Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans

Delphine Dufour; C�line L�vesque

doi:10.21769/bioprotoc.855

False positives in using the zymogram assay for identification of peptidoglycan hydrolases

Analytical Biochemistry ◽

10.1016/j.ab.2017.12.016 ◽

2018 ◽

Vol 543 ◽

pp. 162-166 ◽

Cited By ~ 5

Author(s):

Cristian A. Escobar ◽

Timothy A. Cross

Keyword(s):

False Positives ◽

Peptidoglycan Hydrolases ◽

Zymogram Assay

Download Full-text

Identification of a Broad-Spectrum Peptidoglycan Hydrolase Associated with the Particle of Xanthomonas oryzae Phage Xop411

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000488678 ◽

2018 ◽

Vol 28 (2) ◽

pp. 78-86

Author(s):

Shu-Fen Weng ◽

Yung-Chieh Fu ◽

Juey-Wen Lin ◽

Tsai-Tien Tseng

Keyword(s):

Western Blotting ◽

Inclusion Bodies ◽

Protein S ◽

Bioinformatic Analysis ◽

Immunogold Labelling ◽

Xanthomonas Oryzae ◽

Opportunistic Bacteria ◽

Peptidoglycan Hydrolases ◽

Related Plant ◽

Zymogram Assay

Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in Escherichia coli formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in Pichia pastoris was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of Xanthomonas, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic Pseudomonas aeruginosa and Stenotrophomonas maltophilia causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from Xanthomonas phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.

Download Full-text

Cell Wall Ultrastructure in Three Strains of a Cariogenic Streptococcus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065730 ◽

1971 ◽

Vol 29 ◽

pp. 338-339

Author(s):

M. J. Kramer ◽

Alan L. Coykendall

Keyword(s):

Cell Wall ◽

Dental Caries ◽

Streptococcus Mutans ◽

Microscopic Study ◽

Electron Microscopic Study ◽

Genetic Heterogeneity ◽

Morphological Characteristics ◽

Electron Microscopic ◽

Dna Reassociation ◽

Wall Ultrastructure

During the almost 50 years since Streptococcus mutans was first suggested as a factor in the etiology of dental caries, a multitude of studies have confirmed the cariogenic potential of this organism. Streptococci have been isolated from human and animal caries on numerous occasions and, with few exceptions, they are not typable by the Lancefield technique but are relatively homogeneous in their biochemical reactions. An analysis of the guanine-cytosine (G-C) composition of the DNA from strains K-1-R, NCTC 10449, and FA-1 by one of us (ALC) revealed significant differences and DNA-DNA reassociation experiments indicated that genetic heterogeneity existed among the three strains. The present electron microscopic study had as its objective the elucidation of any distinguishing morphological characteristics which might further characterize the respective strains.

Download Full-text

The role of intracanal medicaments in inhibition of bacteria isolated from root canals of infected primary molars

Mustansiria Dental Journal ◽

10.32828/mdj.v14i1.766 ◽

2019 ◽

Vol 14 (1) ◽

pp. 92

Author(s):

Dr. Maha Abdul- Kareem Mahmood ◽

Dr. Huda Elias Ali ◽

Dr. Haraa Khairi Abdul-Kadher

Keyword(s):

Antimicrobial Activity ◽

Streptococcus Mutans ◽

Normal Saline ◽

Root Canal ◽

Primary Teeth ◽

Primary Molars ◽

Root Canals ◽

Etiologic Agents ◽

Micro Organisms

Microbes are considered as the primary etiologic agents in endodontic diseases.Disinfection of the root canal is obtained by the combined effect of biomechanicalpreparation, irrigation and intra canal medicament. The aim of the present study wasto assess the antimicrobial activity of intracanal medicaments (formocresol andEndosepton) against two micro organisms (Streptococcus mutans and staphylococcusaureus) isolated from 15 necrotic pulps of primary molars indicated for pulpectomyprocedure. The samples were cultured, and purified using microbiological evaluation.Broth dilution test was performed in our study by preparing test tubes containing10 ml of BHI broth (pH. 7) which then inoculated with strains of the tested bacteriaand incubated at 37 C° for 24 h. After over night incubaction, ten fold dilution weremade in test tubes containing 9 ml of normal saline by adding 1 ml of the inoculum tothe first tube . Then from dilution 10-1 , 0.1 ml of cell suspension was added to 9.9 mlof formocresol and endosepton, then 0.1 ml was taken and spread on duplicates ofBHI agar plates at different intervals and incubated aerobically for 24 h. at 37 C°.Colonies on the plates were counted after incubation and CFU/mL (colony formingunit) was calculated. Our results indicating that there were no significant differencesbetween the intracanal medicaments, but there were high significant differencesbetween the intervals time of the study. We concluded that both materials had greatantibacterial effect against the pathogens commonly isolated from necrotic pulpaltissue of primary teeth.

Download Full-text