Ying Liu

Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205.

A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ~10(4) PFU/ml to ~10(11) PFU/ml. Single-step growth curves exhibited a burst size of ~100 PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341 bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane.

Identification and characterization of SNJ2, the first temperate pleolipovirus integrating into the genome of the SNJ1-lysogenic archaeal strain.

Proviral regions have been identified in the genomes of many haloarchaea, but only a few archaeal halophilic temperate viruses have been studied. Here, we report a new virus, SNJ2, originating from archaeal strain Natrinema sp. J7‐1. We demonstrate that this temperate virus coexists with SNJ1 virus and is dependent on SNJ1 for efficient production. Here, we show that SNJ1 is an icosahedral membrane‐containing virus, whereas SNJ2 is a pleomorphic one. Instead of producing progeny virions and forming plaques, SNJ2 integrates into the host tRNAMet gene. The virion contains a discontinuous, circular, double‐stranded DNA genome of 16u2009992u2009bp, in which both nicks and single‐stranded regions are present preceded by a ‘GCCCA’ motif. Among 25 putative SNJ2 open reading frames (ORFs), five of them form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Furthermore, SNJ2‐like proviruses containing the conserved gene cluster were identified in the chromosomes of archaea belonging to 10 different genera. Comparison of SNJ2 and these proviruses suggests that they employ a similar integration strategy into a tRNA gene.

Plasma Hormones Facilitated the Hypermotility of the Colon in a Chronic Stress Rat Model

Objective To study the relationship between brain-gut peptides, gastrointestinal hormones and altered motility in a rat model of repetitive water avoidance stress (WAS), which mimics the irritable bowel syndrome (IBS). Methods Male Wistar rats were submitted daily to 1-h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Plasma hormones were determined using Enzyme Immunoassay Kits. Proximal colonic smooth muscle (PCSM) contractions were studied in an organ bath system. PCSM cells were isolated by enzymatic digestion and IKv and IBKca were recorded by the patch-clamp technique. Results The number of fecal pellets during 1 h of acute restraint stress and the plasma hormones levels of substance P (SP), thyrotropin-releasing hormone (TRH), motilin (MTL), and cholecystokinin (CCK) in WAS rats were significantly increased compared with SWAS rats, whereas vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and corticotropin releasing hormone (CRH) in WAS rats were not significantly changed and peptide YY (PYY) in WAS rats was significantly decreased. Likewise, the amplitudes of spontaneous contractions of PCSM in WAS rats were significantly increased comparing with SWAS rats. The plasma of WAS rats (100 µl) decreased the amplitude of spontaneous contractions of controls. The IKv and IBKCa of PCSMs were significantly decreased in WAS rats compared with SWAS rats and the plasma of WAS rats (100 µl) increased the amplitude of IKv and IBKCa in normal rats. Conclusion These results suggest that WAS leads to changes of plasma hormones levels and to disordered myogenic colonic motility in the short term, but that the colon rapidly establishes a new equilibrium to maintain the normal baseline functioning.

Actions of hydrogen sulfide and ATP-sensitive potassium channels on colonic hypermotility in a rat model of chronic stress.

Objective To investigate the potential role of hydrogen sulphide (H2S) and ATP-sensitive potassium (KATP) channels in chronic stress-induced colonic hypermotility. Methods Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Organ bath recordings, H2S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H2S in repeated WAS-induced hypermotility. Organ bath recordings and western blotting were used to detect the role of KATP channels in repeated WAS. Results Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and decreased the endogenous production of H2S and the expression of H2S-producing enzymes in the colon devoid of mucosa and submucosa. Inhibitors of H2S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats. NaHS concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC50 for the WAS rats was significantly lower than that for the SWAS rats. The inhibitory effect of NaHS was significantly reduced by glybenclamide. Repeated WAS treatment resulted in up-regulation of Kir6.1 and SUR2B of KATP channels in the colon devoid of mucosa and submucosa. Conclusion The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H2S. The increased expression of the subunits of KATP channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H2S donor may have potential clinical utility in treating chronic stress- induced colonic hypermotility.

