The circadian oscillator of the cyanobacterium Synechococcus elongatus, like those in eukaryotes, is entrained by environmental cues. Inactivation of the gene cikA (circadian input kinase) shortens the circadian period of gene expression rhythms in S. elongatus by approximately 2 hours, changes the phasing of a subset of rhythms, and nearly abolishes resetting of phase by a pulse of darkness. The CikA protein sequence reveals that it is a divergent bacteriophytochrome with characteristic histidine protein kinase motifs and a cryptic response regulator motif. CikA is likely a key component of a pathway that provides environmental input to the circadian oscillator in S. elongatus.

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.

In the cyanobacterium Synechococcus elongatus (PCC 7942) the proteins KaiA, KaiB, and KaiC are required for circadian clock function. We deduced a circadian clock function for KaiA from a combination of biochemical and structural data. Both KaiA and its isolated carboxyl-terminal domain (KaiA180C) stimulated KaiC autophosphorylation and facilitated attenuation of KaiC autophosphorylation by KaiB. An amino-terminal domain (KaiA135N) had no function in the autophosphorylation assay. NMR structure determination showed that KaiA135N is a pseudo-receiver domain. We propose that this pseudo-receiver is a timing input-device that regulates KaiA stimulation of KaiC autophosphorylation, which in turn is essential for circadian timekeeping.

In the cyanobacterium Synechococcus elongatus (PCC 7942) the kai genes A, B, and C and the sasA gene encode the functional protein core of the timing mechanism essential for circadian clock regulation of global gene expression. The Kai proteins comprise the central timing mechanism, and the sensor kinase SasA is a primary transducer of temporal information. We demonstrate that the circadian clock also regulates a chromosome compaction rhythm. This chromosome compaction rhythm is both circadian clockcontrolled and kai-dependent. Although sasA is required for global gene expression rhythmicity, it is not required for these chromosome compaction rhythms. We also demonstrate direct control by the Kai proteins on the rate at which the SasA protein autophosphorylates. Thus, to generate and maintain circadian rhythms in gene expression, the Kai proteins keep relative time, communicate temporal information to SasA, and may control access to promoter elements by imparting rhythmic chromosome compaction.cyanobacteria ͉ regulation C ircadian clocks have evolved within the cyanobacteria (an extremely diverse group of oxygenic photosynthesizing bacteria) and many, if not all, eukaryotes. These clocks effectively tune gene expression patterns, and thus metabolic activity and behavior, to distinct daily frequencies (1-3). In each of several well studied model systems, circadian gene expression patterns are generated and maintained by the combined functions of fairly small sets of proteins (1-3). Amazingly, in vitro combination of only three proteins, KaiA, KaiB, and KaiC, from the cyanobacterium Synechococcus elongatus results in a functional circadian timing mechanism (4). This phenomenon underscores recent data demonstrating that a transcription and translation feedback loop, once considered essential for circadian timing, is not required for rhythmic activity in this cyanobacterium (5). Interestingly, demonstration of this simple proteinaceous clock, and presumption of its straightforward transfer to newly formed daughter cells, explains the enigma of how a circadian (24-h) timing mechanism can function in cyanobacteria that have generation times of 8 h or less.Despite those compelling data, questions concerning how this timing mechanism connects circadian clock function to global regulation of gene expression still loom (6). Existent data show functional interactions among the three Kai proteins and the SasA sensor kinase protein as essential for this global regulation (7). For example, the KaiC protein forms a homohexamer upon binding ATP and is an autokinase. It also interacts with double-stranded DNA molecules (8, 9). Overproduction of KaiC represses gene expression on a global scale (8, 9). In an sasA-null strain, except for kai gene expression patterns, all other tested genes are arrhythmically expressed (7). SasA protein thus appears to act as temporal output regulator from the clock. In addition, clock-regulated gene expression rhythms consist of at least two temporal classes (10-12). The major class includes kaiB g...

