Bacteriophages of thermophiles are of great interest due to their important roles in many biogeochemical and ecological processes. However, no virion has been isolated from deep-sea thermophilic bacteria to date. In this investigation, two lytic bacteriophages (termed Bacillus virus W1 and Geobacillus virus E1) of thermophilic bacteria were purified from deep-sea hydrothermal fields in the Pacific for the first time. Bacillus virus W1 (BVW1) obtained from Bacillus sp. w13, had a long tail (300nm in length and 15 nm in width) and a hexagonal head (70 nm in diameter). Another virus, Geobacillus virus E1 (GVE1) from Geobacillus sp. E26323, was a typical Siphoviridae phage with a hexagonal head (130 nm in diameter) and a tail (180 nm in length and 30 nm in width). The two phages contained double-stranded genomic DNAs. The genomic DNA sizes of BVW1 and GVE1 were estimated to be about 18 and 41 kb, respectively. Based on SDS-PAGE of purified virions, six major proteins were revealed for each of the two phages. The findings in our study will be very helpful to realize the effect of virus on thermophiles as well as the communities in deep-sea hydrothermal fields.
A novel xylanase-producing thermophilic strain MT-1 was isolated from a deep-sea hydrothermal field in east Pacific. A xylanase gene encoding 331 amino-acid peptide from this isolate was cloned and expressed in Escherichia coli. The recombinant xylanase exhibited maximum activity at 70 degrees C and had an optimum pH of 7.0. It was active up to 90 degrees C and showed activity over a wide pH ranging from 5.5 to 10.0. The crude xylanase presented similar properties in temperature and pH to those of the recombinant xylanase. The recombinant xylanase was stable in 1 mM of enzyme inhibitors (PMSF, EDTA, 2-ME or DTT) and in 0.1% detergents (Tween 20, Chaps or Triton X-100), whereas, it was strongly inhibited by sodium dodecyl sulfate (SDS) (1 mM). In addition, its catalytic function was stable in the presence of Li(+), Na(+) or K(+). However, it was strongly inhibited by Ni(2+), Mn(2+), Co(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+) and Al(3+) (1 or 0.1 mM). The K (m) and V (max) of the recombinant xylanase for oat spelt xylan were calculated to be 1.579 mg/ml and 289 micromol/(min x mg), respectively. Our study, therefore, presented a rapid overexpression and purification of xylanase from deep-sea thermophile aimed at improving the enzyme yield for industrial applications and scientific research.
Since it was proposed to be a tumor suppressor in 1991, the QM protein has attracted intensive studies in plants, animals and fungi. Up to date, however, the function of QM protein remains unknown. In this investigation, it was found that the shrimp QM gene (designated as PjQM) was significantly up-regulated in virus- resistant shrimp, suggesting that the PjQM was involved in shrimp immunity. The GST pull-down assays showed that the PjQM protein interacted with shrimp hemocyanin and myosin, indicating that the PjQM protein might participate in prophenoloxidation (proPO) activation system of shrimp immunity. As revealed by RNAi assays, it was demonstrated for the first time that the QM protein could regulate the activity of phenol oxidase, a key enzyme in the proPO activation system of invertebrate immunity. This discovery showed a novel aspect of QM protein in arthropod immune response, which contributed a better understanding to the still poorly understood molecular events involved in innate immunity of arthropods.
In total, 170 strains of thermophilic bacteria were isolated from deep-sea hydrothermal fields in the Pacific Ocean and a hot spring in Xiamen of China. To facilitate the identification of thermophilic strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of these strains was first performed. The results showed that there exist four different protein patterns, indicating that the 170 strains might belong to four species or genera. The RAPD (random amplified polymorphic DNA) profiles of nine representative strains were consistent with those of SDS-PAGE. To further identify the species of the nine strains, their 16S rDNA sequences were analyzed. The results showed that the nine strains fell into four species of three genera, which was the same as revealed by SDS-PAGE. Therefore, SDS-PAGE of whole-cell proteins could be used as a rapid and simple method for the discrimination of thermophilic bacteria as the first step of species identification.
Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames (ORFs). Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.
In tailed dsDNA phages, the tail assembly gene cluster is of great importance for phage assembly and infection. Up to date, however, this concern is not addressed to thermophilic bacteriophages from deep-sea hydrothermal vents. In this investigation, a typically organized tail assembly gene cluster, encoding the tail protein, two phage-related proteins and tape measure protein, was revealed in the deep-sea thermophilic lytic phage GVE2 for the first time. As demonstrated by northern and western blots, the vp192 gene was expressed in the late stage of virus infection. The immuno-electron microscopy further indicated that the vp192-encoded polypeptide was the tail protein of GVE2. The results showed that a programmed translational frameshift existed in the tail assembly gene cluster of GVE2, suggesting the existence of a conserved tail assembly pathway in dsDNA tailed phages from deep sea. Our findings would be very helpful for understanding the assembly and infection of thermophilic bacteriophage in deep-sea hydrothermal vent.
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