A strange family,or how a new pleolipovirus reveals its friends and relatives

A new virus of halophilic Archaea is reported by Liu et al., and is remarkable in many ways. SNJ2 is the first temperate, pleomorphic virus (pleolipovirus) that integrates into the genome of its host. Analyses of the virus structure and its genome have provided an unexpected puzzle while at the same time solving another. On the one hand, the study shows a curious relationship exists between SNJ2 and an unrelated provirus (SNJ1) found as a plasmid in the same cell. The presence of SNJ1 appears to allow much higher levels of SNJ2 virus to be produced, although the mechanism involved remains unclear. On the other hand, the curious occurrence of a conserved cluster of pleolipovirus‐related genes found widely distributed among haloarchaeal genomes and known for almost 10 years, now appears to correspond to SNJ2‐related proviruses.     

Resequencing and comparison of whole mitochondrial genome to gain insight into the evolutionary status of the Shennongjia golden snub‐nosed monkey (SNJ R. roxellana)

Shennongjia Rhinopithecus roxellana (SNJ R. roxellana) is the smallest geographical population of R. roxellana. The phylogenetic relationships among its genera and species and the biogeographic processes leading to their current distribution are largely unclear. To address these issues, we resequenced and obtained a new, complete mitochondrial genome of SNJ R. roxellana by next‐generation sequencing and standard Sanger sequencing. We analyzed the gene composition, constructed a phylogenetic tree, inferred the divergence ages based on complete mitochondrial genome sequences, and analyzed the genetic divergence of 13 functional mtDNA genes. The phylogenetic tree and divergence ages showed that R. avunculus (the Tonkin snub‐nosed monkey) was the first to diverge from the Rhinopithecus genus ca. 2.47 million years ago (Ma). Rhinopithecus bieti and Rhinopithecus strykeri formed sister groups, and the second divergence from the Rhinopithecus genus occurred ca. 1.90 Ma. R. roxellana and R. brelichi diverged from the Rhinopithecus genus third, ca. 1.57 Ma. SNJ R. roxellana was the last to diverge within R. roxellana species in 0.08 Ma, and the most recent common ancestor of R. roxellana is 0.10 Ma. The analyses on gene composition showed SNJ R. roxellana was the newest geographic population of R. roxellana. The work will help to develop a more accurate protection policy for SNJ R. roxellana and facilitate further research on selection and adaptation of R. roxellana.     

The thermo‐ and acido‐stable ORF‐99 from the archaeal virus AFV1

Acidianus Filamentous Virus 1 (AFV1), isolated from acidic hot springs, is an enveloped lipid‐containing archaeal filamentous virus with a linear double‐stranded DNA genome. It infects Acidianus, which is a hyperthermostable archaea growing at 85°C and acidic pHs, below pH 3. AFV1‐99, a protein of 99 amino acids of unknown function, has homologues in the archaeal virus families Lipothrixviridae and Rudiviridae. We determined the crystal structure of AFV1‐99 at 2.05 Å resolution. AFV1‐99 has a new fold, is hyperthermostable (up to 95°C) and resists to extreme pH (between pH 0 and 11) and to the combination of high temperature (95°C) and low pH (pH 0). It possesses characteristics of hyperthermostable proteins, such as a high content of charged residues.     

Newly identified proviruses in Thermotogota suggest that viruses are the vehicles on the highways of interphylum gene sharing

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota.     

Genetic Organization of Plasmid pXF51 from the Plant Pathogen Xylella fastidiosa

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.     

Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes

Arabidopsis thaliana represents a valuable and efficient model to understand mechanisms underlying plant susceptibility to viral diseases. Here, we describe the identification and molecular cloning of a new gene responsible for recessive resistance to several isolates of Watermelon mosaic virus (WMV, genus Potyvirus) in the Arabidopsis Cvi‐0 accession. rwm1 acts at an early stage of infection by impairing viral accumulation in initially infected leaf tissues. Map‐based cloning delimited rwm1 on chromosome 1 in a 114‐kb region containing 30 annotated genes. Positional and functional candidate gene analysis suggested that rwm1 encodes cPGK2 (At1g56190), an evolutionary conserved nucleus‐encoded chloroplast phosphoglycerate kinase with a key role in cell metabolism. Comparative sequence analysis indicates that a single amino acid substitution (S78G) in the N‐terminal domain of cPGK2 is involved in rwm1‐mediated resistance. This mutation may have functional consequences because it targets a highly conserved residue, affects a putative phosphorylation site and occurs within a predicted nuclear localization signal. Transgenic complementation in Arabidopsis together with virus‐induced gene silencing in Nicotiana benthamiana confirmed that cPGK2 corresponds to rwm1 and that the protein is required for efficient WMV infection. This work uncovers new insight into natural plant resistance mechanisms that may provide interesting opportunities for the genetic control of plant virus diseases.     

