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.     

The Evolutionary History of Nitrogen Fixation, as Assessed by NifD

The evolutionary history of nitrogen fixation has been vigorously debated for almost two decades. Previous phylogenetic analyses of nitrogen fixation genes (nif) have shown support for either evolution by vertical descent or lateral transfer, depending on the specific nif gene examined and the method of analyses used. The debate centers on the placement and monophyly of the cyanobacteria, proteobacteria, and Gram-positive bacteria (actinobacteria and firmicutes). Some analyses place the cyanobacteria and actinobacteria within the proteobacteria, which suggests that the nif genes have been laterally transferred since this topology is incongruent with ribosomal phylogenies, the standard marker for comparison. Other nif analyses resolve and support the monophyly of the cyanobacteria, proteobacteria, and actinobacteria, supporting vertical descent. We have revisited these conflicting scenarios by analyzing nifD from an increased number of cyanobacteria, proteobacteria, and Gram-positive bacteria. Parsimony analyses of amino acid sequences and maximum likelihood analysis of nucleic acid sequences support the monophyly of the cyanobacteria and actinobacteria but not the proteobacteria, lending support for vertical descent. However, distance analysis of nucleic acid sequences placed the actinobacteria within the proteobacteria, supporting lateral transfer. We discuss evidence for both vertical descent and lateral transfer of nitrogen fixation.     

<Emphasis Type="Italic">EVG</Emphasis>, the Remnants of a Primordial Bilaterian’s Synteny of Functionally Unrelated Genes

Extant genomes are the result of repeated duplications and subsequent divergence of primordial genes that assembled the genomes of the first living beings. Increased information on genome maps of different species is revealing conserved syntenies among different vertebrate taxa, which allow to trace back the history of current chromosomes. However, inferring neighboring relationships between genes of more primitive genomes has proven to be very difficult. Most often, the ancestral arrangements of genes have been lost by multiple histories of internal duplications, chromosomal breaks, and large-scale genomic rearrangements. Here we describe a gene arrangement of nonrelated genes that seems to have endured evolution, at least from the separation of the two major clades of bilateria: deuterostomia and protostomia, approximately 1 billion years ago. In its simplest conception, this gene cluster, named EVG, groups the genes for a glucose transporter, an enolase, and a vesicle-associated membrane protein (VAMP). EVG might represent the evolutionary remnants of the gene organization of an ancient bilaterian genome.     

The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains

The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

The photosynthetic apparatus of Prochlorococcus: Insights through comparative genomics

Within the vast oceanic gyres, a significant fraction of the total chlorophyll belongs to the light-harvesting antenna systems of a single genus, Prochlorococcus. This organism, discovered only about 10 years ago, is an extremely small, Chl b-containing cyanobacterium that sometimes constitutes up to 50% of the photosynthetic biomass in the oceans. Various Prochlorococcus strains are known to have significantly different conditions for optimal growth and survival. Strains which dominate the surface waters, for example, have an irradiance optimum for photosynthesis of 200 μmol photons m⁻² s⁻¹, whereas those that dominate the deeper waters photosynthesize optimally at 30–50 μmol photons m⁻² s⁻¹. These high and low light adapted ‘ecotypes’ are very closely related — less than 3% divergent in their 16S rRNA sequences — inviting speculation as to what features of their photosynthetic mechanisms might account for the differences in photosynthetic performance. Here, we compare information obtained from the complete genome sequences of two Prochlorococcus strains, with special emphasis on genes for the photosynthetic apparatus. These two strains, Prochlorococcus MED4 and MIT 9313, are representatives of high- and low-light adapted ecotypes, characterized by their low or high Chl b/a ratio, respectively. Both genomes appear to be significantly smaller (1700 and 2400 kbp) than those of other cyanobacteria, and the low-light-adapted strain has significantly more genes than its high light counterpart. In keeping with their comparative light-dependent physiologies, MED4 has many more genes encoding putative high-light-inducible proteins (HLIP) and photolyases to repair UV-induced DNA damage, whereas MIT 9313 possesses more genes associated with the photosynthetic apparatus. These include two pcb genes encoding Chl-binding proteins and a second copy of the gene psbA, encoding the Photosystem II reaction center protein D1. In addition, MIT 9313 contains a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate form between the phycobiliproteins of non-Chl b containing cyanobacteria and an extremely modified β phycoerythrin as the sole derivative of phycobiliproteins still present in MED4. Intriguing features found in both Prochlorococcus strains include a gene cluster for Rubisco and carboxysomal proteins that is likely of non-cyanobacterial origin and two genes for a putative and β lycopene cyclase, respectively, explaining how Prochlorococcus may synthesize the α branch of carotenoids that are common in green organisms but not in other cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

Unique properties vs. common themes: the atypical cyanobacterium Gloeobacter violaceus PCC 7421 is capable of state transitions and blue-light-induced fluorescence quenching

The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.     

