Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris

Abstract

Circadian (daily) rhythms are a fundamental and ubiquitous property of eukaryotic organisms. However, cyanobacteria are the only prokaryotic group for which bona fide circadian properties have been persuasively documented, even though homologs of the cyanobacterial kaiABC central clock genes are distributed widely among Eubacteria and Archaea. We report the purple non-sulfur bacterium Rhodopseudomonas palustris (that harbors homologs of kaiB and kaiC) only poorly sustains rhythmicity in constant conditions-a defining characteristic of circadian rhythms. Moreover, the biochemical characteristics of the Rhodopseudomonas homolog of the KaiC protein in vivo and in vitro are different from those of cyanobacterial KaiC. Nevertheless, R. palustris cells exhibit adaptive kaiC-dependent growth enhancement in 24-h cyclic environments, but not under non-natural constant conditions. Therefore, our data indicate that Rhodopseudomonas does not have a classical circadian rhythm, but a novel timekeeping mechanism that does not sustain itself in constant conditions. These results question the adaptive value of self-sustained oscillatory capability for daily timekeepers and establish new criteria for circadian-like systems that are based on adaptive properties (i.e., fitness enhancement in rhythmic environments), rather than upon observations of persisting rhythms in constant conditions. We propose that the Rhodopseudomonas system is a "proto" circadian timekeeper, as in an ancestral system that is based on KaiC and KaiB proteins and includes some, but not necessarily all, of the canonical properties of circadian clocks. These data indicate reasonable intermediate steps by which bona fide circadian systems evolved in simple organisms.

Fig 1. Daily patterns of nitrogen fixation in WT vs. RCKO strains.

A and C, nitrogen fixation activities of the wild-type R. palustris (WT) at 30°C and 23°C. B and D, nitrogen fixation activities of the kaiC^Rp-deletion strain (RCKO) at 30°C. The three traces represent three individual cultures under anaerobic conditions that were tested at different phases of LD 12:12 after growth for ~2 weeks in LD 12:12. The black and white bars underneath represent the light conditions. Nitrogen fixation rates were calculated based on the amount of C₂H₄ (nmol) produced by 10¹⁰ cells per hour. These two-cycle LD experiments were repeated twice, each time with 3 replicate cultures; one representative experiment is shown in this figure (one-cycle LD assays were conducted in five independent experiments, each time with phasing data equivalent to those shown in this figure). The data of Fig 1 are replotted in S3 Fig with all three replicate cultures averaged together (complete time series data appear in S4 Table).

Fig 2. Characterizations of the RCKO and RCKO+kaiC^Rp strains.

A. The mRNA levels of kaiB^Rp and kaiC^Rp were quantified by quantitative PCR from the WT (red) and RCKO (black) strains. The mRNA levels of clpX were included as an internal control. B. immunoblot by anti-FLAG antibody to confirm the expression of FLAG-KaiC^Rp in the rescued strain (RCKO+kaiC^Rp). C. nitrogen fixation of the RCKO+kaiC^Rp strain under LD12:12 cycles at 30°C. Data using the HA-KaiC^Rp rescued strain are equivalent. The three traces represent three individual cultures. These experiments were repeated twice, each time with 3 replicate cultures; one representative experiment is shown in this figure (complete time series data for panel C appear in S5 Table).

Fig 3. Lack of robust persistence of the nitrogen fixation rhythm in LL after transfer from LD.

A, nitrogen fixation activities of the WT (red) and RCKO (green) strains at 30°C. B, nitrogen fixation activities of the WT (red) and RCKO (green) strains at 23°C. Data is plotted as the mean of three individual cultures. Data points are mean +/- S.D. Black-white bars indicate the light/dark conditions: black is dark, white is light. These experiments were repeated five times (five times for WT, and five times for RCKO), each time with three replicate cultures; one representative experiment is shown in this figure (the three replicate cultures of this experiment are plotted separately in S4 Fig, and complete time series data appear in S6 Table).

Fig 4. Phosphorylation patterns of KaiC^Rp in LD and LL.

A & B. Electrophoresis analyses of ³²P-labeled KaiC^Se and KaiC^Rp. Lane 1 is ³²P-labeled KaiC^Se. Lanes 2–5 are purified KaiC^Rp that was mixed with [γ-³²P]ATP. Samples for lanes 2 and 4 were immediately denatured after the addition of [γ-³²P]ATP by mixing with SDS-PAGE sample buffer (time zero samples, lanes 2 and 4). Samples for lanes 3 and 5 were incubated with [γ-³²P]ATP for 24 h at either 4°C (lane 3) or 30°C (lane 5) prior to SDS denaturation and inactivation. The samples were subjected to either regular SDS-PAGE (A) or phosphate-affinity SDS-PAGE with Phos-Tag (B). In both panels, the left portion is a Coomassie-Blue stained gel (CBB) and the right portion is the autoradiogram of P-32 radioactivity. In panel B, three bands of ³²P-labeled KaiC^Rp are indicated as P1, P2, P3 and an unlabeled KaiC^Rp band is indicated as NP (non-phosphorylated). C & D. The strain in which KaiC^Rp has been tagged with HA (RCKO + HA-kaiC^Rp) was grown under LD 12:12 and then transferred to LL. Cells were collected every 6 h. Total protein extracts were separated on SDS-polyacrylamide gels with Phos-tag and analyzed by immunoblotting using an anti-HA antibody. The specificity of the anti-HA antibody against HA-tagged KaiC^Rp was confirmed with extracts from the RCKO cells. C. immunoblot in which the phospho- (P1, P2, P3) and nonphospho- (NP) forms of KaiC^Rp are indicated. D. quantification of phosphorylation states of HA-KaiC^Rp from the immunoblot in panel C. “ZT” = Zeitgeber Time in LD, where ZT0 is lights-on and ZT12 is lights-off (complete time series data for panel D appear in S7 Table).

Fig 5. ATPase activity of KaiC^Rp and KaiC^Rp-EQ1EQ2 in vitro at 30°C.

A. Purified native KaiC^Rp and the KaiC^Rp-EQ1EQ2 mutant (EQ1EQ2) were incubated with1 mM ATP for 0.5, 1, 2, 4, 12, and 24 h at 30°C. The amount of ATP hydrolyzed was quantified by HPLC. Dark blue, buffer only; red, KaiC^Rp; light blue, KaiC^Rp-EQ1EQ2; green, KaiC^Se. B. ATPase hydrolysis rates of native KaiC^Rp at different temperatures. Q₁₀ was calculated based on these rates. These experiments were repeated twice; one representative experiment is shown in this figure.

Fig 6. Growth rate of R. palustris is kaiC^Rp-dependent in cyclic 24-h conditions.

Cell densities (OD₆₀₀) of the WT (red), RCKO (black) and RCKO-kaiC^Rp (green) strains were measured every two days under (A) LL conditions at 30°C; (B) LD 12:12 cycles at 30°C; (C) LL conditions at 23°C; (D) LD 12:12 cycles at 23°C; (E) LD 1:1 cycles at 30°C; (F) LD 12:12 cycles at 30°C in YPA medium. All cultures were grown anaerobically. These experiments were repeated three times, and each time there were three replicate cultures. In this figure, the data from the three separate experiments were pooled and plotted as the mean +/- S.D. (therefore, each datum is the average of n = 9).