Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Phylogenetic Analysis Indicates Evolutionary Diversity and Environmental Segregation of Marine Podovirus DNA Polymerase Gene Sequences

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase (pol) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA pol gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus pol sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.Marine viruses are the most abundant (41) and diverse (2, 6) biological entities in the ocean. They affect community composition by causing the lysis of specific subsets of the microbial community (22, 28, 46, 47) and, by killing numerically dominant host taxa, may influence species evenness and richness (24, 28, 43, 50). Despite the abundance of bacteriophages in marine systems and their important roles in marine microbial composition, little is known about the distribution and diversity of specific groups of marine viruses. However, most marine bacteriophage isolates are tailed phages (3) belonging to the order Caudovirales (27), which comprises the families Myoviridae, Podoviridae, and Siphoviridae.Podoviruses are classified into several groups (e.g., T7-like, P22-like, and phi-29-like) based on genome size, genome arrangement, and shared genes and can be readily isolated from seawater (11, 16, 42, 45). Genomic analysis of roseophage SIO1 (33), cyanophage P60 (7), vibriophage VpV262 (21), and cyanophage PSSP7 (40) suggests that many of the isolates are T7-like. Despite the apparently wide distribution of podoviruses in the sea, and their potential importance as agents of microbial mortality, there has been little effort to explore their diversity.Sequence analysis of representative genes is one approach that has been used to examine the genetic diversity of specific groups of marine viruses. For example, homologues for structural genes (g20 and g23) found in T4-like phages are found in some marine myoviruses (18, 20) and have been used to examine the distribution, diversity, and evolutionary relationships among marine myoviruses (12, 14, 17, 37, 38, 49). Other studies have used DNA polymerase (pol) to examine the diversity of viruses infecting eukaryotic phytoplankton (8, 38) and have shown that phylogenies constructed with this gene are congruent with established viral taxonomy (9, 36, 37).Although it is not universally present, family A DNA pol is a good target for examining the diversity of podoviruses (4). Our study presents a newly designed set of PCR primers that amplify a longer fragment of the DNA polymerase from a much larger suite of podoviruses and shows that the diversity within marine podoviruses as revealed by DNA pol sequences is far greater than previously realized.     

利用叶绿体基因探讨稻属BBCC基因组物种的系统起源

基于9个叶绿体基因片段(atpA、atpB、matK、petA、psaA、psbA、psbB、psbC和rbcL), 深入探讨了稻属(Oryza)3个BBCC基因组异源四倍体和5个与之相关的BB或CC基因组二倍体物种间的谱系关系。进一步的系统发育分析表明: 3个具有相同BBCC基因组的四倍体物种并非同一次物种形成事件的产物, 而是在不同的分布区经历了至少3次分别的物种起源。其中, 四倍体Oryza punctata的母本可能来自同样分布在非洲并具有CC基因组的二倍体物种O. eichingeri; 而四倍体O. malampuzhaensis和O. minuta的母本则可能来自亚洲已经灭绝的具有BB基因组的不同二倍体。研究结果不但为追溯稻属异源四倍体的复杂网状进化提供了重要的分子证据, 而且拓展了我们对有花植物复杂物种形成的深入理解。     

Low Genetic Diversity of the Coat Protein Gene among Blueberry Red Ringspot Virus Isolates

Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.     

Phylogenetic Diversity of New Zealand Gelidiales as Revealed by rbcL Sequence Data

Diversity and phylogenetic relationships of New Zealand representatives of the red algal order Gelidiales have been examined using rbcL sequence data. Extensive field collections have been made from throughout the New Zealand region. Six genera have been reported previously from New Zealand (Capreolia, Gelidium, Pterocladia, Pterocladiella, Pterocladiastrum, Ptilophora). This research has revealed species with very restricted local distributions, as well as the discovery of several undescribed, cryptic taxa. The common and widespread Gelidium caulacantheum is confirmed to be more closely related to Capreolia than to other species of Gelidium. The generic concept of Capreolia, based on life history characters, will need to be modified to accommodate additional species possessing “Gelidium” life histories. A species endemic to New Zealand, Gelidium ceramoides, has been found to differ significantly from all other members of the Gelidiales and requires reclassification in another genus and order. Examination of field collections and herbarium specimens in addition to molecular sequence data have led us to conclude that specimens previously placed in the genera Ptilophora and Pterocladiastrum belong within Pterocladia lucida.     

