Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

Abstract

Holins are phage-encoded hydrophobic membrane proteins that spontaneously and nonspecifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol. The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy. HolGH15 exerted efficient antibacterial activity at 37 8C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus phage holin that exerts antibacterial activity against heterogeneous pathogens.

Complete genomic sequence of the Vibrio alginolyticus bacteriophage Vp670 and characterization of the lysis-related genes, cwlQ and holA

“…The putative HolA contains a transmembrane helix with a hydrophilic C-terminal region inside the cytoplasmic membrane. These features of HolA are similar to the description of common holing characteristics [ 31 ], which further strengthened our speculation that holA encodes for a holin. Holins are produced during the late stages of infection, and once a critical concentration is reached, holins form holes in the cytoplasmic membrane by oligomerization, allowing endolysins, which have accumulated in the cytoplasm, to access their PG substrate [ 32 , 33 ].…”

“…Further TEM observation confirmed that HolA killed the induced V. alginolyticus cells through destroying cellular structures and causing the release of cellular contents. Typical holins spontaneously assemble into oligomers, resulting in the formation of nonspecific and very large pores in the cytoplasmic membrane that allow the nonspecific release of endolysins and other proteins [ 31 , 35 , 36 ]. Though the expression of holA had ability to kill and disrupt the integrity of the bacterial cell wall, it could not rapidly cause large breaches on the cell walls and burst the cells without the synergistic effects of the cwlQ expression.…”

“…This is not the only such case, and similar phenomena have been observed. Heterogenic expression of holin genes from various sources exhibits toxicity towards E. coli cells [ 31 , 37 , 38 ]. It suggests that the integration of holins into cell membranes may lack species specificity [ 31 , 38 ], and therefore, holA and even other holin genes are potentially valuable for some applications.…”

“…Heterogenic expression of holin genes from various sources exhibits toxicity towards E. coli cells [ 31 , 37 , 38 ]. It suggests that the integration of holins into cell membranes may lack species specificity [ 31 , 38 ], and therefore, holA and even other holin genes are potentially valuable for some applications. For instance, holA may be used as a counterselective marker for constructing suicide vectors instead of the unsatisfactory sacB gene, which is not universal enough for use in the manipulation of bacterial genetics [ 21 ].…”

Screening and characterization of prophages in Desulfovibrio genomes

“…While they are present in the bacterial genome, these prophages are directly related to the genome diversity of the host cell, contributing positively or not, to bacterial fitness 7 , 8 . In addition to the better understanding of the involvement of bacteriophages in bacterial development, the identification and isolation of prophages allows, (i) the development of forms of bacterial control based on genetic modifications 9 , 10 , (ii) identification of genes of interest 11 and (iii) infection tests in related and distinct strains 12 – 14 .…”