SummarySignal-responsive components of transmembrane signal-transducing regulatory systems include methyl-accepting chemotaxis proteins and membrane-bound, two-component histidine kinases. Prokaryotes use these regulatory networks to channel environmental cues into adaptive responses. A typical network is highly discriminating, using a speci®c phosphoryl relay that connects particular signals to appropriate responses. Current understanding of transmembrane signal transduction includes periplasmic signal binding with the subsequent conformational changes being transduced, via transmembrane helix movements, into the sensory protein's cytoplasmic domain. These induced conformational changes bias the protein's regulatory function. Although the mutational analyses reviewed here identify a role for the linker region in transmembrane signal transduction, no speci®c mechanism of linker function has yet been described. We propose a speculative, mechanistic model for linker function based on interactions between two putative amphipathic helices. The model attempts to explain both mutant phenotypes and hybrid sensor data, while accounting for recognized features of amphipathic helices.

SummaryAnaerobic respiratory gene expression in Escherichia coli is differentially controlled by nitrate and nitrite through dual interacting two-component regulatory systems. The NarX sensor is one of two membranespanning sensor kinases that control the phosphorylation state of two DNA-binding response regulators. We have studied NarX autophosphor ylation in crude membrane preparations from cells that overexpress NarX protein. The low basal autophosphorylation rate was stimulated about sixfold and threefold by nitrate and nitrite respectively. This demonstrates that nitrate and nitrite differentially activate NarX autokinase activity. We also isolated single-residue substitutions in NarX that affect its ability to respond to or discriminate between nitrate and nitrite. Most of these substitutions affect residues within the conserved P-box sequence in the periplasmic domain. We characterized several of the mutants in vivo, by monitoring ligandregulated gene expression, and in vitro, by monitoring ligand-responsive autophosphor ylation. At least one change, K49I (Lys at position 49 changed to Ile), resulted in a protein with greatly impaired ability to discriminate between nitrate and nitrite. Other changes (H45E and R59K) resulted in proteins that responded normally to nitrate but were unable to respond to nitrite. These results implicate the P-box region in discrimination between subtly different small molecules.

SummaryThe NarX-NarL and NarQ-NarP sensor-response regulator pairs control Escherichia coli gene expression in response to nitrate and nitrite. Previous analysis suggests that the Nar two-component systems form a cross-regulation network in vivo. Here we report on the kinetics of phosphoryl transfer between different sensor-regulator combinations in vitro. NarX exhibited a noticeable kinetic preference for NarL over NarP, whereas NarQ exhibited a relatively slight kinetic preference for NarL. These findings were substantiated in reactions containing one sensor and both response regulators, or with two sensors and a single response regulator. We isolated 21 NarX mutants with missense substitutions in the cytoplasmic central and transmitter modules. These confer phenotypes that reflect defects in phosphoNarL dephosphorylation. Five of these mutants, all with substitutions in the transmitter DHp domain, also exhibited NarP-blind phenotypes. Phosphoryl transfer assays in vitro confirmed that these NarX mutants have defects in catalysing NarP phosphorylation. By contrast, the corresponding NarQ mutants conferred phenotypes indicating comparable interactions with both NarP and NarL. Our overall results reveal asymmetry in the Nar cross-regulation network, such that NarQ interacts similarly with both response regulators, whereas NarX interacts preferentially with NarL.

Cyanobacteria such as Synechococcus elongatus PCC 7942 exhibit 24-h rhythms of gene expression that are controlled by an endogenous circadian clock that is mechanistically distinct from those described for diverse eukaryotes. Genetic and biochemical experiments over the past decade have identified key components of the circadian oscillator, input pathways that synchronize the clock with the daily environment, and output pathways that relay temporal information to downstream genes. The mechanism of the cyanobacterial circadian clock that is emerging is based principally on the assembly and disassembly of a large complex at whose heart are the proteins KaiA, KaiB, and KaiC. Signal transduction pathways that feed into and out of the clock employ protein domains that are similar to those in two-component regulatory systems of bacteria.