蓖麻品种遗传多样性与亲缘关系的SRAP分析

利用SRAP技术对81份蓖麻品种材料亲缘关系进行了分析,实验选用20对SRAP引物组合,在81份蓖麻材料中共扩增出263条带,多态性条带计214条,多态性条带比率（PPB）为81.37%,遗传相似系数变幅范围在0.32558～0.92973,显示了蓖麻品种的遗传多样性较丰富。从分子聚类结果分析表明,在相异系数0.43为阈值时,可将81份蓖麻材料分为4个类群L1-1、L1-2、L1-3和L1-4;若在相异系数0.287为阈值时,又可将L1-4大类群分为两个亚类群L2-1和L2-2。从聚类图得知,聚在同一亚类群的蓖麻品种大多数所处的地域相近或者是由同一育种单位所选育,其类内的品种基因型遗传相似系数较高,类间的品种遗传差异相对较大,该分子聚类树状图可为蓖麻栽培种种质资源遗传多样性与亲缘关系在育种的利用上提供科学依据。     

Phylogenetic Diversity of the Bacillus pumilus Group and the Marine Ecotype Revealed by Multilocus Sequence Analysis

Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments.     

Synthesis and Assembly of Simian Virus 40 II. Synthesis of the Major Capsid Protein and Its Incorporation into Viral Particles

African green monkey kidney cells infected by simian virus 40 were analyzed for the presence of the major capsid protein (capsid protein I) by immunological and radiolabeling techniques. Antisera with different specificities were prepared by immunization with intact or denatured viral particles. Antisera prepared against intact virus reacted by complement fixation with viral particles and with an 8S subunit containing the capsid protein I. Antisera prepared against denatured viral particles reacted with unassembled capsid protein(s) as well as with viral particles. These antisera were used to detect 8S viral subunits or unassembled viral capsid protein in soluble extracts of infected cells after centrifugation at 100,000 x g to remove viral particles. The soluble antigen pool was found to be small during infection with wild-type virus or a temperature-sensitive mutant deficient in the synthesis of viral particles. Pulse-chase experiments, performed at a high multiplicity of infection, also indicated a small pool of nonparticle capsid protein I. Radioactive lysine was incorporated into capsid protein I of virus particles during a 2-hr pulse. A subsequent chase with excess unlabeled lysine resulted in only a slight increase in the radio-activity found in capsid protein I of viral particles. Furthermore, in the same experiments, capsid protein I was incorporated preferentially into empty shells during the pulse with a shift in radioactivity to intact virions during the chase period, indicating a possible precursor relationship between the two types of virus particles.     

RNA Binding by the Brome Mosaic Virus Capsid Protein and the Regulation of Viral RNA Accumulation

Viral capsid proteins (CPs) can regulate gene expression and encapsulate viral RNAs. Low-level expression of the brome mosaic virus (BMV) CP was found to stimulate viral RNA accumulation, while higher levels inhibited translation and BMV RNA replication. Regulation of translation acts through an RNA element named the B box, which is also critical for the replicase assembly. The BMV CP has also been shown to preferentially bind to an RNA element named SLC that contains the core promoter for genomic minus-strand RNA synthesis. To further elucidate CP interaction with RNA, we used a reversible cross-linking-peptide fingerprinting assay to identify peptides in the capsid that contact the SLC, the B-box RNA, and the encapsidated RNA. Transient expression of three mutations made in residues within or close by the cross-linked peptides partially released the normal inhibition of viral RNA accumulation in agroinfiltrated Nicotiana benthamiana. Interestingly, two of the mutants, R142A and D148A, were found to retain the ability to down-regulate reporter RNA translation. These two mutants formed viral particles in inoculated leaves, but only R142A was able to move systemically in the inoculated plant. The R142A CP was found to have higher affinities for SLC and the B box compared with those of wild-type CP and to alter contacts to the RNA in the virion. These results better define how the BMV CP can interact with RNA and regulate different viral processes.     