Halophilic archaea cultivated from surface sterilized middle-late eocene rock salt are polyploid

Live bacteria and archaea have been isolated from several rock salt deposits of up to hundreds of millions of years of age from all around the world. A key factor affecting their longevity is the ability to keep their genomic DNA intact, for which efficient repair mechanisms are needed. Polyploid microbes are known to have an increased resistance towards mutations and DNA damage, and it has been suggested that microbes from deeply buried rock salt would carry several copies of their genomes. Here, cultivable halophilic microbes were isolated from a surface sterilized middle-late Eocene (38–41 million years ago) rock salt sample, drilled from the depth of 800 m at Yunying salt mine, China. Eight unique isolates were obtained, which represented two haloarchaeal genera, Halobacterium and Halolamina. We used real-time PCR to show that our isolates are polyploid, with genome copy numbers of 11–14 genomes per cell in exponential growth phase. The ploidy level was slightly downregulated in stationary growth phase, but the cells still had an average genome copy number of 6–8. The polyploidy of halophilic archaea living in ancient rock salt might be a factor explaining how these organisms are able to overcome the challenge of prolonged survival during their entombment.

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.

Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1

UNLABELLEDnThe temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species.nnnIMPORTANCEnArchaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts.

The complete genome of a viable archaeum isolated from 123‐million‐year‐old rock salt

Live microbes have been isolated from rock salt up to Permian age. Only obligatory cellular functions can be performed in halite-buried cells. Consequently, their genomic sequences are likely to remain virtually unchanged. However, the available sequence information from these organisms is scarce and consists of mainly ribosomal 16S sequences. Here, live archaea were isolated from early Cretaceous (∼ 123 million years old) halite from the depth of 2000 m in Qianjiang Depression, Hubei Province, China. The sample was radiologically dated and subjected to rigorous surface sterilization before microbe isolation. The isolates represented a single novel species of Halobacterium, for which we suggest the name Halobacterium hubeiense, type strain Hbt.u2009hubeiense JI20-1. The species was closely related to a Permian (225-280 million years old) isolate, Halobacterium noricense, originating from Alpine rock salt. This study is the first one to publish the complete genome of an organism originating from surface-sterilized ancient halite. In the future, genomic data from halite-buried microbes can become a key factor in understanding the mechanisms by which these organisms are able to survive in harsh conditions deep underground or possibly on other celestial bodies.

Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging

Archaeal viruses have evolved to infect hosts often thriving in extreme conditions such as high temperatures. However, there is a paucity of information on archaeal virion structures, genome packaging, and determinants of temperature resistance. The rod-shaped virus APBV1 (Aeropyrum pernix bacilliform virus 1) is among the most thermostable viruses known; it infects a hyperthermophile Aeropyrum pernix, which grows optimally at 90u2009°C. Here we report the structure of APBV1, determined by cryo-electron microscopy at near-atomic resolution. Tight packing of the major virion glycoprotein (VP1) is ensured by extended hydrophobic interfaces, and likely contributes to the extreme thermostability of the helical capsid. The double-stranded DNA is tightly packed in the capsid as a left-handed superhelix and held in place by the interactions with positively charged residues of VP1. The assembly is closed by specific capping structures at either end, which we propose to play a role in DNA packing and delivery.The rod-shaped virus APBV1 is among the most thermostable viruses known. Here, Ptchelkine et al. determine its structure at near-atomic resolution, show that the DNA is packed as left-handed superhelix and identify extended hydrophobic interfaces that likely contribute to the extreme thermostability of the capsid.

A novel family of tyrosine integrases encoded by the temperate pleolipovirus SNJ2

Abstract Genomes of halophilic archaea typically contain multiple loci of integrated mobile genetic elements (MGEs). Despite the abundance of these elements, however, mechanisms underlying their site-specific integration and excision have not been investigated. Here, we identified and characterized a novel recombination system encoded by the temperate pleolipovirus SNJ2, which infects haloarchaeon Natrinema sp. J7-1. SNJ2 genome is inserted into the tRNAMet gene and flanked by 14 bp direct repeats corresponding to attachment core sites. We showed that SNJ2 encodes an integrase (IntSNJ2) that excises the proviral genome from its host cell chromosome, but requires two small accessory proteins, Orf2 and Orf3, for integration. These proteins were co-transcribed with IntSNJ2 to form an operon. Homology searches showed that IntSNJ2-type integrases are widespread in haloarchaeal genomes and are associated with various integrated MGEs. Importantly, we confirmed that SNJ2-like recombination systems are encoded by haloarchaea from three different genera and are critical for integration and excision. Finally, phylogenetic analysis suggested that IntSNJ2-type recombinases belong to a novel family of archaeal integrases distinct from previously characterized recombinases, including those from the archaeal SSV- and pNOB8-type families.