Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101

Abstract

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium Trichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in Trichodesmium sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5 degrees C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.

FIG. 1

Circadian rhythm of nitrogenase activity in Trichodesmium sp. strain IMS 101 cultures grown under different L-D regimens at 28.5°C. Measurements were duplicated (open and filled symbols). L-D cultures (open and filled circles) were incubated under a 12-h-L–12-h-D regimen; L-L cultures (open and filled triangles) were incubated under constant illumination beginning at time zero; D-D cultures (open and filled squares) were incubated under constant darkness since the preceding D phase and throughout the experiment. ∗, note that the nitrogenase activities of L-L cultures (triangles) on days 2 and 3 are scaled to the right y axis.

FIG. 2

Circadian rhythm of nifHDK transcript abundance in Trichodesmium sp. strains IMS 101 cultures grown under different L-D regimens at 28.5°C. (A) Three bands hybridized to the probe; they correspond to 1.1-kb (nifH), 2.8-kb (nifHD), and 4.5-kb (nifHDK) transcripts. Numbers at the top indicate time. (B) Densitometric analysis of the Northern blot results of nifH transcript abundance from panel A.

FIG. 3

Circadian rhythm of MoFe protein abundance in Trichodesmium sp. strain IMS 101 cultures grown under different L-D regimens at 28.5°C. Solid boxes indicate the subjective D phase under constant illumination. (A) Western immunoblot analyses were performed with antiserum raised against the MoFe protein of Rhodospirillum rubrum. Numbers at the top indicate time. (B) Densitometric analysis of the Western blot results in panel A.

FIG. 4

Circadian rhythm of nitrogenase activity, as well as densitometric analysis of nifH transcripts and MoFe protein in Trichodesmium sp. strain IMS 101 cultures grown under constant illumination at 24°C. The boldly outlined box indicates the subjective D phase. Measurements were duplicated for nitrogenase activity.

FIG. 5

Circadian rhythm of nifHDK transcripts (A) and MoFe protein abundance (B) in Trichodesmium sp. strain IMS 101 cultures grown under constant illumination at 24°C. The boldly outlined boxes indicate the subjective D phase. Numbers indicate time.

FIG. 6

Entrainment of nitrogenase activity rhythm by 12-h D–12-h L–12-h D pulses in Trichodesmium sp. strain IMS 101. A 6-h phase difference was imposed during the entrainment. Subculture A: subjective L phase, 1600 to 0400; subjective D phase, 0400 to 1600. Subculture B: subjective L phase, 2200 to 1000; subjective D phase, 1000 to 2200. Measurements were duplicated for nitrogenase activity.

FIG. 7

Entrainment of nifH transcription (A) and MoFe protein abundance (B) rhythm by 12-h D–12-h L–-12-h D pulses in Trichodesmium sp. strain IMS 101. A 6-h phase difference was imposed during the entrainment. Subculture A: subjective L phase, 1600 to 0400; subjective D phase, 0400 to 1600. Subculture B: subjective L phase, 2200 to 1000; subjective D phase, 1000 to 2200. The boldly outlined boxes indicate the subjective D phase.

FIG. 8

Northern blot assay of nifH transcript turnover in Trichodesmium sp. strain IMS 101 during L period (1000 to 2200). (A) Treatments started at 1000, the onset of L period. (B) Treatments started at 1600 (above) and 1800 (below). Numbers above blots indicate time.

FIG. 9

Western blot immunoassay of half-lives of MoFe protein in Trichodesmium sp. strain IMS 101. During the L period (1000 to 2200), chloramphenicol (50 μg/ml) was added at 1000 (A) and 1600 (B). Numbers indicate time.

FIG. 10

Nitrogenase activity (B) and Western blot immunoassay of Fe protein (A) in Trichodesmium sp. strain IMS 101 cultures grown in different L-D regimens. L-D culture was incubated under 12-h L–12-h D; L-L cultures were incubated under continuous L for 72 h prior to zero time point (1000 local time). Numbers at top of panel A indicate time.