Fig. S1. Alignment of KaiB sequences of different prokaryotes. In blue, the residues that are well conserved among different species. In bold letter are marked the acidic residues found in the C-terminal described as important for interaction with KaiC for Cyanobacteria [red box (Iwase et al. 2005)]. Legionella pneumophila KaiB misses the acidic residues, although it shares 97% similarity to the KaiB of Cyanobacteria.

Fig. S2. Sequence of the Legionella pneumophila fusion protein KaiB-T. Red, the C-terminal residues from T. elongatus that were fused to KaiB of L. pneumophila.

Fig. S3. Complementation of ΔkaiC and Δlpp1114 mutants under oxidative stress conditions.

A. The sensitivity to 10 mM paraquat of the ΔkaiC strain is complemented by the complete operon (ΔkaiC/pMMB207_kaioperon) in trans.

B. The sensitivity to 10 mM paraquat of the Δlpp1114 strain is complemented by lpp1114lpp1114_pMMB207lpp1114) in trans. Blue, wild-type strain; red ΔkaiC strain; orange ΔkaiC/pMMB207_kaioperon; cyan Δlpp1114; green Δlpp1114_pMMB207lpp1114. Each point represents the mean ± SD of at least two independent experiments.

Fig. S4. Comparison of the survival kinetics of wt Legionella pneumophila and letA, rpoS, stuC and stuA/C mutan strains. Legionella pneumophila ΔrpoS and ΔletA mutants are sensitive to oxidative stress exerted 10 mM paraquat. In agreement with our findings RpoS seems to be regulating the kai operon genes. The ΔrpoS mutant (red curve) has a strong sensitivity to the effect of paraquat in comparison to the wt strain. Interestingly, also the ΔletA mutant shows strong sensitivity to the effects of paraquat. StuC does not show any higher sensitivity to paraquat than the wt, but the ΔstuA/C double mutant is slightly more sensitive as compared with the wt.

Fig. S5. Phylogenetic tree of the aminoacid sequences of KaiB. The tree was obtained by likelihood method. Bootstrap values > 50 are shown in the corresponding nodes.

Fig. S6. Alignment of the KaiC sequence of Legionella and Cyanobacteria. The alignment shows that the Walker motifs in the KaiCI and KaiCII (CI and CII respectively) domains are well conserved in Legionella. The two main phosphorylation residues in cyanobacterial KaiC are serine/threonine (S431 and T432 in Synechococcus sp.) and there is a third phosphorylation site (T426 in Synechococcus). All of them are well conserved in all species (red boxes). Interestingly the Synechocystis KaiC2 shows serine/serine phosphorylation residues like Legionella.

Table. S1. Expression of the kai operon genes in an ΔrpoS mutant.

Table. S1.Kai operon genes are down-regulated in the ΔrpoS mutant.

Table. S2. Primers used for quantitative real-time polymerase chain reaction.

Table. S3.  Primers used for quantitative real-time polymerase chain reaction.

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