Genome-based characterization of a novel prophage of Vibrio parahaemolyticus, VPS05ph1, a novel member of Peduoviridae.

Vibrio parahaemolyticus is a globally important bacterium related to climate warming and health threat to human and marine animals. Yet, there is limited knowledge about its polylysogeny harboring multiple prophages and the genetic information. In this study, two prophages (VPS05ph1 and VPS05ph2) were identified in a V. parahaemolyticus isolate through genomic and transcriptional analyses. Both prophages were determined as HP1-like phages, located in a novel phylogenetic lineage of Peduoviridae. They shared a moderate genome-wide sequence similarity with each other and high synteny with the closest relatives, but showed low identities to the repressor counterparts of the representative phages within the family. In addition, no bacterial virulence genes, antibiotic resistance genes and known phage-encoded lytic proteins were identified on both prophage genomes. Moreover, the V. parahaemolyticus isolate was induced with mitomycin, which caused aberrant cellular morphology and nonviability of bacterial cells and excision of prophage VPS05ph1, accompanied by the respective inhibition and promotion of transcriptions of the cI-like and cox-like regulator genes for phage decision making. Results in this study provide the genetic context of polylysogeny in the V. parahaemolyticus isolate, support the diversity and prevalence of HP1-like phages in vibrios, and promote to explore interactions between the HP1-like prophage and its vibrio host.

Spontaneous excision and facilitated recovery as a control for phenotypes arising from RNA interference and other dominant transgenes.

An essential control for genetic manipulation of microbes is the regeneration of the wild-type state and phenotype to validate that any mutant phenotypes are 'on target'. For Leishmania gene knockouts, this is often done by re-expression of the target gene from episomal vectors, often bearing counter-selectable markers. Methods for similarly validating the outcomes from dominant mutations such as those arising from RNA interference (RNAi) are needed. We present here such an approach, relying on facilitated recovery after spontaneous excision - or 'popouts' - of dominant transgenes stably inserted into the ribosomal RNA array, utilizing GFP as a marker and single cell sorting to recover regenerated WT controls. We validate its utility using RNA interference knockdowns of the paraflagellar rod gene PFR2 of L. (Viannia) braziliensis. The method yields stably modified lines suitable for long term studies of Leishmania virulence, relies solely on host rather than introduced genetic machinery, and is thus readily applied in many species and circumstances including functional genetic testing.