Cyanobacterial Two-Component Proteins: Structure, Diversity, Distribution, and Evolution

A survey of the already characterized and potential two-component protein sequences that exist in the nine complete and seven partially annotated cyanobacterial genome sequences available (as of May 2005) showed that the cyanobacteria possess a much larger repertoire of such proteins than most other bacteria. By analysis of the domain structure of the 1,171 potential histidine kinases, response regulators, and hybrid kinases, many various arrangements of about thirty different modules could be distinguished. The number of two-component proteins is related in part to genome size but also to the variety of physiological properties and ecophysiologies of the different strains. Groups of orthologues were defined, only a few of which have representatives with known physiological functions. Based on comparisons with the proposed phylogenetic relationships between the strains, the orthology groups show that (i) a few genes, some of them clustered on the genome, have been conserved by all species, suggesting their very ancient origin and an essential role for the corresponding proteins, and (ii) duplications, fusions, gene losses, insertions, and deletions, as well as domain shuffling, occurred during evolution, leading to the extant repertoire. These mechanisms are put in perspective with the different genetic properties that cyanobacteria have to achieve genome plasticity. This review is designed to serve as a basis for orienting further research aimed at defining the most ancient regulatory mechanisms and understanding how evolution worked to select and keep the most appropriate systems for cyanobacteria to develop in the quite different environments that they have successfully colonized.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.     

Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis

Receptor-like kinases (RLKs) are a family of transmembrane proteins with versatile N-terminal extracellular domains and C-terminal intracellular kinases. They control a wide range of physiological responses in plants and belong to one of the largest gene families in the Arabidopsis genome with more than 600 members. Interestingly, this gene family constitutes 60% of all kinases in Arabidopsis and accounts for nearly all transmembrane kinases in Arabidopsis. Analysis of four fungal, six metazoan, and two Plasmodium sp. genomes indicates that the family was represented in all but fungal genomes, indicating an ancient origin for the family with a more recent expansion only in the plant lineages. The RLK/Pelle family can be divided into several subfamilies based on three independent criteria: the phylogeny based on kinase domain sequences, the extracellular domain identities, and intron locations and phases. A large number of receptor-like proteins (RLPs) resembling the extracellular domains of RLKs are also found in the Arabidopsis genome. However, not all RLK subfamilies have corresponding RLPs. Several RLK/Pelle subfamilies have undergone differential expansions. More than 33% of the RLK/Pelle members are found in tandem clusters, substantially higher than the genome average. In addition, 470 of the RLK/Pelle family members are located within the segmentally duplicated regions in the Arabidopsis genome and 268 of them have a close relative in the corresponding regions. Therefore, tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK/Pelle family in Arabidopsis.     

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.     

Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome

Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions.     

Strain-specific genes of Helicobacter pylori: genome evolution driven by a novel type IV secretion system and genomic island transfer

The availability of multiple bacterial genome sequences has revealed a surprising extent of variability among strains of the same species. The human gastric pathogen Helicobacter pylori is known as one of the most genetically diverse species. We have compared the genome sequence of the duodenal ulcer strain P12 and six other H. pylori genomes to elucidate the genetic repertoire and genome evolution mechanisms of this species. In agreement with previous findings, we estimate that the core genome comprises about 1200 genes and that H. pylori possesses an open pan-genome. Strain-specific genes are preferentially located at potential genome rearrangement sites or in distinct plasticity zones, suggesting two different mechanisms of genome evolution. The P12 genome contains three plasticity zones, two of which encode type IV secretion systems and have typical features of genomic islands. We demonstrate for the first time that one of these islands is capable of self-excision and horizontal transfer by a conjugative process. We also show that excision is mediated by a protein of the XerD family of tyrosine recombinases. Thus, in addition to its natural transformation competence, conjugative transfer of genomic islands has to be considered as an important source of genetic diversity in H. pylori.     

Signal transduction pathways in Synechocystis sp. PCC 6803 and biotechnological implications under abiotic stress

Cyanobacteria have developed various response mechanisms in long evolution to sense and adapt to external or internal changes under abiotic stresses. The signal transduction system of a model cyanobacterium Synechocystis sp. PCC 6803 includes mainly two-component signal transduction systems of eukaryotic-type serine/threonine kinases (STKs), on which most have been investigated at present. These two-component systems play a major role in regulating cell activities in cyanobacteria. More and more co-regulation and crosstalk regulations among signal transduction systems had been discovered due to increasing experimental data, and they are of great importance in corresponding to abiotic stresses. However, mechanisms of their functions remain unknown. Nevertheless, the two signal transduction systems function as an integral network for adaption in different abiotic stresses. This review summarizes available knowledge on the signal transduction network in Synechocystis sp. PCC 6803 and biotechnological implications under various stresses, with focuses on the co-regulation and crosstalk regulations among various stress-responding signal transduction systems.     