Phylogenetics of New World Astragalus: Screening of novel nuclear loci for the reconstruction of phylogenies at low taxonomic levels

This study explores methods to use information gathered from genomics technology to understand evolutionary relationships in the hyperdiverse legume group Neo-Astragalus. These species inhabit deserts and mountains of North and South America, and even though the monophyly of the group is well established, relationships within it are still poorly understood. Plastid genes, commonly used to infer phylogenies in plants, are usually not useful for closely related taxa because of low levels of genetic variation. TheMedicago truncatula genome project provided a suite of candidate nuclear loci with high levels of variation that might prove suitable for low-level phylogenetics. This paper reports the development of methods for screening a large number of these nuclear loci, and detailed analysis of four of them. Four different patterns of phylogenetic diversification occur in the loci sampled from these genomes ofAstragalus species. One locus (CNGC4) was single copy and could be directly used in phylogenetic analyses. Two loci (ARG10 and FENR) showed patterns strongly suggestive of duplication events in some taxa, and one locus (tRALS) has apparently undergone a cryptic duplication, making it very difficult to diagnose. Potential methods for using the information provided by these loci are discussed.     

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d_N/d_S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.     

Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the <Emphasis Type="Italic">amoCAB</Emphasis> operon

The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Pilar Junier and Ok-Sun Kim contributed equally to this work.     

Phylogenetic Distribution of Unusual Triheme to Tetraheme Cytochrome Subunit in the Reaction Center Complex of Purple Photosynthetic Bacteria

To understand the evolutionary relationship between triheme and tetraheme cytochrome subunits in the reaction center complex, genes located downstream of that coding for the M subunit of the reaction center complex (pufM) were amplified by PCR and analyzed in six established and two unidentified species of the genus Rhodovulum and five species of the genus Rhodobacter. All the Rhodovulum species tested had the pufC gene coding for the reaction-center-bound cytochrome subunit, while all the Rhodobacter species were found to have the pufX gene at the corresponding position. Analyses of the amino acid sequences of the pufC gene products showed that the cytochrome subunits of all the Rhodovulum species have three heme-binding-motifs and lack a methionine residue probably working as the sixth axial-ligand to one of the three hemes. Phylogenetic relationships among Rhodovulum species based on the pufC gene products were basically consistent with those based on 16S rRNA sequences, suggesting that the basic characteristics of the triheme cytochrome subunit have been conserved during the evolutionary process of the Rhodovulum species.     

Carotenoid biosynthesis in <Emphasis Type="Italic">Gloeobacter violaceus</Emphasis> PCC4721 involves a single crtI-type phytoene desaturase instead of typical cyanobacterial enzymes

Gloeobacter violaceus is a cyanobacterium isolated from other groups by lack of thylakoids and unique structural features of its photosynthetic protein complexes. Carotenoid biosynthesis has been investigated with respect to the carotenoids formed and the genes and enzymes involved. Carotenoid analysis identified ss-carotene as major carotenoid and echinenone as a minor component. This composition is quite unique and the cellular amounts are up to 10-fold lower than in other unicellular cyanobacteria. Carotenoid biosynthesis is up-regulated in a light-dependent manner. This enhanced biosynthesis partially compensates for photooxidation especially of ss-carotene. The sequenced genome of G. violaceus was analyzed and several gene candidates homologous to carotenogenic genes from other organisms obtained. Functional expression of all candidates and complementation in Escherichia coli led to the identification of all genes involved in the biosynthesis of the G. violaceus carotenoids with the exception of the lycopene cyclase gene. An additional diketolase gene was found that functioned in E. coli but is silent in G. violaceus cells. The biggest difference from all other cyanobacteria is the existence of a single bacterial-type 4-step desaturase instead of the poly cis cyanobacterial desaturation pathway catalyzed by two cyanobacterial-type desaturases and an isomerase. The genes for these three enzymes are absent in G. violaceus.     

Historical ecology meets conservation and evolutionary genetics: a secondary contact zone between Carabus violaceus (Coleoptera, Carabidae) populations inhabiting ancient and recent woodlands in north-western Germany

Only very few cases have documented that an increase in connectivity after a period of fragmentation in ecological time has had an effect on the distribution, genetic structure and morphology of stenotopic species. In this study we present an example of clinal variability in a woodland ground beetle as a result of changes in the connectivity of a landscape during the last two centuries. The study area hosts both the nominate form Carabus violaceus s. str. and the subspecies Carabus violaceus purpurascens, which is ranked as a distinct species by some authors. We studied 12 Carabus violaceus populations from a 30 km transect of ancient and recent forests in north-western Germany. We analyzed three polymorphic enzyme loci, classified the elytron sculpture and measured the shape of the aedeagus tip of the specimens. Carabus violaceus showed secondary gradients both in allozyme markers and morphometric characters in our study area. A genetic differentiation of 16% between the populations is high but lies within the range of intraspecific variability in habitat specialists of the genus Carabus. Populations had no significant deficit of heterozygotes. We found many hybrid populations in terms of morphological properties. This study highlights the conservation value of ancient woodland and the consequences of landscape connectivity and defragmentation on the genetic setting of a ground beetle. Moreover, it shows that differences in the external shape of male genitalia do not prevent gene flow within the genus Carabus. Thus, the establishment of species status should not exclusively be based on this property.