Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712

Abstract

The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis DftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the DftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.Large-scale dairy fermentations can be seriously compromised by bacteriophages infecting lactic acid bacteria (LAB), and particularly Lactococcus lactis, which is a commonly used starter culture for cheese making (Garneau & Moineau, 2011). Lactococcal phages are currently classified in 10 groups, eight of them belonging to the Siphoviridae family of the order Caudovirales. Those of the c2, 936 and P335 groups are the most commonly found in dairy plants (Deveau et al., 2006).A considerable effort has been made to improve and select phage-resistant starter strains based on the knowledge of resistance mechanisms developed by LAB and a better understanding of phage-host interactions (Sturino & Klaenhammer, 2006). Described mechanisms of phage resistance expand from blocking adsorption, restriction/ modification enzymes, to abortive infection systems that interfere with phage DNA replication, morphogenesis and release. Clustered regularly interspaced short palindromic repeats (CRISPRs) located in the genomes of host bacteria together with a group of associated proteins can also confer resistance to phages (Labrie et al., 2010). Recently, the response to phage infection in L. lactis has been approached by genome-wide transcriptomics (Fallico et al., 2011;Ainsworth et al., 2013). L. lactis appears to sense bacteriophage infection as a perturbation of its cell envelope and mounts a response targeted to the cell wall, activating regulators of the cell envelope stress response such as the two-component system CesSR.One of the members of the CesSR regulon in L. lactis is the ftsH gene (Martínez et al., 2007). ftsH encodes a conserved AAA (ATPase associated with various cellular activities)-type membrane protease involved in stress response and protein quality control (Ito & Akiyama, 2005;Narberhaus et al., 2009). In this work, we analysed the impact of the ftsH null mutation on the life cycle of the P335 temperate phage TP712, which is able to infect and lysogenize L. lactis MG1363 (Gasson, 1983) and derivatives thereof. The complete TP712 nt sequence has been determined (GenBank accession number AY766464) essentially as described by Wegmann et al. (2012).Preliminary experiments revealed that in standard plaque assays (Lillehaug, 1997), the efficiency of plaquing (EOP) of this phage on an available non-polar and in-frame L. lactis mutant lacking ftsH (Pinto et al., 2011) was reduced to 1.5610 25 when compared to its parent L. lactis NZ9000. Upon complementation with the plasmid pUK200 : : ftsH, ...

Characterization of prophages of Lactococcus garvieae

“…3). Interestingly, they are all related to the temperate phage ɸTP712 found in the widely used plasmid-free laboratory strain L. lactis MG1363 and derived from the dairy L. lactis strain NCDO 712 32, 33 . Phage ɸTP712 is also related to the sequenced temperate genome, PLgT-1, isolated from a marine environment (Fig.…”

Clostridium difficile phages: still difficult?

“…AbiF is a protein involved in phage abortive infection [see review by Labrie et al (2010)]. Homologs of this gene are not present in any of the other C. difficile phages and while Abi genes are typically encoded by their bacterial hosts, they have been found on a prophage element in Lactococcus lactis (Ventura et al, 2007) and in the genome of L. lactis phage TP712 (Roces et al, 2013). Although the mechanism by which AbiF performs its function is not fully known (Garvey et al, 1995), the unusual presence of this gene in the genome of phage φC2 suggests it could inhibit secondary phage infection or modulate φC2 replication and may contribute to the low frequencies reported for isolating free phages.…”

Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis

“…Causing ablation of this promoter was aimed at preventing t712 prophage induction and virion production. 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 ]. This expectation was indeed verified, as mutagenesis of the deduced promoter sequence for t712 Cro protein caused an approximate 100-fold increase in the number of produced infective TP901-1 erm phage particles obtained following mitomycin C induction from the NZ9000-Cro t712 background as compared to that obtained when strain NZ9000 was used (data not shown; indicator strain used for TP901-1 erm phage titre determination was L .…”

“…Inducible prophage t712 of NZ9000 [ 58 ], called TP712 by Roces et al . (2013) [ 59 ], was identified using the PHAge Search Tool (PHAST) [ 60 ]. BLAST, Pfam and HHpred analyses were used for functional annotations of proteins [ 61 – 64 ].…”