Entrainment of the Circadian Clock of the Enteric Bacterium Klebsiella aerogenes by Temperature Cycles

Abstract

The gastrointestinal bacterium Klebsiella (née Enterobacter) aerogenes expresses an endogenously generated, temperature-compensated circadian rhythm in swarming motility. We hypothesized that this rhythm may be synchronized/entrained in vivo by body temperature (T_B). To determine entrainment, cultures expressing bioluminescence were exposed to temperature cycles of 1°C (35°C-36°C) or 3°C (34°C-37°C) in amplitude at periods (T-cycles) of T = 22, T = 24, or T = 28 h. Bacteria entrained to all T-cycles at both amplitudes and with stable phase relationships. A high-amplitude phase response curve (PRC) in response to 1-h pulses of 3°C temperature spike (34°C-37°C) at different circadian phases was constructed, revealing a Type-0 phase resetting paradigm. Furthermore, real-time bioluminescence imaging revealed a spatiotemporal pattern to the circadian rhythm. These data are consistent with the hypothesis that the K. aerogenes circadian clock entrains to its host via detection of and phase shifting to the daily pattern of T_B.

Keywords: Biological Sciences; Chronobiology; Microbiology.

Graphical abstract

Figure 1

K. aerogenes motA Expression Entrains to a 24-h T-Cycle Bioluminescence from swarming cultures entrained to (A) 3°C or (B) 1°C cycles of 12H:12L. High temperature is indicated by white rectangles and low temperature by black rectangles above entrainment traces in (A) and (B) Representative traces are in black, and other samples determined as rhythmic by MetaCycle are in gray. Rhythms were driven equally well by 3°C in the absence (A, left) or presence (A, right) of 1 nM melatonin. After release into constant low temperature, circadian rhythms persisted in both vehicle-treated (C, left) or melatonin-treated (C, right) cultures. Period analysis (E, top) showed no significant difference in period length in entrained or free-running cultures. Phases of entrained and free-running cultures showed no significant differences (F, top). Amplitudes between entrained and free-running cultures were significantly different in both melatonin-treated and non-treated cultures (G, top). Cycles of temperature of 1°C also entrained cultures (B) and rhythms persisted in constant low temperature (D); however, amplitude was significantly lower than that of 3°C cycling conditions (G, bottom). Period length showed higher variation compared with higher-amplitude temperature cycles (E, bottom), but no significant difference between entrained and free-running cultures was observed. Phases of entrained and free-running cultures under 1°C variation were not significantly different (F, bottom). Amplitudes between entrained and free-running cultures were significantly different under 1°C variation (G, bottom). Asterisks indicate p < 0.001 as determined by one-way ANOVA on ranks with Dunn's post hoc test.

Figure 2

K. aerogenes motA Expression Entrains to a 22-h T-Cycle Bioluminescence from swarming cultures entrained to (A) 3°C or (B) 1°C cycles of 11H:11L. High temperature is indicated by white rectangles and low temperature by black rectangles above entrainment traces in (A) and (B) Representative traces are in black, and other samples determined as rhythmic by MetaCycle are in gray. Rhythms were driven equally well by 3°C in the absence (A, left) or presence (A, right) of 1 nM melatonin. After release into constant low temperature, circadian rhythms persisted in both vehicle-treated (C, left) or melatonin-treated (C, right) cultures. Period analysis (E, top) showed no significant difference in period length in entrained or free-running cultures. Similarly, the phases of entrained and free-running cultures showed no significant differences (F, top). Amplitudes between entrained and free-running cultures were significantly different in both melatonin-treated and non-treated cultures (G, top). Cycles of temperature of 1°C also entrained cultures (B), and rhythms persisted in constant low temperature (D). Period length variability was increased compared with cultures under higher temperature variation (E, bottom), but no significant difference between entrained and free-running cultures was observed. Phases of entrained and free-running cultures under 1°C variation were not significantly different (F, bottom). Amplitudes between entrained and free-running cultures were significantly different under 1°C variation (G, bottom). Asterisks indicate p < 0.001 as determined by one-way ANOVA on ranks with Dunn's post hoc test.

Figure 3

K. aerogenes motA Expression Entrains to a 28-h T-Cycle Bioluminescence from swarming cultures entrained to (A) 3°C or (B) 1°C cycles of 14H:14L. High temperature is indicated by white rectangles and low temperature by black rectangles above entrainment traces in (A) and (B) Representative traces are in black, and other samples determined as rhythmic by MetaCycle are in gray. Rhythms were driven equally well by 3°C in the absence (A, left) or presence (A, right) of 1 nM melatonin. After release into constant low temperature, circadian rhythms persisted in both vehicle-treated (C, left) or melatonin-treated (C, right) cultures. Period analysis (E, top) showed complete entrainment to 28-h T-cycles that decreased upon release into constant low temperature. After aligning peak phase to onset of higher temperature, no significant difference in phase was observed (F, top). Amplitudes between entrained and free-running cultures were not significantly different in either melatonin-treated or non-treated cultures (G, top). Cycles of temperature of 1°C also entrained cultures (B), and rhythms persisted in constant low temperature (D); however, amplitude was significantly lower than that of 3°C cycling conditions (G, bottom). Period length showed higher variation compared with higher-amplitude temperature cycles (E, bottom), but no significant difference between entrained and free-running cultures was observed. Phases of entrained and free-running cultures under 1°C variation were not significantly different (F, bottom). Asterisks indicate p < 0.001 as determined by one-way ANOVA on ranks with Dunn's post hoc test.

Figure 4

K. aerogenes Exhibits “Type 0” Phase Resetting Behavior in Response to Temperature Schematic (A) describing the experimental design of recording 3 days of free running (after 3 days of 3°C 12H:12L entrainment) followed by a 1-h pulse of 3°C and return to free-running conditions for an additional 3 days. PRC (B) generated by plotting the magnitude of phase shift across 10 phases. Each data point represents a single culture, with multiple experiments comprising the entire dataset. CT6 here is defined as the pre-pulse peak of bioluminescence. A Phase Transition Curve (C) was generated by plotting the pre-pulse phase against the post-pulse phase from the PRC. Linear regression analysis is indicated by the solid line segment. The dashed line segment represents a slope equal to 1.

Figure 5

Real-Time Bioluminescence Imaging of K. aerogenes Bioluminescence was measured from a 24-well cell culture plate containing swarming cultures of K. aerogenes initially high-amplitude rhythm that damps over time (A). Two representative wells (B) are shown with inset images of the quantified signal at 4-h intervals after baseline correction. The full plate can be seen in Video S1. Average period of bioluminescence from all intact cultures was ∼25.0 h. Cultures that cracked during the imaging were not included in the analysis.