Fig. 6

luxS 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.