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Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.

ABSTRACT: Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0-6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-l-Ala-d-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities.

SUBMITTER: Bustamante N

PROVIDER: S-EPMC3466307 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.

Bustamante Noemí N Rico-Lastres Palma P García Ernesto E García Pedro P Menéndez Margarita M

PloS one 20121008 10

Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live- ...[more]

PMID: 23056389

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Project description:Cp-1, a bacteriophage infecting Streptococcus pneumoniae, has a linear double-stranded DNA genome, with a terminal protein covalently linked to its 5' ends, that replicates by the protein-priming mechanism. We describe here the complete DNA sequence and transcriptional map of the Cp-1 genome. These analyses have led to the firm assignment of 10 genes and the localization of 19 additional open reading frames in the 19,345-bp Cp-1 DNA. Striking similarities and differences between some of these proteins and those of the Bacillus subtilis phage phi 29, a system that also replicates its DNA by the protein-priming mechanism, have been revealed. The genes coding for structural proteins and assembly factors are located in the central part of the Cp-1 genome. Several proteins corresponding to the predicted gene products were identified by in vitro and in vivo expression of the cloned genes. Mature major head protein from the virion particles results from hydrolysis of the primary gene product at the His-49 residue, whereas the phage gene is expressed in Escherichia coli without modification. We have also identified two open reading frames coding for proteins that show high degrees of similarity to the N- and C-terminal regions, respectively, of the single tail protein identified in phi 29. Sequencing and primer extension analysis suggest transcription of a small RNA showing a secondary structure similar to that of the prohead RNA required for the ATP-dependent packaging of phi 29 DNA. On the basis of its temporal expression, transcription of the Cp-1 genome takes place in two stages, early and late. Combined Northern (RNA) blot and primer extension experiments allowed us to map the 5' initiation sites of the transcripts, and we found that only three genes were transcribed from right to left. These analyses reveal that there are also noticeable differences between Cp-l and phi 29 in transcriptional organization. Considered together, the observations reported here provide new tangible evidence on phylogenetic relationships between B. subtilis and S. pneumoniae.

Dataset Information

Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.

Publications

Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.

Similar Datasets

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