Phylogenetic diversity and metagenomics of candidate division OP3

Except for environmental 16S rRNA gene sequences, no information is available for members of the candidate division OP3. These bacteria appear to thrive in anoxic environments, such as marine sediments, hypersaline deep sea, freshwater lakes, aquifers, flooded paddy soils and methanogenic bioreactors. The 16S rRNA phylogeny suggests that OP3 belongs to the Planctomycetes/Verrucomicrobia/Chlamydiae (PVC) superphylum. Metagenomic fosmid libraries were constructed from flooded paddy soil and screened for 16S rRNA gene‐containing fragments affiliated with the PVC superphylum. The screening of 63 000 clones resulted in 23 assay‐positive fosmids, of which three clones were affiliated with OP3. The 16S rRNA gene sequence divergence between the fragments OP3/1, OP3/2 and OP3/3 ranges from 18% to 25%, indicating that they belong to different OP3 subdivisions. The 23S rRNA phylogeny confirmed the membership of OP3 in the PVC superphylum. Sequencing the OP3 fragments resulted in a total of 105 kb of genomic information and 90 ORFs, of which 47 could be assigned a putative function and 11 were conserved hypothetical. Using BLASTP searches, a high proportion of ORFs had best matches to homologues from Deltaproteobacteria, rather than to those of members of the PVC superphylum. On the fragment OP3/3, a cluster of nine ORFs was predicted to encode the bacterial NADH dehydrogenase I. Given the high proportion of homologues present in deltaproteobacteria and anoxic conditions in the natural environment of OP3 bacteria, the detection of NADH dehydrogenase I may suggest an anaerobic respiration mode. Oligonucleotide frequencies calculated for OP3/1, OP3/2 and OP/3 show high intraphylum correlations. This novel sequence information could therefore be used to identify OP3‐related fragments in large metagenomic data sets using marker gene‐independent procedures in the future. In addition to the OP3 fragments, a single metagenomic fragment affiliated with the candidate division BRC1 was obtained and analysed.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

A new method for rapid identification of ansamycin compounds by inactivating KLM gene clusters in potential ansamycin-producing actinomyces

Aims: In this study, we explored the possibility of construction of a ‘universal targeting vector’ by Red/ET recombination to inactivate L gene encoding 3‐amino‐5‐hydroxybenzoic acid (AHBA)‐oxidoreductase in AHBA biosynthetic gene cluster to facilitate the detection of ansamycins production in actinomycetes. Methods and Results: Based on the conserved regions of linked AHBA synthase (K), oxidoreductase (L) and phosphatase (M) gene clusters, degenerate primers were designed and PCR was performed to detect KLM gene clusters within 33 AHBA synthase gene‐positive actinomycetes strains. Among them, 22 KLM gene cluster‐positive strains were identified. A ‘universal targeting vector’ was further constructed using the 50‐nt homologous sequences chosen from four strains internal L gene in KLM gene clusters through Red/ET recombination. The L gene from nine of the KLM gene cluster‐positive actinomycetes strains was inactivated by insertion of a kanamycin (Km) resistance marker into its internal region from the ‘universal targeting vector’. By comparison of the metabolites produced in parent strains with those in L gene‐inactivated mutants, we demonstrated the possible ansamycins production produced by these strains. One strain (4089) was proved to be a geldanamycin producer. Three strains (3‐20, 7‐32 and 8‐32) were identified as potential triene‐ansamycins producers. Another strain (3‐27) was possible to be a streptovaricin C producer. Strains 24‐100 and 4‐124 might be served as ansamitocin‐like producers. Conclusions: The results confirmed the feasibility that a ‘universal targeting vector’ could be constructed through Red/ET recombination using the conserved regions of KLM gene clusters to detect ansamycins production in actinomycetes. Significance and Impact of the Study: The ‘universal targeting vector’ provides a rapid approach in certain degree to detect the potential ansamycin producers from the 22 KLM gene cluster‐positive actinomycetes strains.     

