Sequence of the two operons encoding the four core subunits of the cytochrome b(6)f complex from the thermophilic Cyanobacterium synechococcus elongatus

The genes encoding cytochrome f (petA), cytochrome b(6) (petB), the Rieske FeS-protein (petC), and subunit IV (petD) of the cytochrome b(6)f complex from the thermophilic cyanobacterium Synechococcus elongatus were cloned and sequenced. Similar to other cyanobacteria, the structural genes are arranged in two short, single-copy operons, petC/petA and petB/petD, respectively. In addition, five open reading frames with homology to known orfs from the cyanobacterium Synechocystis PCC 6803 were identified in the immediate vicinity of these two operons.     

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.     

An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.     

Mutations of cytochrome b6 in Chlamydomonas reinhardtii disclose the functional significance for a proline to leucine conversion by petB editing in maize and tobacco

We have introduced a proline codon in place of a leucine codon at position 204 of the petB gene of Chlamydomonas reinhardtii. This gene modification mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observed no editing at this position in C. reinhardtii, independent of the type of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototrophic and displayed a block in photosynthetic electron transfer, consistent with a lack of cytochrome b6f activity. Thus the presence of a proline residue at position 204 in cytochrome b6 is detrimental to photosynthesis. We show that the mutant phenotype arose from a defective assembly of cytochrome b6f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical comparison of the proline-containing transformants with a cytochrome b6 mutant deficient in heme-attachment indicates that their primary defect is at the level of assembly of apocytochrome b6 with the bh heme, thereby preventing assembly of the whole cytochrome b6f complex.     

Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.     

Effect of temperature and light on growth of and photosynthesis by Synechococcus isolates typical of those predominating in the octopus spring microbial mat community of Yellowstone National Park

Previous molecular analysis of the Octopus Spring cyanobacterial mat revealed numerous genetically distinct 16S rRNA sequences from predominant Synechococcus populations distantly related to the readily cultivated unicellular cyanobacterium Synechococcus lividus. Patterns in genotype distribution relative to temperature and light conditions suggested that the organisms contributing these 16S rRNA sequences may fill distinct ecological niches. To test this hypothesis, Synechococcus isolates were cultivated using a dilution and filtration approach and then shown to be genetically relevant to natural mat populations by comparisons of similarities of 16S rRNA genes and 16S-23S internal transcribed spacer (ITS) regions. Most isolates were identical or nearly identical at both loci to predominant mat genotypes; others showed 1- to 2-nucleotide differences at the 16S rRNA locus and even greater difference in ITS sequences. Isolates with predominant mat genotypes had distinct temperature ranges and optima for growth that were consistent with their distributions in the mat. Isolates with genotypes not previously detected or known to be predominant in the mat exhibited temperature ranges and optima that were not representative of predominant mat populations and also grew more slowly. Temperature effects on photosynthesis did not reflect temperature relations for growth. However, the isolate with the highest temperature optimum and upper limit was capable of performing photosynthesis at a higher temperature than other isolates. Growth rate and photosynthetic responses provided evidence for light acclimation but evidence of, at best, only subtle light adaptation.     

Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.     

Genetic relationships and origin of two geographic groups of the freshwater threespine stickleback, 'hariyo'

'Hariyo' comprise the southernmost freshwater populations of the threespine stickleback, Gasterosteus aculeatus species complex, distributed in central Honshu, Japan. Two geographic groups (Gifu and Shiga) of the 'hariyo' have been recognized as differing from each other in some morphological and ecological features. In order to elucidate the genetic characteristics and phylogenetic position of these populations, partial sequences of the mitochondrial cytochrome b gene were compared in 123 specimens from 10 'hariyo' populations, and eight other freshwater and anadromous populations of threespine stickleback in Japan. Phylogenetic analysis resolved 22 haplotypes in a single most-parsimonious tree. In contrast to previous allozyme analyses, the haplotype tree indicated that 'hariyo' populations are monophyletic. Each of two geographic groups of the 'hariyo' was also shown to be nearly monophyletic. The two geographic groups differed from each other in sequence by an average of 0.47% and from other populations by 0.70%. Based on a molecular clock, constructed from fossil records and mtDNA genetic distances of Gasterosteus and Pungitius, it was estimated that the two groups of 'hariyo' differentiated from the other threespine stickleback populations and also from each other in the middle Pleistocene. The results suggested that each of the two groups of 'hariyo' is a distinct evolutionarily significant unit with unique genetic features, as well as morphological and ecological characteristics.     

