The GGDEF protein Dgc2 suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana.

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 (LBDG_02920) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation. IMPORTANCE Self-propelled bacteria often exhibit complex collective behaviors, such as formation of dense-moving clusters, which are exemplified by wandering comet-like and rotating disk-like colonies; however, the molecular details of how these structures are formed are scant. We found that a strain of the filamentous cyanobacterium Leptolyngbya deficient in the GGDEF protein gene dgc2 elicits motility and complex and dynamic colony pattern formation, including comet-like and disk-like clusters. Although c-di-GMP has been reported to activate biofilm formation in some bacterial species, disruption of dgc2 unexpectedly enhanced it, suggesting a novel role for this GGDEF protein for inhibiting both colony pattern formation and biofilm formation.

Quantification of cyanobacterial cyclic di-guanosine monophosphate (c-di-GMP) by liquid chromatography electrospray ionization tandem mass spectrometry.

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

Analysis of the Fine-Tuning of Cyanobacterial Circadian Phase by Monochromatic Light and Long-Day Conditions.

The cyanobacterial circadian-related protein, Pex, accumulates in the dark period of the diurnal light-dark cycle. After the diurnal cycle, an approximately 3 h advance in the phase of the circadian bioluminescence rhythm is observed in pex-deficient mutants, as compared with the wild type. However, it is unclear what type of photosensing mechanism regulates the accumulation and the phase change. In monochromatic light irradiation experiments, Pex accumulation was strongly repressed under blue light conditions; however, only small reductions in Pex accumulation were observed under red or green light conditions. After the diurnal cycle of 12 h of white fluorescent light and 12 h of blue light, the phase advance was repressed more than that of the cycle of 12 h red (or green) light. The phase advance also occurred after 16 h light/8 h dark cycles (long-day cycles) but did not occur after 8 h light/16 h dark cycles (short-day cycles). While Pex is a unique winged helix transcription factor harboring secondary structures (α0 and α4 helices), the importance of the structures is not understood. In in vivo experiments with site-directed mutations in the α0 helix, the obtained mutants, in which Pex was missing the hydrophobic side chain at the 28th or 32nd amino acid residue, exhibited no phase delay after the light/dark cycle. In in vitro DNA binding assays, the mutant proteins showed no binding to the promoter region of the clock gene kaiA. From these results, we propose a molecular model which describes the phase delay in cyanobacteria.

CmpR is important for circadian phasing and cell growth.

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status.

The circadian clock-related gene pex regulates a negative cis element in the kaiA promoter region.

In the cyanobacterium Synechococcus sp. strain PCC 7942, a circadian clock-related gene, pex, was identified as the gene prolonging the period of the clock. A PadR domain, which is a newly classified transcription factor domain, and the X-ray crystal structure of the Pex protein suggest a role for Pex in transcriptional regulation in the circadian system. However, the regulatory target of the Pex protein is unknown. To determine the role of Pex, we monitored bioluminescence rhythms that reported the expression activity of the kaiA gene or the kaiBC operon in pex deficiency, pex constitutive expression, and the wild-type genotype. The expression of kaiA in the pex-deficient or constitutive expression genotype was 7 or 1/7 times that of the wild type, respectively, suggesting that kaiA is the target of negative regulation by Pex. In contrast, the expression of the kaiBC gene in the two pex-related genotypes was the same as that in the wild type, suggesting that Pex specifically regulates kaiA expression. We used primer extension analysis to map the transcription start site for the kaiA gene 66 bp upstream of the translation start codon. Mapping with deletion and base pair substitution of the kaiA upstream region revealed that a 5-bp sequence in this region was essential for the regulation of kaiA. The repression or constitutive expression of the kaiA transgene caused the prolongation or shortening of the circadian period, respectively, suggesting that the Pex protein changes the period via the negative regulation of kaiA.

Structural and biochemical characterization of a cyanobacterium circadian clock-modifier protein.

Circadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an (alpha+beta) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor.

Expression of the circadian clock-related gene pex in cyanobacteria increases in darkness and is required to delay the clock.

The time measurement system of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is analogous to the circadian clock of eukaryotic cells. Circadian clock-related genes have been identified in this strain. The clock-related gene pex is thought to maintain the normal clock period because constitutive transcription or deficiency of this gene causes respectively longer (approximately 28 h) or shorter (approximately 24 h) circadian periods than that of the wild type (approximately 25 h). Here, the authors report other properties of pex in the circadian system. Levels of pex mRNA increased significantly in a 12-h exposure to darkness. Western blotting with a GST-Pex antibody revealed a 13.5-kDa protein band in wild-type cells that were incubated in the dark, while this protein was not detected in pex-deficient mutant cells. Therefore, the molecular weight of the Pex protein appears to be 13.5 kDa in vivo. The PadR domain, which is conserved among DNA-binding transcription factors in lactobacilli, was found in Pex. In the pex mutant, several 12-h light/12-h dark cycles reset the phase of the clock by 3 h earlier (phase advance) compared to wild-type cells. The degree of the advance in the pex mutant was proportional to the number of exposed light-dark cycles. In addition, ectopic induction of pex with an inducible Escherichia coli promoter, Ptrc, delayed the phase in the examined recombinant cells by 2.5 h (phase delay) compared to control cells. These results suggest that the dark-responsive gene expression of pex delays the circadian clock under daily light-dark cycles.

Transcriptional regulation of the circadian clock operon kaiBC by upstream regions in cyanobacteria.

In the cyanobacterium, Synechococcus elongatus PCC 7942, the kaiBC operon is upregulated by the KaiA protein and downregulated by the KaiC protein to generate circadian oscillation. We investigated the regulation of kaiBC transcription. A primer extension and deletion analyses of the upstream region mapped the sufficient promoter region (SPR) to base pairs -55 to +1 (the transcription start site, TSS) and identified a constitutive negative regulatory region upstream of the SPR (base pairs -897 to -56) that extended into the coding sequence of kaiA. Base-pair substitution within the SPR identified a sequence from -52 to -28 that was the essential element for transcription. Most of the examined sequences drove rhythmic expression of a luxAB reporter that was similar to the expression driven by the kaiBC promoter (PkaiBC) and responded to the overexpression of kaiA or kaiC, even in a promoter activity range of 1-8000%. These results indicate that circadian feedback regulation by KaiA and KaiC is addressed to a global step preceding transcription driven by PkaiBC. However, increasing or decreasing the intrinsic activity of PkaiBC greatly affected the rhythm, suggesting that constitutive adjustment of PkaiBC activity by the sequences identified here is essential for the oscillator.

Global gene repression by KaiC as a master process of prokaryotic circadian system.

A kaiABC clock gene cluster was previously identified from cyanobacterium Synechococcus elongatus PCC 7942, and the feedback regulation of kai genes was proposed as the core mechanism generating circadian oscillation. In this study, we confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria. We probed the nature of this regulation and found that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome. This result strongly suggests that the KaiC protein primarily coordinates genomewide gene expression, including its own expression. We also found that a promoter derived from E. coli is feedback controlled by KaiC and restores the complete circadian rhythm in kaiBC-inactivated arrhythmic mutants, provided it can express kaiB and kaiC genes at an appropriate level. Unlike eukaryotic models, specific regulation of the kaiBC promoter is not essential for cyanobacterial circadian oscillations.

Bipolar localization of putative photoreceptor protein for phototaxis in thermophilic cyanobacterium Synechococcus elongatus.

We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp. PCC6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The mRNA of S. elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon. SePixJ was detected exclusively in the membrane fraction after cell fractionation. Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell; probably bound with the cytoplasmic membrane.