Transcriptional Regulation in Synechocystis sp. PCC 6803 by Duplicated Hik31 Operons as Master Regulators of Central Metabolism

Sowmya Nagarajan, Purdue University

Abstract

Nagarajan, Sowmya. Ph.D., Purdue University, May 2013. Transcriptional Regulation in Synechocystis sp. PCC 6803 By Duplicated Hik31 Operons As Master Regulators of Central Metabolism. Major Professor: Louis Allen Sherman. 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 were not redundant and regulated common and separate targets in the light and the dark. The chromosomal operon is involved in the negative control of autotrophic events, whereas the plasmid operon is 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-CO2 conditions and the plasmid operon under low-O2 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 determined 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 in the light 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. A P3 mutant (deltaP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light-dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, and energy metabolism genes, and an upregulation of cell envelope and transport and binding genes in the mutant. DeltaP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism, cell division and the circadian cycle in the dark. The C3 mutant, on the other hand, did not show a strong growth defect and transcriptional impact in the same conditions beyond the first light period and recovered in later time points. We compared the transcriptome data from both mutants and determined many potential target genes that were commonly affected, oppositely affected and uniquely affected, revealing the importance of these operons as master regulators of key genes in the light and the dark. The re-sequencing results for our WT lab strain revealed the genotype and the plasmid copy number that was comparable to the chromosome for the large plasmids, and varied from 4-8 times that of the chromosome for the small plasmids. This sheds light on the importance of plasmid genes in complementing the functions of the chromosomal genes. Taken together, our results reveal new connections between gene regulation in the diurnal cycle of this organism and carbon processing metabolic pathways, and have implications for improved growth for biofuel production. Consequently, we believe that this research will provide valuable information that will impact the work of several researchers in the field and advance earlier models on gene signaling.

Degree

Ph.D.

Advisors

Sherman, Purdue University.

Subject Area

Biology|Molecular biology|Microbiology

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