Functions of the duplicated hik31 operons in central metabolism and responses to light, dark, and carbon sources in Synechocystis sp. strain PCC 6803

Abstract

There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO(2) conditions and the plasmid operon under low-O(2) conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose.

FIG 1

Construction of the operon and hik mutants and separation of transcripts for the two operons. (A) Diagrammatic representation of the construction of hik31 mutants. We constructed deletion mutants that lacked all three genes in the operon on either the chromosome (the ΔC3 mutant) or the plasmid (the ΔP3 mutant) and on both the chromosome and the plasmid (the ΔC3P3 mutant). The dotted portions were deleted and replaced with various antibiotic resistance cassettes—spectinomycin (Sp^r), kanamycin (Kan^r), and chloramphenicol (Cl^r). Similarly, we also made deletion mutants that lack hik31 alone on the chromosome (the ΔhikC mutant), on the plasmid (the ΔhikP mutant), and on both the chromosome and the plasmid (the ΔhikCP mutant). Shown are the portions of the genes that were replaced (dotted clear area), deleted extra (+), and left behind (−), as well as the restriction enzymes used. The promoter and the direction of transcription are indicated for each construct. (B) Gels showing PCR confirmation of the mutations and complete segregation. Separate primers were used to selectively amplify the chromosomal and plasmid copies. Sizes of the bands are indicated for the WT and mutants. (C) PCR primer and restriction digest design to differentiate the expression of the two operons. The restriction enzymes KpnI and MlyI cut at unique sites in the plasmid rre and hypo genes, respectively.

FIG 2

Transmission electron microscopy under PA conditions. Transmission electron micrographs of WT Synechocystis sp. strain PCC 6803 (A, D, and G), the ΔC3P3 mutant (B, E, and H), and the ΔP3 mutant (C, F, and I) grown under PA conditions under LL (A, B and C), LD (12 h light/12 h dark; D, E, and F), or HL (G, H, and I). Details are described in Materials and Methods. PM, photosynthetic membranes; PP, polyphosphate granules; S, septum. Magnifications: ×28,500 to ×29,400.

FIG 3

Transmission electron microscopy under MT and HT conditions. Transmission electron micrographs of WT Synechocystis sp. strain PCC 6803 (A, D, and G) and the ΔP3 mutant (B, C, E, F, H, and I) grown under MT conditions with 5 mM glucose under LL (A, B, and C) and LD (12 h light/12 h dark; D, E, and F) and under HT conditions in DD (G, H, and I). CG, cyanophycin granules; PHB, polyhydroxybutyrate granules; S, septum. Magnifications, ×20,000 to ×21,000.

FIG 4

Transmission electron microscopy under MT high-light conditions. Transmission electron micrographs of WT Synechocystis sp. strain PCC 6803 (A) and the ΔC3 mutant (B and C) under MT HL. Magnifications: A, ×18,600; B and C, ×12,000.

FIG 5

Regulatory functions of the proteins of the chromosomal and plasmid hik31 operons. Regulatory relationship of the hik31 operons as shown by a Venn diagram representing the different and overlapping functions that are regulated by the hik31 operon on the chromosome (C3) and the plasmid (P3). The operons are represented as bars on the chromosome and plasmid. Both operons regulate major metabolic processes in the light and the dark and show both positive (P3 to C3) and negative control (C3 to P3) to their targets and to each other. ETS, electron transport system.

FIG 6

Transcription of the operon and hik genes of both hik31 operons in the WT under various growth conditions. (A) Expression of the hik31 operon genes in different stages of growth. WT cells were grown under PA LL conditions for 6 days. RNA was extracted just before (t = 0) and after each day (days 1 to 6) of growth for RT-PCR. (B) Expression of the hik31 operon genes after addition of glucose in LL. WT cells were grown for about a day (30 h) under PA LL conditions, and then 5 mM glucose was added and the labeled time points (2, 6, 18, 24, and 48 h) were analyzed in LL. Dotted lines above sample times indicate those taken after the addition of glucose. The 30-h time point represents RNA from cells just before the addition of glucose (t = 0). (C) Expression of the hik31 operon genes after addition of glucose under LD conditions. WT cells were grown for about a day (30 h) under PA LL conditions, 5 mM glucose was added, and samples taken at the labeled time points (2L, 6L, 2D, and 6D) were analyzed in 12 h light, 12 h dark. Dotted lines above sample times indicate samples taken after the addition of glucose. The 30-h time point represents RNA from cells just before the addition of glucose. −RTase, without reverse transcriptase.

FIG 7

Transcription of the hik and rre genes of both hik31 operons in the WT and mutants under various growth conditions. (A) Expression of hik31 operon genes in the WT and operon and hik mutants. Cells were grown for about 3 days under PA LL (lanes 1), PA LD (lanes 2), MT LL (lanes 3), and MT LD (lanes 4) conditions. PA, MT, LL (24 h light), and LD (12 h light/12 h dark) conditions. (B) Expression of the operon and hik genes in all of the strains after growth under high-light conditions Cells were grown for 1 day in PA LL and then exposed to HL at 150 μE m⁻² s⁻¹ for 2 h in the presence (MT) or absence (PA) of 5 mM glucose. The zero time point was taken after growth for about 1 day (26 h) under PA LL conditions to serve as the control. The RnpB and −RTase (without reverse transcriptase) controls were similar to those in the experiments in panel A (data not shown).

FIG 8

Working model of the regulatory relationship between the two hik31 operons. The direction of transcription, expression conditions, and promoters are indicated for each operon, along with the effects of the Hik proteins on the Rre proteins and, in turn, those of the Rre proteins on the targets. The plasmid operon promoter is shown smaller to represent the lower expression results.