Fremyella diplosiphon is a freshwater filamentous cyanobacterium that possesses the ability to sense and adapt to changes in ambient light. In a process called complementary chromatic adaptation (CCA), which is predominantly responsive to red light (RL) and green light (GL), the cyanobacterium enhances its photosynthesis by altering the phycobiliprotein composition of its light-harvesting antennae. RcaE, a phytochrome-class photoreceptor, is required for CCA to occur (Kehoe and Grossman, 1996, Science, 273:1409–12). In addition to the pigmentation phenotype associated with CCA, early micrograph studies showed that wild-type (WT) F. diplosiphon displays different cell morphologies under GL and RL conditions (Bennett and Bogorad, 1973, J Cell Biol, 58:419–35).
Microscopic and biochemical analyses confirmed that WT F. diplosiphon strains maintain distinct RL and GL morphologies. Further, analyses of an RcaE null mutant strain (FdBk14) showed that RcaE regulates filament length and cell shape in response to RL and GL. Light-shifting experiments demonstrated that RcaE regulation of light-dependent morphology is photoreversible. Lysozyme-sensitivity experiments with WT and FdBk14 strains established a light-dependent alteration in cell wall integrity associated with the observed morphology differences, thus establishing that RcaE-regulated changes in cellular morphology are correlated with modifications of cell wall structure or composition. Identification and mRNA expression analyses of the cell-shape-determining mre genes from F. diplosiphon demonstrated that mre expression is RcaE-regulated. RT-PCR analyses showed that the expression of mre genes was down-regulated in the FdBk14 strain, indicating that RcaE controls expression of the gene encoding bacterial actin MreB, a cytoskeletal component involved in the regulation of cell shape in many prokaryotic systems.
Sequence analysis of RcaE indicates similarity to plant phytochromes in its N-terminus, as well as to two-component histidine kinases in its C-terminus (Kehoe and Grossman, 1996). In addition, RcaE contains conserved GAF, PAS and Hbox domains which have been associated with chromophore attachment, signal sensing, and phospho-transfer, respectively (Kehoe and Gutu, 2006, Annu Rev Plant Biol, 57:127–50). To determine the role of these domains in RcaE's regulation of CCA, a mutational analysis approach was taken. Mutation of residues within the GAF domain resulted in defects in both pigmentation and cellular morphology. Mutating a cluster of conserved residues within the PAS domain showed that this domain was essential for GL-regulated cellular morphology. Further, mutating a conserved histidine within the Hbox domain confirmed that this residue contributes to the in vivo biochemical activity of RcaE, as both pigmentation and morphology were affected. These studies established that the GAF, PAS and Hbox domains all contribute to the regulation of CCA. Therefore, the analyses in this dissertation work have contributed significantly towards understanding the molecular basis of the photoregulation of cellular morphology in F. diplosiphon, as well as advanced our knowledge of the biochemical mechanisms utilized by RcaE in its regulation of CCA.