Mutually-induced Conformational Switching of RNA and Coat Protein Underpins Efficient Assembly of a Viral Capsid

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T = 3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T = 3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.     

Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities.     

军曹鱼淋巴囊肿病毒主衣壳蛋白基因全序列分析

军曹鱼(Rachycentron canadum)亦称海鲡,是我国南方沿海一带的重要海水网箱养殖对象。2005年8月,广东省海水网箱养殖的军曹鱼首次暴发类似的淋巴囊肿病,病鱼的口唇、鳃、鳍、尾及体表等处,可看到大小不一的单个或成群的肿瘤,个别网箱的感染率在80%以上,死亡率近30%。病鱼形象丑陋,严重影响其市场价值,造成了较大的经济损失。淋巴囊肿病(Lymphocystis disease)发现于1874年,1965年正式确认该病病原为淋巴囊肿病毒[1],现已知可感染9目34科140种以上鱼类。我国在20世纪90年代陆续在养殖石斑鱼、鲈鱼、牙鲆中发现淋巴囊肿病[2-5],随后对其病原…     

Detection,Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

A species‐specific Polymerase Chain Reaction (sPCR) method was developed to identify and detect isolates of Ralstonia solanacearum, the cause of bacterial wilt disease in chilli. PCR primers for R. solanacearum were identified by alignment of hrpB gene sequences and selection of sequences specific for R. solanacearum at their 3′ ends. The primers were shown to be specific for R. solanacearum, as no PCR product was obtained when genomic DNA from other bacterial species including closely related Ralstonia species, were used as test species. Lone pair of primers (RshrpBF and RshrpBR) was designed using hrpB gene sequence, unique to R. solanacearum which amplified a predicted PCR product of 810 bp from 20 different isolates. Phylogenetic analysis was also attempted to understand the evolutionary divergence of Indian R. solanacearum isolates. Based on phylogenetic analysis, Indian isolates showed homology with the standard reference isolates from other countries but, interestingly, one new isolate showed complete evolutionary divergence by forming an out‐group.     

Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the Major Capsid Protein

A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and -sensitive strains. In a plaque reduction assay under conditions in which the 50% effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25 μM, the 50% effective concentration of 35B2 was 0.75 μM. The selective index of the compound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open reading frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corresponding to the “floor” domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electron microscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are required to clarify the precise antiviral mechanism.     

Intestinal Origin of Sourdough Lactobacillus reuteri Isolates as Revealed by Phylogenetic, Genetic, and Physiological Analysis

Lactobacillus reuteri is both a gut symbiont and a stable member of sourdough microbiota. This study employed multilocus sequence analysis and an analysis of host-specific physiological and genetic traits to assign five sourdough isolates to rodent- or human-specific lineages. Comparative genome hybridization revealed that the model sourdough isolate LTH2584 had a genome content very similar to that of the model rodent isolate 100-23. These results demonstrate that sourdough isolates of L. reuteri are of intestinal origin.     

Protist Diversity and Seasonal Dynamics in Skagerrak Plankton Communities as Revealed by Metabarcoding and Microscopy

Protist community composition and seasonal dynamics are of major importance for the production of higher trophic levels, such as zooplankton and fish. Our aim was to reveal how the protist community in the Skagerrak changes through the seasons by combining high‐throughput sequencing and microscopy of plankton collected monthly over two years. The V4 region of the 18S rRNA gene was amplified by eukaryote universal primers from the total RNA/cDNA. We found a strong seasonal variation in protist composition and proportional abundances, and a difference between two depths within the euphotic zone. Highest protist richness was found in late summer‐early autumn, and lowest in winter. Temperature was the abiotic factor explaining most of the variation in diversity. Dinoflagellates was the most abundant and diverse group followed by ciliates and diatoms. We found about 70 new taxa recorded for the first time in the Skagerrak. The seasonal pattern in relative read abundance of major phytoplankton groups was well in accordance with microscopical biovolumes. This is the first metabarcoding study of the protist plankton community of all taxonomic groups and through seasons in the Skagerrak, which may serve as a baseline for future surveys to reveal effects of climate and environmental changes.