Genome Characterization of Lipid-Containing Marine Bacteriophage PM2 by Transposon Insertion Mutagenesis

Abstract

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed-and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

“…Interestingly, the organization of the two early PM2 operons was found to be similar to those required for the maintenance of Pseudoalteromonas plasmid pAS28 (124,173). One of these two operons shares significant sequence similarity with the corresponding region in pAS28 and was therefore suggested to represent a recent horizontal acquisition in the PM2 genome from a pAS28-like plasmid (153,173). Notably, the replication proteins in PM2 and pAS28 are nonhomologous, although their genes occupy equivalent positions within the two replicons.…”

Archaeal virus with exceptional virion architecture and the largest single-stranded DNA genome

“…However, the functional complexity of the ACV genome is far greater than that observed for other known ssDNA-and even certain dsDNA-viruses. Indeed, there are examples of bacterial (33), archaeal (18), and eukaryotic (34) viruses with dsDNA genomes that are considerably smaller than that of ACV. The ability to maintain a relatively large genome allows ACV to encode auxiliary functions that, although not typical for ssDNA viruses, are rather frequent in complex dsDNA viruses (SI Text).…”

Genetics for Pseudoalteromonas Provides Tools To Manipulate Marine Bacterial Virus PM2

“…The four genomic fragments were cut out from the plasmids, separated by agarose gel electrophoresis, mixed together, and ligated back to a circular form. The purified ligation mixture was transferred into ER72M2 by electroporation (30). Plaques were purified by three subsequent single-plaque purifications.…”

“…The resulting plasmid, pSM96, was amplified in E. coli HMS174 and selected with tetracycline. The PM2V1 genome was cut out from the plasmid (BamHI), ligated into a circular form, and transferred to ER72M2 cells by electroporation (30) to confirm the viability of the phage after DNA manipulations.…”

“…The construction was transformed into HB101 using tetracycline selection, and the resulting plasmid was named pJB140. The phage genome was digested from the vector by PstI endonuclease, ligated into a circular form, and transferred into ER72M2 cells by electroporation (30).…”