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RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

Fig 1

Cys128 is required for growth and regulation of the Piu operon and colonization.

(A) Western blot analysis representative of three independent experiments of PiuA iron transporter lipoprotein levels in the R800 ritR genetic background variants grown in 5% CO2. RitR and NanA are used as controls. Ukn, an unknown cross-reacting protein. (B) Piu promoter activity in the genetic background of R800 ritR variants in 5% CO2 as measured by beta-galactosidase activity and expressed in Miller units. (C) Growth comparison of wild-type D39 cells plus ritR variants under static 5% CO2 and aeration (O2) conditions. (D) Streptonigrin killing assay representative of three biological repeats. (E) Bar graph of three independent experiments as shown in D. (F) Ability of D39 variants to colonize the murine nasopharynx. Mice were inoculated and the colonization let run for 7 days before cells were collected and plated to determine CFUs. Statistics. Colonization data were analyzed by analysis of variance followed by Tukey’s multiple comparisons test. Statistical significance was considered to be a p-value of < 0.05. Each point indicates the CFUs retained in a single animal. Graphs in B and E represent the mean of three independent experiments. Error bars represent +/- standard deviation. One asterisk indicates a p-value of ≤0.05, two of ≤0.01, three of ≤0.001, and four of ≤ 0.0001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test in B, and a two-tailed students t-test in E. ns, not significant.

Fig 1