Population genetic structure of the striped silverside, Atherinomorus endrachtensis (Atherinidae, Atheriniformes, Teleostei), inhabiting marine lakes and adjacent lagoons in Palau: marine lakes are "Islands" for marine species

Although evidence for the evolution of terrestrial species on islands continues to rapidly accumulate, little is known about the evolution of marine species in geographically isolated environments such as islands as ocean currents often facilitate gene flow among populations. In this study, we focused on marine lakes of the Palau Islands, which are considered to be true analogues of terrestrial islands for marine species. To examine evolutionary processes in marine lakes, we conducted population genetic analyses on marine lake and lagoon populations of the striped silverside, Atherinomorus endrachtensis, using two mitochondrial DNA markers differing in evolutionary rate, the cytochrome b gene and the control region. The analyses revealed that the amount of genetic diversity of marine lake populations is much lower than that of lagoon populations and high levels of genetic differentiation occur among marine lake and lagoon populations. The present study has shown that marine lake populations have been completely isolated and have differentiated from lagoon populations, and each marine lake population is experiencing different evolutionary processes. These findings clearly demonstrate that marine lakes are excellent environments for the evolutionary study of marine species.     

Population structure at two geographic scales in the burrowing crustacean Callichirus islagrande (Decapoda, Thalassinidea): historical and contemporary barriers to planktonic dispersal

There has been much recent interest in the extent to which marine planktonic larvae connect local populations demographically and genetically. Uncertainties about the true extent of larval dispersal have impeded our understanding of the ecology and evolution of marine species as well as our attempts to effectively manage marine populations. Because direct measurements of larval movements are difficult, genetic markers have often been used for indirect measurements of gene flow among marine populations. Here we examine data from allozymes, mitochondrial DNA sequences, and microsatellite length polymorphisms to assess the extent of gene flow among populations of the burrowing crustacean Callichirus islagrande. All three types of markers revealed a genetic break between populations separated by the Louisiana Chenier Plain. The extent of mitochondrial sequence divergence across this break indicates that the nominal species, C. islagrande, consists of at least two lineages that have been reproductively isolated for about a million years. Within the eastern lineage microsatellite allele frequencies were significantly heterogeneous among populations as little as 10 km apart. Maximum likelihood estimates of gene flow and effective population size based on a coalescent model for the microsatellite data indicated that local populations are nearly closed. A model-based clustering method identified four or five groups from the microsatellite data, although individuals sampled from each location generally consisted of mixtures of these groups. This suggests a mechanism that would lead to genetic differentiation of open populations: gene flow from different source populations that are themselves genetically distinct.     

Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances

Microbial ecology examines the diversity and activity of micro-organisms in Earth's biosphere. In the last 20 years, the application of genomics tools have revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world. This review first introduces the basic concepts in microbial ecology and the main genomics methods that have been used to examine natural microbial populations and communities. In the ensuing three specific sections, the applications of the genomics in microbial ecological research are highlighted. The first describes the widespread application of multilocus sequence typing and representational difference analysis in studying genetic variation within microbial species. Such investigations have identified that migration, horizontal gene transfer and recombination are common in natural microbial populations and that microbial strains can be highly variable in genome size and gene content. The second section highlights and summarizes the use of four specific genomics methods (phylogenetic analysis of ribosomal RNA, DNA-DNA re-association kinetics, metagenomics, and micro-arrays) in analysing the diversity and potential activity of microbial populations and communities from a variety of terrestrial and aquatic environments. Such analyses have identified many unexpected phylogenetic lineages in viruses, bacteria, archaea, and microbial eukaryotes. Functional analyses of environmental DNA also revealed highly prevalent, but previously unknown, metabolic processes in natural microbial communities. In the third section, the ecological implications of sequenced microbial genomes are briefly discussed. Comparative analyses of prokaryotic genomic sequences suggest the importance of ecology in determining microbial genome size and gene content. The significant variability in genome size and gene content among strains and species of prokaryotes indicate the highly fluid nature of prokaryotic genomes, a result consistent with those from multilocus sequence typing and representational difference analyses. The integration of various levels of ecological analyses coupled to the application and further development of high throughput technologies are accelerating the pace of discovery in microbial ecology.