Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

Abstract

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these speciesspecific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.Phages of oral bacteria have been studied in this laboratory for a number of years since we believe they are likely to play a significant ecological role in regulating the oral microflora. We are also interested in phage-encoded lytic enzymes as potential diagnostic tools (10,19,23) and as therapeutic agents (1,6,7,13) to control specific oral pathogens. Actinomyces naeslundii is a gram-positive, facultative anaerobe that is found in high numbers in dental plaque and is believed to be causally involved in gingivitis and root surface caries in the oral cavity, in addition to actinomycosis in other regions of the body (22). The cell wall of this organism is resistant to salivary lysozyme and most other peptidoglycan-hydrolyzing enzymes, even though it is chemically similar to many other gram-positive cell walls that are lysozyme sensitive (18). Phage Av-1, originally isolated in this laboratory (3), is a small group I phage that is lytic for several human strains of A. naeslundii (2). This implies that its genome encodes an endolysin that can degrade the cell wall of this species. Preliminary experiments in this laboratory demonstrated that phage lysates of Av-1 contained a lytic enzyme that is specific for A. naeslundii. To clone the Av-1 lysin gene in Escherichia coli, which is also resistant to the enzyme, we devised an indirect strategy in which the functional activity of a plasmid-encoded endolysin is restored via complementation of a defective holin. Since holins are not species specific, we constructed a cloning vector to isolate plasmids containing phage inserts that express lethal, holin-like activity in E. coli. By sequencing these inserts, identifying potential open reading frames (ORFs), and analyzing their deduced amino acid sequences, putative holins could be tentatively i...

Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

“…It is known that holins must oligomerize to achieve their lethal membrane effect (9). To identify the oligomeric states of Ms6 Gp4 and Gp5, the membrane fractions from E. coli expressing Gp4 or Gp5 (fused to an S tag at the N terminus and a His 6 tag at the C terminus) from a derivative plasmid of pET29a were collected 60 min after induction; proteins were extracted from the membranes with Triton X-100 and subjected to crosslinking with the water-soluble membrane-impermeant, homobifunctional sulfo-N-hydroxy-succinimide ester, BS 3 . The presence of a band of 24.2 kDa in the absence of the cross-linker shows that Gp4 forms SDS-resistant dimers during membrane extraction with Triton X-100 (Fig.…”

The lysis cassette of bacteriophage фKMV encodes a signal-arrest-release endolysin and a pinholin

“…A holin-selective vector (pAD330, 22 derived from pS105 23 ) was used to analyze the holin function of KMV44. This vector provides all components of the lysis cassette of phage  (endolysin, Rz and Rz1), except the necessary holin.…”

Recombinant bacteriophage lysins as antibacterials

“…Along with the lysins targeting the Gram-positive pathogens discussed above, a number of similar enzymes [99][100][101][102][103][104] which also have potential to eliminate Gram-positive pathogens including Streptococcus, Staphylococcus, Actinomyces, Micrococcus and Enterococcus are under investigation in different laboratories. These are also included in Table 1.…”