Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis

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.

Identification and Analysis of a Novel Group of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus

“…The defined tail morphogenesis gene cluster in phage 9871 commences with ORF11 9871 , which encodes a putative tail component (based on the presence of domains detected using CDD and Pfam). ORF12 9871 was not confirmed as a structural proteinencoding gene; however, a homologue of this gene product present in lactococcal phage TP901-1 has recently been annotated as the tail terminator protein (45), with apparently unchecked tail extension observed in mutant phages containing a stop codon in this gene. ORF13 9871 specifies the presumed major tail protein, with ORF14 9871 and ORF15 9871 encoding putative tail assembly chaperone proteins (46,47) of ORF16 9871 (predicted to encode the tail tape measure protein).…”

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

“…To this end, intact tails (produced from a mutant that does not produce the major head protein31) were added exogenously to the crude lysate of non-plaque forming mutants and in all cases, (almost) full infectious activity was restored in terms of lysogeny conversion at levels comparable to that of the parent phage (Table 1). …”

“…Phage lysates for the various biological characterisation assays were induced from (at least) three separate overnights of NZ9000-Cro t712 lysogenized with TP901-1 erm , or a particular mutant derivative using 0.5 μg/ml mitomycin C when the growing cultures reached an optical density at 600 nm (OD600) of approximately 0.2. Polyethylene glycol 8000 (PEG 8000) precipitation of phage particles (or component parts) of the TP901-1 erm phage or a particular mutant derivative, and their subsequent purification, was performed as described previously31.…”

Genomics of tailless bacteriophages in a complex lactic acid bacteria starter culture

“…Temperate lactococcal phages of Siphoviridae family, belonging to the P335 group, are usually characterised by a long non-contractile tail -a structure responsible for host recognition, adsorption and the initiation of phage infection by envelope penetration and DNA ejection (Mc Grath et al, 2006). Despite the fact that all inducible prophages found in strains from the complex starter culture Ur so far are essentially tailless, phage sequencing revealed the presence of genes encoding most of the tail structural elements: head-tail connector, major tail protein (MTP), tail length tape-measure protein (TMP), distal tail protein, tail associated lysin, upper and lower base plate protein (Vegge et al, 2005;Veesler and Cambillau, 2011;Veesler et al, 2012;Stockdale et al, 2015). However, detailed sequence analysis identified the presence of insertions of mobile genetic elements in the tail module in most of the phage genomes analysed in this study.…”