Widespread Adaptive Evolution in the Human Immunodeficiency Virus Type 1 Genome

We investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured by using the nonsynonymous/synonymous substitution rate ratio ( = d_N/d_S). To identify amino acid sites under positive selection with > 1, we applied maximum likelihood models that allow variable ratios among sites to analyze genomic sequences of 26 HIV-1 lineages including subtypes A, B, and C. Likelihood ratio tests detected sites under positive selection in each of the major genes in the genome: env, gag, pol, vif, and vpr. Positive selection was also detected in nef, tat, and vpu, although those genes are very small. The majority of positive selection sites is located in gp160. Positive selection was not detected if was estimated as an average across all sites, indicating the lack of power of the averaging approach. Candidate positive selection sites were mapped onto the available protein tertiary structures and immunogenic epitopes. We measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. Our results demonstrate genomewide diversifying selection acting on the HIV-1.     

The Tsetse Fly Obligate Mutualist Wigglesworthia morsitans Alters Gene Expression and Population Density via Exogenous Nutrient Provisioning

The obligate mutualist Wigglesworthia morsitans provisions nutrients to tsetse flies. The symbiont''s response to thiamine (B₁) supplementation of blood meals, specifically towards the regulation of thiamine biosynthesis and population density, is described. Despite an ancient symbiosis and associated genome tailoring, Wigglesworthia responds to nutrient availability, potentially accommodating a decreased need.     

Genome Sequence of Azotobacter vinelandii,an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.Azotobacter vinelandii is a free-living nitrogen-fixing bacterium of the gammaproteobacteria. It is found in soils worldwide, with features of nitrogen and energy metabolism relevant to agriculture (41, 42). This organism has been studied for more than 100 years by numerous scientists throughout the world. Prior to Joshua Lederberg''s discovery of sexuality in Escherichia coli (47), A. vinelandii was the experimental organism of choice for many investigators during the emergence of biochemistry as a dominant discipline within the life sciences. Examples include the classical Lineweaver-Burk kinetic parameters, developed using enzymes from A. vinelandii (51), and the isolation by Severo Ochoa of polynucleotide phosphorylase from A. vinelandii, which was used in studies that contributed to the elucidation of the genetic code (62).A. vinelandii is able to adapt its metabolism to diverse sources of nutrients. If no carbon source is present, A. vinelandii will undergo a differentiation process to form cysts that are resistant to desiccation and other chemical and physical challenges (74). While the process of encystment has been known for many years and studied at the physiological and morphological levels, there is little knowledge about the unique biosynthetic pathways that are involved and how they are regulated. Previous work has implicated the alternative sigma factors AlgU and RpoS in the differentiation process (13, 57, 64). Alginate polymers with different monomer compositions are an important structural component of the cyst, and at the end of exponential growth, A. vinelandii cells accumulate poly-beta-hydroxybutyrate (PHB) as a reserve carbon and energy source (81). The physiology of PHB formation has been well studied in a variety of different systems, and the PHB biosynthetic operon has been described (67, 77). A. vinelandii can also produce copolymers of hydroxybutyrate and hydroxyvalerate, known to improve the flexibility and stretch of bioplastics (63).A. vinelandii has long served as a model for biochemical and genetic studies of biological nitrogen fixation, the conversion of N₂ into NH₃ by a nitrogenase enzyme. The best-studied nitrogenase consists of two oxygen-sensitive metalloproteins that, in the case of the molybdenum nitrogenase, are denominated the Fe protein and the MoFe protein. A. vinelandii is unusual in that it is one of the few bacteria that contain three nitrogenases with different subunit and metal cofactor compositions, namely, the molybdenum nitrogenase, the vanadium nitrogenase, and the iron-only nitrogenase. Expression of these nitrogenases is differentially regulated by metal availability from the medium (27).Here we present the complete genome sequence of A. vinelandii DJ and discuss what the genome has revealed about the organism''s ability to protect oxygen-sensitive processes. A. vinelandii has been cited as having one of the highest respiratory rates of any known bacterium (10). Diazotrophic growth under aerobic conditions is possible because A. vinelandii can adjust oxygen consumption rates to help maintain low levels of cytoplasmic oxygen, which is otherwise detrimental not only to nitrogenase but also to other oxygen-sensitive enzymes expressed by A. vinelandii. This phenomenon is called respiratory protection. In this work, we identify unique features of the A. vinelandii genome that help to explain the coexistence of oxygen-sensitive reactions and strict aerobic metabolism. The genome sequence and annotation allowed identification of the genes involved in respiration, including key players in respiratory protection. In addition, we have identified unexpected gene clusters encoding a carbon monoxide dehydrogenase (CODH), a formate dehydrogenase (FDH), and a second hydrogenase, all of which are also oxygen-sensitive enzymes.     

