Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells

Abstract

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.

FIG. 1

Lysis genes of Cp-1. (A) Localizations of ORF21 and ORF22, which encode holin and lysozyme, respectively. PL8 and PL9, represented by carets, are the late tandem promoters preceding the holin gene and are named according to the nomenclature given to the Cp-1 genome (14). (B) Amino acid sequence, translational start region, and predicted secondary structure of the Cph1 holin. RBS, ribosome-binding site. Filled and open bars represent putative transmembrane domains and β-turns, respectively. Positive (+) and negative (−) charges of amino acids are indicated above the sequence. Asterisks indicate the charged C-terminal domain. (C) Modular organization of Cpl1 lysozyme. The hatched region represents the N-terminal domain, and the six dotted boxes, P1 to P6, indicate choline-binding domains.

FIG. 2

Schematic representation of the construction of pAMR11 and pAMR12. Plasmids are drawn with the relevant elements and restriction sites indicated. Thin and thick lines represent vector-derived sequences and Cp-1-derived sequences, respectively. Arrows represent the direction of transcription of the genes. Pm indicates an inducible promoter of the TOL plasmid, and xylS encodes the cognate regulator of Pm. Oligonucleotides used for isolation of the genes by PCR are marked as bent arrows. Sm and Km indicate the genes conferring streptomycin and kanamycin resistance, respectively, and RBS indicates the ribosome-binding sites.

FIG. 3

Effects of expression of the cph1 and cpl1 genes on the growth of the E. coli. (A) Cultures of HB101(pNM185) (○ and •), HB101(pAMR11) (□ and ▪), and HB101(pAMR12) (▵ and ▴) that were uninduced (open symbols) or induced at time zero with 2 mM 3-MB (filled symbols). Cultures were incubated at 30°C. OD₆₀₀, optical density at 600 nm. (B) Viabilities of the same cultures obtained after plating two appropriate dilutions on LB medium.

FIG. 4

Effects of cph1 and lytA genes on the growth of E. coli. Shown are optical densities at 600 nm (OD₆₀₀) of cultures of HB101(pAMR11, pGL80) that were uninduced (○) or induced at time zero with 2 mM 3-MB (•). Cultures were incubated at 30°C.

FIG. 5

Schematic representation of the construction of pAMR21 and pAMR22. Plasmids are drawn with the relevant elements and restriction sites indicated. Thin lines represent vector-derived sequences, and thick lines represent Cp-1-derived sequences. Arrows indicate the direction of transcription of the genes. P represents the promoter of the tetracycline resistance gene. Oligonucleotides used for isolation of the genes by PCR are indicated as bent arrows. Tc and Ery indicate the genes conferring tetracycline and erythromycin resistance, respectively, and RBS indicates the ribosome-binding sites.

FIG. 6

In vitro transcription-translation of plasmids pLSE1, pAMR21, and pAMR22. Samples containing pAMR22 (lane 1), pLSE1 (lane 2), and pAMR21 (lane 3) were loaded onto a sodium dodecyl sulfate–15% polyacrylamide gel. Arrows indicate the positions of the holin (Hol) and the lysozyme (Lys). Standard size markers, in kilodaltons, are shown on the left.

FIG. 7

Effects of cph1 and cpl1 genes on the growth of S. pneumoniae. (A) Cultures of M31(pLSE1) (•), M31(pAMR21) (▪), and M31(pAMR22) (▴) were incubated at 37°C. Pneumolysin release to the periplasmic fraction was measured, as explained in Materials and Methods, for M31(pLSE1) (○) and M31(pAMR21) (□) cultures. N, nephelometric units; OD₅₄₁, optical density at 541 nm. (B) Viabilities of the same cultures obtained after plating two appropriate dilutions on C medium.