Alteromonas is a ubiquitous, abundant, copiotrophic and phytoplankton-associated marine member of the Gammaproteobacteria with a range extending from tropical waters to polar regions and including hadal zones. Here, we describe a novel Alteromonas phage, ZP6, that was isolated from surface coastal waters of Qingdao, China. ZP6 contains a linear, double-stranded, 38,080-bp DNA molecule with 50.1% G+C content and 47 putative open reading frames (ORFs). Three auxiliary metabolic genes were identified, encoding metal-dependent phosphohydrolase, diaminopurine synthetase, and nucleotide pyrophosphohydrolase. The first two ORFs facilitate the replacement of adenine (A) by diaminopurine (Z) in phage genomes and help phages to evade attack from host restriction enzymes. The nucleotide pyrophosphohydrolase enables the host cells to stop programmed cell death and improves the survival rate of the host in a nutrient-depleted environment. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis revealed that ZP6 is most closely related to Enhodamvirus but with low similarity (shared genes, <30%, and average nucleotide sequence identity, <65%); it is distinct from other bacteriophages. Together, these results suggest that ZP6 could represent a novel viral genus, here named Mareflavirus. Combining its ability to infect Alteromonas, its harboring of a diaminopurine genome-biosynthetic system, and its representativeness of an understudied viral group, ZP6 could be an important and novel model system for marine virus research. IMPORTANCE Alteromonas is an important symbiotic bacterium of phytoplankton, but research on its bacteriophages is still at an elementary level. Our isolation and genome characterization of a novel Alteromonas podovirus, ZP6, identified a new viral genus of podovirus, namely, Mareflavirus. The ZP6 genome, with a diaminopurine genome-biosynthetic system, is different from those of other isolated Alteromonas phages and will bring new impetus to the development of virus classification and provide important insights into novel viral sequences from metagenomic data sets.
Matched MeSH terms: Bacteriophages/growth & development
A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.
Matched MeSH terms: Bacteriophages/growth & development