Molecular Characterizations Of Thermophilic Bacteriophages And Thermostable Maltogenic Amylase

The ocean is the most abundant and the last underutilized biology resources on earth. When the terrestrial resources have been developed fully, halobios have become new focuses for their importance. Marine microbes, especially the thermophiles have become a study hotspot. Current theory and circumstancial evidence suggest that the thermophiles are the first lifes on earth, which will be helpful for understanding the origin of life. Thermophiles and their enzymes are also the important resources in industry and biotechnology.Recently years, viruses from thermophiles have attracted intensive investigation because they can serve as model systems to study the biochemistry and molecular biology of marine microbes, and they could influence many biogeochemical and ecological processes, including nutrient cycling, taxon of biology, bacterial biodiversity and species distribution, genetic transfer and evolution of biology. Therefore, thermophilic viruses are not only helpful for understanding the life, but also have great potential usese in industry and biotechnology.In total, 170 strains of thermophilic bacteria were isolated from deep-sea hydrothermal fields in Pacific and a hot spring in Xiamen of China. To facilitate the identification of thermophilic strains, SDS-PAGE of whole-cell proteins of these strains was firstly performed. The results showed that there existed 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.During the isolation of thermophiles, a maltogenic amylase-producing thermophilic strain WPD616, assigned to Bacillus sp. WPD616 based on 16S rRNA sequence, was isolated from a deep-sea hydrothermal field in west Pacific. Subsequently a maltogenic amylase gene encoding 588 amino acids from this isolate was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The results showed that the recombinant maltogenic amylase had an activity optimum at 50℃and pH at 6．0．It was active up to 70℃at pH 6．0 and stable at pH ranging from 6．0 to 8．0．The recombinant enzyme was active when Chaps, DTT and Tween 20 (0．1% or 1%) were used. However, it can be partially inhibited by 1 mM of EDTA, PMSF or SDS, as well as 0．1% of Triton X-100 or 2-ME, and completely inhibited by 10 mM of PMSF and SDS (10mM). Its catalytic function was stable in the presence of Li⁺ and K⁺ (1mM or 10mM), but its activity decreased when Ba²⁺, Ca²⁺, Mg²⁺, Mn²⁺ (1mM or 10mM) and Fe²⁺ (1mM) were used. In the presence of Zn²⁺, Cu², Fe³⁺(1mM and 10mM) and Fe²⁺ (10mM), no activity was detected.During the cultures of thermophiles, a thermophilic bacteriophage Geobacillus sp.Virus 1 (GBSV1) was obtained from an offshore hot spring in Xiamen of China, and three thermophilic bacteriophages Bacillus Virus W13 (BVW13),Geobacillus Virus E26323 (GVE26323)å’ŒGeobacillus sp. Virus E1 (GSVE1) were isolated from a deep-sea hydrothermal field. The GBSV1 and GSVE1were further characterized. GBSV1 was isolated from a thermophilic bacterium Geobacillus sp. 6k51．GBSV1 was a typical Myoviridae phage with a hexagonal head (60 in diameter) and a tail (120-135 nm in length). As revealed by PCR detection, the GBSV1 could infect 5 species of Geobacillus or Bacillus thermophiles. The GBSV1 contained a double-stranded circular DNA of 34579 bp, which had the capacity encoding 55 open reading frames (ORFs). Surprisingly, eight of the 55 GBSV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. This was the first report that human disease-relevant bacterial genes were found in thermophilic phage. The different morphology and genome size from reported thermophilic viruses suggested that the GBSV1 was a novel thermophilic bacteriophage. Five proteins of the purified GBSV1 virions were identified by proteomic analysis. Among them, the vp425 gene was characterized to be a viral late gene. Determination of GBSV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.Bacteriophage GSVE1 infecting thermophilic Geobacillus sp. E263 was purified in this study. The results revealed that GSVE1 was a typical Siphoviridae phage containing a 40863-bp linear double-stranded genomic DNA with 62 presumptive open reading frames (ORFs). Surprisingly, 8 ORFs shared sequence similarities to genes from human disease-relevant bacteria and their bacteriophages. Microarray analysis indicated that 74．2% of the presumptive ORFs were expressed. The purified virions contained six protein bands, which were identified by mass spectrometric analysis. Of the newly retrieved proteins, VP371 (derived from band 4) was further characterized. Western blot analysis showed that the anti-GST-VP371 antibody reacted strongly with band 4．The immuno-electron microscopy indicated that VP371 protein was a component of the viral capsid. As the first characterized thermophlic bacteriophage from deep-sea hydrothermal field with functional genomics, the molecular characterizations of GSVE1 will be very helpful to elucidate the role of thermophilic virus in deep-sea hydrothermal vents.