The N-Terminal Region of the Oenococcus oeni Bacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide in Escherichia coli and as a cis -Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells

Abstract

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.All tailed bacteriophages with double-stranded DNA genomes appear to accomplish lysis of the host cell by the concerted action of a peptidoglycan hydrolase (referred to as endolysin or lysin) and a small hydrophobic protein (holin) presumed to form nonspecific lesions upon oligomerization in the membrane (for a review, see reference 41). The latter function seems essential to allow access of the lytic enzyme to the cell wall compartment since in the phage lysins examined so far, the presence of a signal peptide (SP) that would target them to the translocase of the general secretion pathway (GSP) has never been demonstrated.We have recently described the sequences of the lysin and holin genes from the Oenococcus oeni bacteriophage fOg44 and noted that the N-terminal region of its putative lysin (Lys44) was highly hydrophobic (23). A similar observation was made earlier concerning a related enzyme from the lactococcal phage Tuc2009 (2). In spite of its hydrophobic character, the function of the N-terminal sequence of the Tuc2009 lysin as a possible SP was not considered, presumably because the presence of a holin gene upstream of lys argued for a standard holin-dependent lysin export mechanism. This assumption was strengthened by the observation that the expression of an almost identical lysin in Escherichia coli (LysB from the Lactococcus lactis phage LC3) did not result in a decrease in culture absorbance unless the corresponding holin was simultaneously induced (3).Interestingly, however, during an attempt to overproduce Lys44 in an easily purifiable form (as a histidine-tagged fusi...

Molecular Aspects and Comparative Genomics of Bacteriophage Endolysins

“…The presence of N-terminal signal sequences in endolysins suggests an alternative to the lambda paradigm and thus provides a mode of action different from that of the great majority of endolysins to access the PG at the end of the lytic phage cycle. Signal sequences may enable transport of the endolysin through the inner membrane to achieve the periplasm in a holin-independent manner by making use of the host Sec machinery (24,27,77).…”

“…Subsequently, a small hydrophobic membranespanning protein called holin is expressed at a genetically predetermined time and accumulates in clusters producing homo-oligomeric pores in the inner membrane, thereby exposing the PG layer to the endolysin (17,25,26). Some endolysins, such as the Bacillus cereus phage TP21-L, Oenococcus oeni phage fOg44, and Lactobacillus plantarum phage Øg1e endolysins, present different intrinsic signal sequences that allow them to pass the cytoplasmic membrane and reach the PG in a holin-independent manner (27,28). The referred lytic systems are reported exclusively for Caudovirales phages, and not much is known about lytic systems present in cassettes of phages outside this order.…”

Bacillus subtilisOperon Encoding a Membrane Receptor for Bacteriophage SPP1

“…To obtain this recombinant protein, the corresponding sequence of yueB, obtained by PCR with the primer pair yueB-4/yueB-5, was cloned into pIVEX2.3d (Table 1) after digestion of both the product and the vector with the enzymes NcoI and XmaI. The resulting plasmid (pCSJ65) was used to transform E. coli strain CG61, which produces T7 RNA polymerase upon temperature upshift (from 28 to 42°C) (31). This constructed strain was used to overproduce YueB780, which was then purified from E. coli extracts by affinity chromatography (C. São-José and S. Lhuillier, unpublished data).…”

Sizing the Holin Lesion with an Endolysin-β-Galactosidase Fusion

“…Holin and endolysin genes can be identified by analysis of the genomic sequences of most dsDNA phages, although only a few have been characterized in terms of in vivo function. For phages tested to date, the endolysin requires the holin to achieve access to the peptidoglycan, except in some phages of gram-positive bacteria, where the endolysins use the sec system (20). The best-characterized example of holin-endolysin systems is that of phage , where the S gene encodes the holin and R encodes the endolysin, a transglycosylase.…”