Cyanobacteria: photosynthetic factories combining biodiversity,radiation resistance,and genetics to facilitate drug discovery

Cyanobacteria are ancient, abundant, and widely diverse photosynthetic prokaryotes, which are viewed as promising cell factories for the ecologically responsible production of chemicals. Natural cyanobacteria synthesize a vast array of biologically active (secondary) metabolites with great potential for human health, while a few genetic models can be engineered for the (low level) production of biofuels. Recently, genome sequencing and mining has revealed that natural cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The corresponding panoply of enzymes (polyketide synthases and non-ribosomal peptide synthases) of interest for synthetic biology can still be increased through gene manipulations with the tools available for the few genetically manipulable strains. In this review, we propose to exploit the metabolic diversity and radiation resistance of cyanobacteria, and when required the genetics of model strains, for the production and radioactive (¹⁴C) labeling of bioactive products, in order to facilitate the screening for new drugs.

     

A genomic perspective of protein kinases in Plasmodium falciparum

Protein kinases are central to regulation of cellular signaling in the eukaryotes. Well-conserved and lineage-specific protein kinases have previously been identified from various completely sequenced genomes of eukaryotes. The current work describes a genome-wide analysis for protein kinases encoded in the Plasmodium falciparum genome. Using a few different profile matching methods, we have identified 99 protein kinases or related proteins in the parasite genome. We have classified these kinases into subfamilies and analyzed them in the context of noncatalytic domains that occur in these catalytic kinase domain-containing proteins. Compared to most eukaryotic protein kinases, these sequences vary significantly in terms of their lengths, inserts in catalytic domains, and co-occurring domains. Catalytic and noncatalytic domains contain long stretches of repeats of positively charged and other polar amino acids. Various components of the cell cycle, including 4 cyclin-dependent kinase (CDK) homologues, 2 cyclins, 1 CDK regulatory subunit, and 1 kinase-associated phosphatase, are identified. Identification of putative mitogen-activated protein (MAP) Kinase and MAP Kinase Kinase of P. falciparum suggests a new paradigm in the highly conserved signaling pathway of eukaryotes. The calcium-dependent kinase family, well represented in P. falciparum, shows varying domain combinations with EF-hands and pleckstrin homology domains. The analysis reveals a new subfamily of protein kinases having limited sequence similarity with previously known subfamilies. A new transmembrane kinase with 6 membrane-spanning regions is identified. Putative apicoplast targeting sequences have been detected in some of these protein kinases, suggesting their export to the apicoplast.     

Comparative analysis of NBS domain sequences of NBS-LRR disease resistance genes from sunflower, lettuce, and chicory

Plant resistance to many types of pathogens and pests can be achieved by the presence of disease resistance (R) genes. The nucleotide binding site-leucine rich repeat (NBS-LRR) class of R-genes is the most commonly isolated class of R-genes and makes up a super-family, which is often arranged in the genome as large multi-gene clusters. The NBS domain of these genes can be targeted by polymerase chain reaction (PCR) amplification using degenerate primers. Previous studies have used PCR derived NBS sequences to investigate both ancient R-gene evolution and recent evolution within specific plant families. However, comparative studies with the Asteraceae family have largely been ignored. In this study, we address recent evolution of NBS sequences within the Asteraceae and extend the comparison to the Arabidopsis thaliana genome. Using multiple sets of primers, NBS fragments were amplified from genomic DNA of three species from the family Asteraceae: Helianthus annuus (sunflower), Lactuca sativa (lettuce), and Cichorium intybus (chicory). Analysis suggests that Asteraceae species share distinct families of R-genes, composed of genes related to both coiled-coil (CC) and toll-interleukin-receptor homology (TIR) domain containing NBS-LRR R-genes. Between the most closely related species, (lettuce and chicory) a striking similarity of CC subfamily composition was identified, while sunflower showed less similarity in structure. These sequences were also compared to the A. thaliana genome. Asteraceae NBS gene subfamilies appear to be distinct from Arabidopsis gene clades. These data suggest that NBS families in the Asteraceae family are ancient, but also that gene duplication and gene loss events occur and change the composition of these gene subfamilies over time.     

Domain combinations in archaeal, eubacterial and eukaryotic proteomes.

There is a limited repertoire of domain families that are duplicated and combined in different ways to form the set of proteins in a genome. Proteins are gene products, and at the level of genes, duplication, recombination, fusion and fission are the processes that produce new genes. We attempt to gain an overview of these processes by studying the evolutionary units in proteins, domains, in the protein sequences of 40 genomes. The domain and superfamily definitions in the Structural Classification of Proteins Database are used, so that we can view all pairs of adjacent domains in genome sequences in terms of their superfamily combinations. We find 783 out of the 859 superfamilies in SCOP in these genomes, and the 783 families occur in 1307 pairwise combinations. Most families are observed in combination with one or two other families, while a few families are very versatile in their combinatorial behaviour; 209 families do not make combinations with other families. This type of pattern can be described as a scale-free network. We also study the N to C-terminal orientation of domain pairs and domain repeats. The phylogenetic distribution of domain combinations is surveyed, to establish the extent of common and kingdom-specific combinations. Of the kingdom-specific combinations, significantly more combinations consist of families present in all three kingdoms than of families present in one or two kingdoms. Hence, we are led to conclude that recombination between common families, as compared to the invention of new families and recombination among these, has also been a major contribution to the evolution of kingdom-specific and species-specific functions in organisms in all three kingdoms. Finally, we compare the set of the domain combinations in the genomes to those in the RCSB Protein Data Bank, and discuss the implications for structural genomics.     

