Fig 1.
Comparison of hemolytic activity and colony formation to elucidate influence of deletion CylK.
Colony size and hemolytic activity of a GBS ATCC strain (BAA-611), recombinant strains (ΔcylK), and complemented strains (ΔcylK+ pDL278 and ΔcylK+pCylK) on 5% sheep blood agar. The strains were grown at 37°C in 5% CO2 for 16 h. ΔcylK is a recombinant strain based on ATCC BAA-611, harboring the G379T substitution in cylK, resulting in premature termination at amino acid 127 in CylK. pDL278 is a gram-positive and gram-negative shuttle vector. pCylK is a complementation plasmid used to express intact TPK in the ΔcylK strain.
Table 1.
Nucleic acid differences between MRY11-004 and MRY11-004L, MRY11-005 and MRY11-005L, NUBL-2449 and NUBL-2449L using CLC workbench ver. 9 (>80%; Frequency).
Fig 2.
Comparison of hemolytic activity and colony formation to elucidate influence of deletion TPK.
Colony morphology of BAA-1138, Δtpk, Δtpk+pDL278, and Δtpk+pTPK grown on sheep blood agar. BAA-1138 and Δtpk+pTPK were grown at 37°C in 5% CO2 for 16 h. Δtpk and Δtpk+pDL278 were grown at 37°C in 5% CO2 for 72 h. Δtpk is a recombinant strain based on ATCC BAA-1138, harboring the 276_277insG insertion in tpk, resulting in a deletion in premature termination at amino acid 103 in thiamin pyrophosphokinase (TPK). pDL278 is a gram-positive and gram-negative shuttle vector. pTPK is a complementation plasmid used to express intact TPK in the Δtpk strain.
Fig 3.
Comparison of growth rate of GBS ATCC strain, recombinant strain and complemented strains.
Growth curves (OD600nm) of the clinical isolates (MRY11-004, MRY11-005, and NUBL-2449), GBS ATCC strain (BAA-1138), recombinant strain (Δtpk), and complemented strains (Δtpk+pDL278 and Δtpk+pTPK) at 37°C in ambient air (A,B). OD600nm = 0–2.5 (A), OD600nm = 0–0.2 (B). Data are presented as averages of five independent experiments. Data are the mean ± standard deviation OD600nm. *,**Statistically significant difference: *P < 0.05; **P < 0.01. Δtpk indicates a recombinant strain based on ATCC BAA-1138, harboring the 276_277insG insertion in tpk, resulting in premature termination at amino acid 103 in thiamin pyrophosphokinase (TPK). pDL278 is a gram-positive and gram-negative shuttle vector. pTPK is a complementation plasmid used to express intact TPK in the Δtpk strain. Δtpk+TPP indicates Δtpk grown in Todd–Hewitt broth containing thiamine pyrophosphate (500 μg/L).
Fig 4.
Comparison of auxotrophy of GBS ATCC strain, recombinant strain and complemented strains.
Colony morphology of the clinical isolate MRY11-004 (A–D) and recombinant strain Δtpk (E–H) grown on sheep blood agar plate (A, B, E, F) and Mueller Hinton Agar with 5% sheep blood (C, D, G, H) after incubation in 5% CO2 overnight. In B, D, F, and H, the left disc contained 20 μg of thymidine (thy) and the right disc contained 20 μg of thiamin pyrophosphate (TPP). In A, C, E, and G, both the left and right discs were empty (E). Δtpk is a recombinant strain based on ATCC BAA-1138, harboring the 276_277insG insertion in the thiamin pyrophosphokinase (tpk) gene, resulting in premature termination at amino acid 103 in TPK.
Fig 5.
Comparison of TEM analysis of GBS ATCC strain, recombinant strain and complemented strains.
TEM of BAA-1138 (A and F), Δtpk with TPP 500 μg/L (B and G), Δtpk (C and H), and MRY11-004 (D, E and I). Bars: 200 nm (A–E). Bars: 1 μm (F–I) Δtpk is a recombinant strain based on ATCC BAA-1138, harboring the 276_277insG insertion in tpk, resulting in premature termination at amino acid 103 in thiamin pyrophosphokinase.