Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC, KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.
The mammalian protein CHRONO was previously identified to be a rhythmically expressed repressor of the circadian transcriptional activator complex CLOCK:BMAL1. Mice and cells lacking CHRONO display a lengthened circadian period and altered circadian gene expression. Currently, however, we lack specific mechanistic understanding of CHRONO′s activity and function. Here we define an evolutionarily conserved minimal repressive domain (MRD) of CHRONO and demonstrate this domain′s capacity to repress CLOCK:BMAL1 activity through interaction with the BMAL1 C-terminal transactivation domain (TAD). Notably, this binding region overlaps with the binding site for CRY and coactivators CBP/p300, with CHRONO capable of competing with both of these classical regulators of BMAL1 for TAD binding, highlighting this as a hotspot for BMAL1 regulation. Additionally, we investigate the previously unexplored interaction between CHRONO and another major circadian repressor, PER2. We show that CHRONO reduces PER2 stability through interaction between the CHRONO C-terminus and the Casein Kinase 1 (CK1)-binding domain of PER2. This results in competition between CHRONO and CK1 for binding at this site on PER2, adding another layer to our understanding of PERIOD protein regulation. Taken together, these data show a more substantive role for CHRONO within molecular circadian timekeeping than previously posited and provide a platform for further investigation into CHRONO′s role within the circadian repressive complex.
The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPS), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts, and thus for formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation, and can facilitate invasion in the absence of succinoglycan on some host plants. Biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to: identify putative pyruvyltransferase and acetyltransferase genes; construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan; and analyze EPS from mutant bacterial strains. EPS samples were examined by 13 C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enable detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains, with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through ectopic expression of plasmid-encoded wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPS) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. Biosynthesis of galactoglucan is not as well characterized as succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into biosynthesis of an important S. meliloti EPS and expands knowledge of enzymes that modify polysaccharides.
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