IDENTIFICATION OF THE FGL‐AMIDE ALLATOSTATIN GENE OF THE PRIMITIVE TERMITE Mastotermes darwiniensis AND THE WOODROACH Cryptocercus darwini

Allatostatins with the C‐terminal ending Tyr/Phe‐Xaa‐Phe‐Gly‐Leu/Ile‐amide (FGLa/ASTs) are widespread neuropeptides with multiple functions. The gene encoding the FGLa/AST polypeptide precursor was first isolated from cockroaches and since then could be identified in many insects and crustaceans. With its strictly conserved regions in combination with variable regions the gene seems to be a good candidate for phylogenetic analyses between closely and distantly related species. Here, the structure of the FGLa/AST gene of the most primitive termite, the giant northern termite Mastotermes darwiniensis Froggatt, was identified. The FGLa/AST gene of the woodroach Cryptocercus darwini was also determined. Precursor sequences of both species possess the general organization of dictyopteran FGLa/AST precursors containing 14 putative FGLa/AST peptides. In M. darwiniensis, only 11 out of the 14 FGLa/AST‐like peptides possess the C‐terminal conserved region Y/FXFGL/I/V/M and four of the putative peptide structures are not followed by a Gly residue that would lead to nonamidated peptides. Phylogenetic analyses show the high degree of similarity of dictyopteran FGLa/AST sequences. The position of termites, nested within the Blattaria, confirms that termites have evolved from primitive cockroaches.     

Salinity Regulation of the Interaction of Halovirus SNJ1 with Its Host and Alteration of the Halovirus Replication Strategy to Adapt to the Variable Ecosystem

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.     

Historical geographic dispersal of the golden snub-nosed monkey (Rhinopithecus roxellana) and the influence of climatic oscillations

Current understanding of historic climate oscillations that have occurred over the past few million years has modified scientific views on evolution. Major climatic events have caused local and global extinction of plants and animals and have impacted the spatial distribution of many species. The endangered golden snub‐nosed monkey (Rhinopithecus roxellana) currently inhabits three isolated regions of China: the Sichuan and Gansu provinces (SG), the Qinling Mountains in Shaanxi province (QL), and the Shennongjia Forestry District in Hubei province (SNJ). However, considerable uncertainty still exists about their historical dispersal routes under the influence of environment change. To date, two dispersal routes have been proposed: (1) the QL and SNJ populations originated from the SG population; and (2) the SG population recolonized from the QL and SNJ populations. We used the mitochondrial DNA complete control region to perform statistical assessments of the relative probability of alternative migration scenarios and the role of environmental change on the geographic dispersal of Rhinopithecus roxellana. Thirty haplotypes were identified from the three geographic regions and a high degree of genetic structure was observed. The most recent common ancestor among the mitochondrial DNA haplotypes was estimated to live around 0.47–1.88 million years ago and five notable haplotype clusters were found. Phylogenetic analysis and historical gene flow estimates suggested that the QL and SNJ populations originated from the SG population, with at least two dispersal events from the SG population occurring during the Pleistocene (1.17±0.70 and 0.53±0.30 Ma). Composite dispersal history of the golden snub‐nosed monkey can be explained by both environmental change inducing global climate change and the influence of the Tibetan Plateau uplift. Such range shifts involved considerable demographic changes, as revealed in the dramatic decreases in population size during the last 25,000 years.     

A novel RNA‐binding peptide regulates the establishment of the Medicago truncatula–Sinorhizobium meliloti nitrogen‐fixing symbiosis

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen‐fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so‐called nodulin genes are induced, including large families that encode small peptides. Using a three‐hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA‐binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single‐stranded RNA‐binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA‐binding peptides.     

AtTRB1, a telomeric DNA‐binding protein from Arabidopsis,is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double‐stranded DNA‐binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N‐terminus, a central H1/H5‐like domain and a C‐terminally located coiled‐coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double‐stranded DNA in vitro, while the central H1/H5‐like domain interacts non‐specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5‐like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle‐regulated, as the level of nuclear‐associated GFP diminishes during mitotic entry and GFP progressively re‐associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell‐cycle progression, with the other involving rapid exchange.     

The rulB gene of plasmid pWW0 is a hotspot for the site‐specific insertion of integron‐like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site‐specific integration of related integron‐like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site‐specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILE_FH1 and ILE_FH5) and the NR‐II virulence region of genomic island PAGI‐5 (ILE_FH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.