Precise circadian clocks in prokaryotic cyanobacteria

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle. This biological clock orchestrates global regulation of gene expression and controls the timing of cell division. Proteins that may be involved in input pathways have been identified. Mutational screening has identified three clock genes that are organized as a gene cluster. The structure of cyanobacterial clock proteins, their phosphorylation, and regulation is described. A new model for the core clockwork in cyanobacteria proposes that rhythmic changes in the status of the chromosome underlie the rhythms of gene expression. Mixed-strain experiments demonstrate that this timekeeper confers adaptive value when different strains compete against each other.     

蓝藻生物钟分子机理的研究进展

蓝藻是具有内源性生物钟的简单生物.虽然蓝藻生物钟具有跟真核生物同样的基础特征,但其相关基因和蛋白质与真核生物没有同源性.蓝藻生物钟的核心是kai基因簇及其编码的蛋白KaiA,KaiB和KaiC.这三种Kai蛋白相互作用调节KaiC的磷酸化状态,从而产生昼夜节律信息.KaiC的磷酸化循环是昼夜节律的起博器,调控包括kai基因在内的相关基因的节律性表达.组氨酸蛋白激酶的磷酸化传递可将环境信息输入和将节律信息输出生物钟核心.     

Cyanobacterial circadian clockwork: roles of KaiA,KaiB and the kaiBC promoter in regulating KaiC

Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not. One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter. On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo. The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA. Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC. These observations are integrated into a model proposing rhythmic changes in chromosomal status.     

Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.     

Circadian timing mechanism in the prokaryotic clock system of cyanobacteria

Cyanobacteria are the simplest organisms known to exhibit circadian rhythms and have provided experimental model systems for the dissection of basic properties of circadian organization at the molecular, physiological, and ecological levels. This review focuses on the molecular and genetic mechanisms of circadian rhythm generation in cyanobacteria. Recent analyses have revealed the existence of multiple feedback processes in the prokaryotic circadian system and have led to a novel molecular oscillator model. Here, the authors summarize current understanding of, and open questions about, the cyanobacterial oscillator.     

湖泊蓝藻水华发生机理研究进展

蓝藻水华是富营养化湖泊常见的生态灾害,通过产生毒素、死亡分解时使水体缺氧和破坏正常的食物网威胁到饮用水安全、公众健康和景观,会造成严重的经济损失和社会问题,揭示其发生机理是进行防治的基础。综述了蓝藻水华发生机理的主要假说和证据,主要分为环境因子(营养盐、氮磷比、温度、微量元素、浮游动物牧食、水文和气象条件等)和生理生态特性(伪空泡、胶质鞘、CO2浓缩机制、适应低光强、贮藏营养物质、防晒、产毒素和固氮等)两个方面;评述了主要新理论,展望了今后的研究。到目前为止的研究表明寻找一两个关键因子并不能阐明蓝藻水华的发生机理。现存的理论或假说尽管已经在蓝藻水华的防治实践中产生重要作用,但仍然未能清楚地阐释其发生的客观规律。认为蓝藻水华是在各种环境因子(外因)的耦合驱动下,水华蓝藻由于其独特的生理生态特性(内因),产生巨大的生物量而在浮游植物群落中占绝对优势,在合适的水文气象条件下集聚于水表而形成。因此水华机理的研究应同时关注水华蓝藻的生理生态学规律和蓝藻水华发生的各种环境条件。不同环境因子协同影响水华蓝藻的不同生理生态特性的表达,从而影响水华的发生过程,将可能是以后研究的重点。蓝藻水华机理的研究在微观方面正趋向于应用分子生物学手段分析蓝藻生理过程,宏观方面则将广泛应用遥感遥测技术观测全湖蓝藻的变化规律。今后加强对水华蓝藻生理生态特性的基因表达与调控和环境多因子耦合作用于蓝藻水华过程的研究将有重要意义。蓝藻水华的机理研究包括现象、过程和原因3个层次的问题,通过大量的现象和过程的研究,不断揭示其发生过程中水华蓝藻的群落演替、种群发展、细胞活性和分子机理等变化规律,才能找到其发生的真正原因,为其防治提供理论依据和更好的治理措施。在蓝藻水华防治方面,控制营养盐和生态修复可能将是今后很长时间内最根本最有效和最具操作性的方法。     

The current state and problems of circadian clock studies in cyanobacteria

Circadian rhythms have been observed in innumerable physiological processes in most of organisms. Recent molecular and genetic studies on circadian clocks in many organisms have identified and characterized several molecular regulatory factors that contribute to generation of such rhythms. The cyanobacterium is the simplest organism known to harbor circadian clocks, and it has become one of most successful model organisms for circadian biology. In this review, we will briefly summarize physiological observations and consideration of circadian rhythms in cyanobacteria, molecular genetics of the clock using Synechococcus, and current knowledge of the input and output pathways that support the cellular circadian system. Finally, we will document some current problems in the studies on the cyanobacterial circadian clock.     

