Fig. 6From: luxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operonluxS indirectly regulates the expression of fruRKI operon through the cre region in the fruRKI promoter. A The sequence alignment of cre from eight gram-positive bacteria. The conserved regions of cre are highlighted. B Competitive binding of the luxS promoter and the fruRKI promoter to CcpA by EMSA analysis. The 129-bp fragment of luxS promoter was tested for its ability to bind with CcpA (0.4, 0.6 and 0.8 μM) in presence of 188-bp fragment of wild type fruRKI promoter (lane 4, 7 and 10) or fruRKI promoter with mutations on cre (lane 6, 9 and 12); the 188-bp fragment of fruRKI promoter was tested for its ability to bind with CcpA (0.4, 0.6 and 0.8 μM) in presence of 129-bp fragments of wild type luxS promoter (lane 4, 7 and 10) or luxS promoter with mutations on cre (lane 5, 8 and 11); Lane 1 to 3. Different components of internal controls. C The DNA-binding capacity of CcpA was measured with grayscale analysis of the blots. D The fruRKI promoter activity in the WT and ΔluxS strains containing the PMfruRKI-lacZ reporter plasmid. The WT and ΔluxS strains containing T1PfruRKI-lacZ or T2PfruRKI-lacZ reporter plasmids serve as control groups. The β-galactosidase activity was expressed as relative miller units. E Relative mRNA levels of the fruRKI genes determined by real-time PCR. WT-G1: eight-base substitution in the cre conserve region of fruRKI promoter in WT; ΔluxS-G1: eight-base substitution in the cre conserve region of fruRKI promoter in ΔluxS; WT-G2: eight-base substitution in the cre non-conserve region of fruRKI promoter in WT; ΔluxS-G2: eight-base substitution in the cre non-conserve region of fruRKI promoter in ΔluxS. F Intracellular survival rates of the WT, ΔluxS, WT-G1, ΔluxS-G1, WT-G2 and ΔluxS-G2 strains in macrophages. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, or ***P < 0.001.Back to article page