Genome of Sindbis Virus

³²P-labeled ribonucleic acid (RNA) from purified Sindbis virus was examined for the presence of hidden breaks. Viral RNA was treated with acid at pH 2.9 or with formaldehyde and was analyzed on sucrose gradients or by polyacrylamide gel electrophoresis. The sedimentation pattern and mobility on polyacrylamide gels of the 42S RNA was unaffected by heating and quick cooling and was not altered by denaturing agents such as dimethyl sulfoxide and urea. No evidence that Sindbis RNA is a polyaggregate of fragments was obtained. It is concluded that the genome consists of a continuous length of single-stranded polynucleotide.     

Allelic Skewing of DNA Methylation Is Widespread across the Genome

DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change ≥0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM (∼90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.     

The Genome of Swinepox Virus

Swinepox virus (SWPV), the sole member of the Suipoxvirus genus of the Poxviridae, is the etiologic agent of a worldwide disease specific for swine. Here we report the genomic sequence of SWPV. The 146-kbp SWPV genome consists of a central coding region bounded by identical 3.7-kbp inverted terminal repeats and contains 150 putative genes. Comparison of SWPV with chordopoxviruses reveals 146 conserved genes encoding proteins involved in basic replicative functions, viral virulence, host range, and immune evasion. Notably, these include genes with similarity to genes for gamma interferon (IFN-gamma) receptor, IFN resistance protein, interleukin-18 binding protein, IFN-alpha/beta binding protein, extracellular enveloped virus host range protein, dUTPase, hydroxysteroid dehydrogenase, superoxide dismutase, serpin, herpesvirus major histocompatibility complex inhibitor, ectromelia virus macrophage host range protein, myxoma virus M011L, variola virus B22R, four ankyrin repeat proteins, three kelch-like proteins, five vaccinia virus (VV) A52R-like family proteins, and two G protein-coupled receptors. The most conserved genomic region is centrally located and corresponds to the VV region located between genes F9L and A38L. Within the terminal 13 kbp, colinearity is disrupted and multiple poxvirus gene homologues are absent or share a lower percentage of amino acid identity. Most of these differences involve genes and gene families with likely functions involving viral virulence and host range. Three open reading frames (SPV018, SPV019. and SPV020) are unique for SWPV. Phylogenetic analysis, genome organization, and amino acid identity indicate that SWPV is most closely related to the capripoxvirus lumpy skin disease virus, followed by the yatapoxvirus yaba-like disease virus and the leporipoxviruses. The gene complement of SWPV better defines Suipoxvirus within the Chordopoxvirinae subfamily and provides a basis for future genetic comparisons.     

The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite

We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular α-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin–antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as β-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.     

The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.     

Comparative Genome Analysis of Wheat Blue Dwarf Phytoplasma,an Obligate Pathogen That Causes Wheat Blue Dwarf Disease in China

Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ∼94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms.     

Genome Sequencing Reveals Widespread Virulence Gene Exchange among Human Neisseria Species

Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genus Neisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenic Neisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged among Neisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, other Neisseria. Our findings strongly suggest that commensal Neisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.     

Species-Specific Seed Dispersal in an Obligate Ant-Plant Mutualism

Throughout lowland Amazonia, arboreal ants collect seeds of specific plants and cultivate them in nutrient-rich nests, forming diverse yet obligate and species-specific symbioses called Neotropical ant-gardens (AGs). The ants depend on their symbiotic plants for nest stability, and the plants depend on AGs for substrate and nutrients. Although the AGs are limited to specific participants, it is unknown at what stage specificity arises, and seed fate pathways in AG epiphytes are undocumented. Here we examine the specificity of the ant-seed interaction by comparing the ant community observed at general food baits to ants attracted to and removing seeds of the AG plant Peperomia macrostachya. We also compare seed removal rates under treatments that excluded vertebrates, arthropods, or both. In the bait study, only three of 70 ant species collected P. macrostachya seeds, and 84% of observed seed removal by ants was attributed to the AG ant Camponotus femoratus. In the exclusion experiment, arthropod exclusion significantly reduced seed removal rates, but vertebrate exclusion did not. We provide the most extensive empirical evidence of species specificity in the AG mutualism and begin to quantify factors that affect seed fate in order to understand conditions that favor its departure from the typical diffuse model of plant-animal mutualism.     

Complete Genome Sequence of an Infectious Bronchitis Virus Chimera between Cocirculating Heterotypic Strains

To date, multiple serotypes and genotypes of infectious bronchitis virus (IBV) have been isolated and identified. In order to provide more information on the viral evolution of IBVs, a new virulent strain named GX-NN09032, isolated from Guangxi, China, in 2009, was sequenced, and phylogenetic and recombination analyses were conducted. Furthermore, potential recombination events associated with GX-NN09032 were found in four IBV strains, including GX-YL5, DY07, CK/CH/SD09/005, TC07-2. The present study suggested that GX-NN09032 might contribute to the emergence of modern IBV variants through recombination.     

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design.     

Complete Genome Sequence of an Amur Virus Isolated from Apodemus peninsulae in Northeastern China

Amur virus was recently identified as the causative agent of hemorrhagic fever with renal syndrome. Here we report the complete genome sequence of an Amur virus isolated from Apodemus peninsulae in Northeastern China. The sequence information provided here is critical for the molecular epidemiology and evolution of Amur virus in China.