Anabaena circadian clock proteins KaiA and KaiB reveal a potential common binding site to their partner KaiC

The cyanobacterial clock proteins KaiA and KaiB are proposed as regulators of the circadian rhythm in cyanobacteria. Mutations in both proteins have been reported to alter or abolish circadian rhythmicity. Here, we present molecular models of both KaiA and KaiB from the cyanobacteria Anabaena sp PCC7120 deduced by crystal structure analysis, and we discuss how clock-changing or abolishing mutations may cause their resulting circadian phenotype. The overall fold of the KaiA monomer is that of a four-helix bundle. KaiB, on the other hand, adopts an alpha-beta meander motif. Both proteins purify and crystallize as dimers. While the folds of the two proteins are clearly different, their size and some surface features of the physiologically relevant dimers are very similar. Notably, the functionally relevant residues Arg 69 of KaiA and Arg 23 of KaiB align well in space. The apparent structural similarities suggest that KaiA and KaiB may compete for a potential common binding site on KaiC.     

Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data

Taxonomic affiliations and molecular diversity of 41 heterocystous cyanobacteria representing 12 genera have been assessed on an evolutionary landscape using rbcl gene sequence data-based phylogenomics and evogenomics approaches. Phylogenetic affiliations have clearly demonstrated the polyphyly of the true branching cyanobacteria, along with a frequent intermixing amongst the heterocystous cyanobacteria. The monophyletic origin of the heterocystous cyanobacteria was also quite evident from maximum parsimony and neighbor joining analyses. Incongruency with the traditional scheme of cyanobacterial taxonomy was frequently observed, thus advocating towards some re-amendments in the cyanobacterial classificatory schemes. Evogenomics analyses of gene sequence data gave a clear indication about the greater evolutionary pace of the unbranched cyanobacteria as compared to the branched forms. It was evident that the order Nostocales would be controlling the future pace of evolution of heterocystous cyanobacteria. The cyanobacteria Nostoc was found to have the greatest genetic heterogeneity amongst the studied genera, along with some evidence towards events of lateral gene transfer amongst the heterocystous cyanobacteria in case of the rbcl gene. Thus, heterocystous cyanobacteria were found to be a fast evolving group, with estimates of gene conversion tracts pointing towards the unbranched heterocystous cyanobacteria being at the base of evolutionary diversifications of the complete heterocystous lineage.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

Engineered cyanobacteria: teaching an old bug new tricks

Cyanobacteria have played an important role in the development of the Earth and have long been studied as model organisms for photosynthesis and the circadian rhythm. Recent developments have led to increased interest in the use of engineered cyanobacteria for the production of protein and chemical products. This review highlights the genetic tools and strategies for manipulation of cyanobacteria as well as previous accomplishments in the development of engineered cyanobacteria for applied use. Particular attention is given to the engineering of cyanobacteria for biofuel production, including both hydrocarbon and hydrogen fuels. Genetic engineering efforts to enhance cyanobacterial fitness are reviewed with an emphasis on physiological improvements for large-scale production. Lastly, a future outlook on engineered cyanobacteria is presented, highlighting the future areas of focus and technical challenges in this field. With the uncertainty of future energy security, it is an exciting time in applied cyanobacterial research, but we must take the time to learn from these past accomplishments before we can capitalize on the potential of these photosynthetic microorganisms.     

Circadian clock-protein expression in cyanobacteria: rhythms and phase setting

The cyanobacterial gene cluster kaiABC encodes three essential circadian clock proteins: KaiA, KaiB and KaiC. The KaiB and KaiC protein levels are robustly rhythmical, whereas the KaiA protein abundance undergoes little if any circadian oscillation in constant light. The level of the KaiC protein is crucial for correct functioning of the clock because induction of the protein at phases when the protein level is normally low elicits phase resetting. Titration of the effects of the inducer upon phase resetting versus KaiC level shows a direct correlation between induction of the KaiC protein within the physiological range and significant phase shifting. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. When the metabolism is repressed by either translational inhibition or constant darkness, the rhythm of KaiC abundance persists; therefore, clock protein expression has a preferred status under a variety of conditions. These data indicate that rhythmic expression of KaiC appears to be a crucial component of clock precession in cyanobacteria.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Transgenic plants with cyanobacterial genes

Over the years, cyanobacteria have been regarded as ideal model systems for studying fundamental biochemical processes like oxygenic photosynthesis and carbon and nitrogen assimilation. Additionally, they have been used as human foods, sources for vitamins, proteins, fine chemicals, and bioactive compounds. Aiming to increase plant productivity as well as nutritional values, cyanobacterial genes involved in carbon metabolism, fatty acid biosynthesis, and pigment biosynthesis have been intensively exploited as alternatives to homologous gene sources. In this short review, transgenic plants with cyanobacterial genes generated over the last two decades are examined, and the future prospects for transgenic crops using cyanobacterial genes obtained from functional genomics studies of numerous cyanobacterial genomes information are discussed.     

Gene copy number variation and its significance in cyanobacterial phylogeny

ABSTRACT: BACKGROUND: In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. RESULTS: We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic istances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions: A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria.