Abstract
Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin–pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate.
Highlights
Bacteriophages are obligate parasites that infect and, in most cases, eventually kill bacteria
The TP712 Lysis Cassette Features a Holin Gene With a Functional Dual Start Motif and Both Holin Proteins are Synthesized in L. lactis NZ9000 and L. lactis ftsH
The lysis cassette of the phage TP712 comprises a putative pinholin gene holTP712 and the lysin gene lysTP712 (Figure 1). holTP712 encodes a 74-aa membrane protein Hol74TP712 with two transmembrane domains (TMD), a topology that resembles that of the class II pinholin S21 of the E. coli phage 21 (Park et al, 2006)
Summary
Bacteriophages are obligate parasites that infect and, in most cases, eventually kill bacteria. Lysis of the host is a crucial step for survival of lytic phages and sophisticated mechanisms have evolved to maximize phage propagation. This is exemplified by the enrolment of several proteins dedicated to coordinate and time lysis such as holin/antiholins to disrupt the cytoplasmic membrane, endolysins to degrade the peptidoglycan layer, and spanins (in phages infecting Gram-negative hosts) required to overcome the outer membrane (Young, 2013). Most dsDNA phages possess a typical holin–endolysin system, where a hydrophobic membrane protein, the holin, inserts into the cytoplasmic membrane forming holes These holes are large enough to provide access to the cell wall for the endolysin that has up to this point been
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