Clear Plaque Mutants of Lactococcal Phage TP901-1.

Witold Kot,Karin Hammer,Finn K Vogensen,Mogens Kilstrup

doi:10.1371/journal.pone.0155233

Abstract

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.

Highlights

The birth of phage genetics was based on the visual inspection of the morphology of plaques
The model for temperate phages, bacteriophage lambda, forms turbid plaques on its Escherichia coli host, and this phenotype was used for isolation of clear plaque mutants (CI) more than fifty years ago[1]
Addition of 200 mM MOPS pH 7.2 and lowering of the glucose concentration from 1% to 0.5% and anaerobic incubation had the required effect and we obtained large turbid plaques upon infection of L. lactis strain 3107 with the wild type bacteriophage TP901-1 as well as the TP901-BC1034 labelled with an ErmR cassette

Summary

Introduction

The birth of phage genetics was based on the visual inspection of the morphology of plaques. The model for temperate phages, bacteriophage lambda, forms turbid plaques on its Escherichia coli host, and this phenotype was used for isolation of clear plaque mutants (CI) more than fifty years ago[1]. The isolation and analysis of such clear plaque mutants has been used to identify additional genes involved in the maintenance of the lysogenic state or in the decision process leading to lysogenisation. Mutations in three different genes cI, cII and cIII[1] were found to result in clear plaques. The lactococcal temperate phage TP901-1 has an interesting and simple switch mechanism consisting of a repressor CI and a small protein, Mor, which apparently is counteracting CI repression of the lytic promoter PL[3]. When a 1kb DNA fragment containing the entire switch region is inserted into a plasmid, PLOS ONE | DOI:10.1371/journal.pone.0155233 June 3, 2016

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Conclusion