Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of Listonella pelagia

Abstract

The genome for the marine pseudotemperate member of the Siphoviridae HSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. HSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10 9 cells/ml and >10 11 phage/ml. The genome (37,966 bp; G؉C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a ␤ subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), HSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.

Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1

“…A blue-white band containing the phage was removed (2 ml total volume) and dialysed (Pierce Slide-A-Lyzer 10 K MWCO, Rockford, IL, USA) twice in 1 l buffer (10 mM NaCl, 50 mM Tris-HCl (pH 8), 10 mM MgCl 2 ) to remove CsCl. Phages were concentrated 10 Â (Microcon 30 kD; Millipore, Billerica, MA, USA) and proteins denatured by five freeze-thaw (96 1C) cycles and 12% sodium dodecyl sulphate, then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as described by Paul et al (2005). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry peptide mass fingerprint spectra were generated from trypsindigested bands excised from polyacrylamide gel electrophoresis gel (TOPLAB GmbH; Martinsried, Germany).…”

The Temperate Marine Phage ΦHAP-1 of Halomonas aquamarina Possesses a Linear Plasmid-Like Prophage Genome

“…on May 10, 2018 by guest http://jvi.asm.org/ rine bacteria (46,56). These marine phages most likely have lytic genes, but they remain unknown due to the bias of genomic databases toward terrestrial and medical phages.…”

“…This results in DNA of different lengths with no specific sequence at the ends of the majority of the packaged phage genomes. These types of tailed phages have circularly permuted genomes; thus, the terminase small subunits are often selected as the arbitrary start points for genomic maps to ease comparison between phages (13,46). This convention was followed when constructing the ⌽HAP-1 genomic map.…”

“…Protein-containing bands were numbered and excised from the gel with a scalpel. The excised samples were sent to the Proteomics Core Facility at Moffitt Cancer Research Center (Tampa, FL) for de novo peptide analysis by matrix-assisted laser desorption ionization-time-offlight-time-of-flight (MALDI-TOF-TOF) mass spectroscopy as described in Paul et al (46). The tandem mass spectral analysis produces a collision-induced dissociation spectrum that can be used for protein identification based on peptide sequence (68).…”

“…Most of the DNA sequence of the ⌽HAP genome obtained was from two types of libraries. The first was made by the Nanoclone method at Lucigen (Middleton, WI); this entails physical shearing of phage DNA purified from mitomycin C-induced cultures of the Halomonas host, end repair, selection of fragments of between 1 and 2 kb, and cloning into the pSMART vector (46). The second library was made by complete restriction digestion with NheI of phage DNA from a mitomycin C-induced Halomonas lysogen culture and cloning into pBR322 cut with NheI.…”