investigate whether RyhB acts as a transcriptional activator for the promoter activity of orf1 orf3, and orf16, the reporter plasmids pOrf12 (P orf1-2 ::lacZ), pOrf315 (P orf3-15 ::lacZ), and pOrf1617 (P orf16-17 ::lacZ), each carrying a lacZ reporter gene transcriptionally fused to the putative promoter region of the K2 cps gene Selleck OICR-9429 cluster , were used to transform the K. pneumoniae Temsirolimus concentration strains CG43S3ΔlacZΔfur and ΔlacZΔfurΔryhB. The promoter activity measurements shown in Figure 3C revealed that the deletion of ryhB in ΔlacZΔfur reduced activity of P orf1-2 ::lacZ by at least 50%, while no obvious change was detected in the activity of P orf3-16 ::lacZ. The activity of P orf16-17 ::lacZ was reduced by more than 75% in ΔlacZΔfurΔryhB as compared to the ΔlacZΔfur strain. These results imply that RyhB enhances CPS biosynthesis in K. pneumoniae by boosting the transcriptional level of the orf1 and orf16 gene clusters. Figure 3 RyhB activates the transcriptional level of the orf1 and orf16 . (A) qRT-PCR analyses of the expression of the K2 cps genes (orf1, orf3, and orf16) were measured in Δfur and ΔfurΔryhB strains. (B)
WT strain carrying the IPTG inducible vector pETQ and pETQ-ryhB in response to 100 μM IPTG. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZΔfur and ΔlacZΔfurΔryhB carrying the reporter plasmid pOrf12 (P orf1-2 ::lacZ), Cytidine deaminase pOrf315 (P orf3-15 ::lacZ) or pOrf1617 (P orf16-17 ::lacZ) were determined using log-phased cultures grown in LB broth. The MK-0457 mw results shown are an average of triplicate samples. Error bars indicate standard deviations. RyhB does not affect the rcsA, rmpA2,
and rmpA mRNA expression level In previous studies, K. pneumoniae Fur was found to repress the expression of genes encoding the cps regulatory proteins RcsA, RmpA, and RmpA2 [21, 22]. To investigate whether RyhB affects the expression of rcsA rmpA, and rmpA2 to increase the orf1 and orf16 transcripts, the mRNA levels were measured by qRT-PCR after inducing the expression of ryhB in WT. However, qRT-PCR results did not show a significant effect of ryhB on the mRNA levels of rmpA rmpA2, and rcsA (Data not shown), suggesting that the activation of RyhB on the orf1 and orf16 expression is not via RmpA, RmpA2, and RcsA. Deletion of ryhB attenuated the higher serum resistance in Δfur strain In addition to the roles played by RyhB and Fur in regulating the CPS amount, we suggest that RyhB and Fur may also affect the ability of the strain to resist the bactericidal effects of serum. In a human serum resistance assay, we found that the deletion of fur in WT increased the survival rate in treatment with 75% normal human serum from 63.3% to 87.9% (Figure 4).