Two-component signal transduction pathways in Arabidopsis

The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.     

Distribution, structure and diversity of "bacterial" genes encoding two-component proteins in the Euryarchaeota

The publicly available annotated archaeal genome sequences (23 complete and three partial annotations, October 2005) were searched for the presence of potential two-component open reading frames (ORFs) using gene category lists and BLASTP. A total of 489 potential two-component genes were identified from the gene category lists and BLASTP. Two-component genes were found in 14 of the 21 Euryarchaeal sequences (October 2005) and in neither the Crenarchaeota nor the Nanoarchaeota. A total of 20 predicted protein domains were identified in the putative two-component ORFs that, in addition to the histidine kinase and receiver domains, also includes sensor and signalling domains. The detailed structure of these putative proteins is shown, as is the distribution of each class of two-component genes in each species. Potential members of orthologous groups have been identified, as have any potential operons containing two or more two-component genes. The number of two-component genes in those Euryarchaeal species which have them seems to be linked more to lifestyle and habitat than to genome complexity, with most examples being found in Methanospirillum hungatei, Haloarcula marismortui, Methanococcoides burtonii and the mesophilic Methanosarcinales group. The large numbers of two-component genes in these species may reflect a greater requirement for internal regulation. Phylogenetic analysis of orthologous groups of five different protein classes, three probably involved in regulating taxis, suggests that most of these ORFs have been inherited vertically from an ancestral Euryarchaeal species and point to a limited number of key horizontal gene transfer events.     

The phytochromes: A biochemical mechanism of signaling in sight?

The biochemical mechanism by which the phytochrome family of plant sensory photoreceptors transmit perceived informational light signals downstream to transduction pathway components is undetermined. The recent sequencing of the entire genome of the cyanobacterium Synechocystis, however, has revealed a protein that has an NH₂-terminal domain with striking sequence similarity to the photosensory NH₂-terminal domain of the phytochromes, and a COOH-terminal domain strongly related to the transmitter histidine kinase module of bacterial two-component sensors. The Synechocystis protein is capable of autocatalytic chromophore ligation and exhibits photoreversible light-absorption changes analogous to the phytochromes, indicating its capacity to function as an informational photoreceptor. Together with earlier observations that the COOH-terminal domains of the plant phytochromes also have sequence similarity to the histidine kinases, these data suggest that the cyanobacteria utilize photoregulated histidine kinases as a sensory system and that the plant phytochromes may be evolutionary descendants of these photoreceptors.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Cyanobacterial genomics for ecology and biotechnology

Cyanobacteria are the only prokaryotes that directly convert solar energy and CO(2) into organic matter by oxygenic photosynthesis, explaining their relevance for primary production in many ecosystems and the increasing interest for biotechnology. At present, there are more than 60 cyanobacteria for which a total genome sequence is publicly available. These cyanobacteria belong to different lifestyles and origins, coming from marine and freshwater aquatic environments, as well as terrestrial and symbiotic habitats. Genome sizes vary by a factor of six, from 1.44 Mb to 9.05 Mb, with the number of reported genes ranging from 1241 to 8462. Several studies have demonstrated how these sequences could be used to successfully infer important ecological, physiological and biotechnologically relevant characteristics. However, sequences of cyanobacterial origin also comprise a significant portion of certain metagenomes. Moreover, genome analysis has been employed for culture-independent approaches and for resequencing mutant strains, a very recent tool in cyanobacterial research.     

Statistical analyses of protein sequence alignments identify structures and mechanisms in signal activation of sensor histidine kinases

Statistical analyses of genome sequence‐derived protein sequence data can identify amino acid residues that interact between proteins or between domains of a protein. These statistical methods are based on evolution‐directed amino acid variation responding to structural and functional constraints in proteins. The identified residues form a basis for determining structure and folding of proteins as well as inferring mechanisms of protein function. When applied to two‐component systems, several research groups have shown they can be used to identify the amino acid interactions between response regulators and histidine kinases and the specificity therein. Recently, statistical studies between the HisKA and HATPase‐ATP‐binding domains of histidine kinases identified amino acid interactions for both the inactive and the active catalytic states of such kinases. The identified interactions generated a model structure for the domain conformation of the active state. This conformation requires an unwinding of a portion of the C‐terminal helix of the HisKA domain that destroys the inactive state residue contacts and suggests how signal‐binding determines the equilibrium between the inactive and active states of histidine kinases. The rapidly accumulating protein sequence databases from genome, metagenome and microbiome studies are an important resource for functional and structural understanding of proteins and protein complexes in microbes.     

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.     

Chemotaxis and flagellar genes of Chromobacterium violaceum

The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.