Abstract
Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.
Highlights
Myoviridae, Podoviridae and Siphoviridae comprise the three familial divisions of Caudovirales, or tailed phages
Several improvements were made to optimize the host background for analysing mutations made in TP901-1erm, a derivative of TP901-1 that is marked with an erythromycin-resistance (Emr) cassette[76], which had been used to select a TP901-1erm lysogenized derivative of strain L. lactis NZ9000 [13]
Induction of prophage t712 was identified as a potential problem as; (i) t712 particle production would interfere with TP901-1 phage particle study, (ii) t712 induction would increase the metabolic burden on the host when only TP901-1 induction was desired, and (iii) it was deemed possible that, due to the conserved nature of Siphoviridae proteins, t712 may supply TP901-1 with structural proteins being analysed during this study in trans [59]
Summary
Myoviridae, Podoviridae and Siphoviridae comprise the three familial divisions of Caudovirales, or tailed phages. Siphoviridae phages represent the dominant viral morphology, frequently outnumbering other viral morphotypes in environments such as the human gut and oceanic waters [1,2,3,4]. Despite the prevalence of phages with long non-contractile tails, only a handful of Siphoviridae have been extensively studied at structural and functional levels (for a review, see [5]). The prototypical and related Lactococcus lactis-infecting phages TP901-1 and Tuc2009 have been important in understanding Siphoviridae phages due to the genetic accessibility of their Gram-positive hosts, the various molecular tools available for manipulating their genomes and the conserved nature of phage structural proteins [6,7,8,9,10,11,12,13].
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