. (F) qRT-PCR amplification of hp0197 (ssu98_0197) flanking genes (ssu98_0195 to ssu98_0199) yielded identical results in WT and Δhp0197 while hp0197 could only be amplified from WT.