Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Scheme for the construction of plasmid pMDHK.
Primer pairs P1/P2 and P3/P4 were used to amplify the upstream and downstream regions of the htrA gene respectively, each with 20-bp end sequences identical to the kan fragment (using primer P2 and P3) and 20-bp end sequences identical to vector pMD19-T (using primer P1 and P4). Primers P5/P6 were used to amplify the kan fragment from the plasmid pKD4. The resulting three PCR products and the cloning vector pMD19-T were joined by In-Fusion cloning. Two USSs required for efficient transformation were introduced into the plasmid pMDHK by using primer P1 and P4.
Fig 2.
Scheme for the construction of the unmarked SC1401 ΔhtrA strain.
The plasmid pMDHKS was generated by inserting the sacB fragment (amplified from the plasmid pEMOC2 using primer pairs P7/P8) into the plasmid pMDHK by inverse PCR (using primer P9 and P10) and In-Fusion cloning. The plasmid pMDH was obtained by inverse PCR (using primer P11 and P12) and In-Fusion cloning. To construct the unmarked SC1401 ΔhtrA strain, the kan-sacB cassette was integrated into the SC1401 chromosome and the htrA gene was replaced by the first allelic replacement. Next, the linearized plasmid pMDH was employed in the second allelic replacement to remove the kan-sacB cassette from the SC1401ΔhtrA::kan-sacB chromosome, leaving an unmarked htrA deletion strain.
Fig 3.
PCR confirmation of the SC1401 ΔhtrA ΔpotD strain.
PCR amplification was performed with templates of SC1401 genome (lanes 1, 3, 5, 7) and of SC1401 ΔhtrA ΔpotD chromosomal DNA (lanes 2, 4, 6, 8) using primers: P1/P4 (lanes 1, 2); P13/P16 (lanes 3, 4); P5/P6 (lanes 5, 6); P7/P8 (lanes 7, 8). Lane 9, PCR fragments were amplified using primers P5/P6 with template of plasmid pKD4 (positive control for kan gene). Lane 10, PCR fragments were amplified using primers P7/P8 with template of plasmid pEMOC2 (positive control for sacB gene). Lane M, DNA molecular weight markers.
Fig 4.
Western blotting analyses of the SC1401 ΔhtrA ΔpotD strain.
(A) The lysates of the SC1401 (lane 1) and SC1401ΔhtrA ΔpotD strain (lane 2) were analyzed using anti-rHtrA antibodies. The wild strain displayed the expected specific band while no band detected accordingly in the ΔhtrA ΔpotD strain. (B) The lysates of the SC1401 (lane 1) and SC1401ΔhtrA ΔpotD strain (lane 2) were analyzed using anti-rPotD antibodies. The expected specific band was detected in the wild strain but not in the ΔhtrA ΔpotD strain.