Light-dependent expression of four cryptic archaeal circadian gene homologs

Abstract

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. As an initial investigation of this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during balanced growth. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

Keywords: archaea; gene expression; halophiles; mutants.

Figure 1

Amino acid alignments of the N-terminal region of S. elongatus KaiC (SeKaiC) with H. volcanii (HV) CirA, CirB, CirC, and CirD. Predicted Walker A and B motifs, as well a conserved pair of catalytic glutamate residues,are boxed. Identical residues are shaded in black; similar residues are shaded in gray.

Figure 2

Phylogenetic analysis of various known and predicted KaiC sequences from select cyanobacteria and archaea as compared with H. volcanii Cir sequences. Sequence alignments and tree construction, using Maximum Likelihood, were performed using MEGA6 (Tamura et al., 2013). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Accession numbers for the sequences used in the trees are provided in the figure.

Figure 3

Quantitative RT-PCR analysis of cir gene expression. Expression of (A) cirA, (B) cirB, (C) cirC, or (D) cirD during synchronous growth in LD 12:12 cycles. Relative gene expression, as indicated by fold-change, was calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Results given are pooled data collected from three independently-conducted experiments.

Figure 4

Quantitative RT-PCR analysis of cir gene expression in select Δcir mutants. Expression of (A) cirD in JN1; (B) cirB in JN2; (C) cirC in JN3; and (D) cirB in JN1 (pMM2) during synchronous growth in LD 12:12 cycles. Loss of any single cir gene results in arrhythmic gene expression. Complementation of JN1 with an in trans copy of wild-type cirB (pMM2) restores rhythmic gene expression. Relative gene expression, as indicated by fold-change, was calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Results given are pooled data collected from two independently-conducted experiments.