scholarly journals Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

2008 ◽  
Vol 74 (24) ◽  
pp. 7490-7496 ◽  
Author(s):  
Yu Pei Tan ◽  
Philip M. Giffard ◽  
Daniel G. Barry ◽  
Wilhelmina M. Huston ◽  
Mark S. Turner
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Transmembrane Protein ◽  
Random Mutagenesis ◽  
Lytic Enzyme ◽  
Control Strain ◽  
Lytic Enzymes ◽  
Positive Bacterium ◽  
Peptidoglycan Biosynthesis ◽  
Cell Wall Lytic Enzymes

ABSTRACT Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

A Computational Method for the Identification of Endolysins and Autolysins

2020 ◽  
Vol 27 (4) ◽  
pp. 329-336 ◽  
Author(s):  
Lei Xu ◽  
Guangmin Liang ◽  
Baowen Chen ◽  
Xu Tan ◽  
Huaikun Xiang ◽  
...  
Keyword(s):  
Support Vector Machine ◽  
Cell Wall ◽  
Experimental Results ◽  
Computational Method ◽  
Lytic Enzyme ◽  
Support Vector ◽  
Lytic Enzymes ◽  
Data Set ◽  
Optimal Feature ◽  
Better Than

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. Objective: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. Method: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. Results: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. Conclusion: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

Searching for the Evolutionary Design of the Pneumococcal Cell Wall Lytic Enzymes

1993 ◽  
pp. 253-259
Author(s):  
Rubens López ◽  
José L. García ◽  
Eduardo Díaz ◽  
Jesús M. Sanz ◽  
José M. Sánchez-Puelles ◽  
...  
Keyword(s):  
Cell Wall ◽  
Evolutionary Design ◽  
Lytic Enzymes ◽  
Cell Wall Lytic Enzymes

scholarly journals Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation

2020 ◽  
Vol 295 (27) ◽  
pp. 9171-9182 ◽  
Author(s):  
Danielle L. Sexton ◽  
Francesca A. Herlihey ◽  
Ashley S. Brott ◽  
David A. Crisante ◽  
Evan Shepherdson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Spore Germination ◽  
Lytic Enzymes ◽  
Lytic Transglycosylases ◽  
Signaling Function ◽  
Dormant Spore ◽  
The Subject ◽  
Resuscitation Promoting Factors ◽  
Insight Into ◽  
Cell Wall Lytic Enzymes

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

Lysis ofPediococcusandBrevibacteriumCells by Cell-Wall Lytic Enzymes, L3, L11, ALE and Lysozyme

1966 ◽  
Vol 30 (11) ◽  
pp. 1097-1101
Author(s):  
Kenji Sakaguchi ◽  
Shozo Kotani ◽  
Hidekazu Suginaka ◽  
Yoshiyuki Hirachi ◽  
Shuzo Kashiba ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lytic Enzymes ◽  
Cell Wall Lytic Enzymes

scholarly journals Lysis of Pediococcus and Brevibacterium Cells by Cell-Wall Lytic Enzymes, L3, L11, ALE and Lysozyme

1966 ◽  
Vol 30 (11) ◽  
pp. 1097-1101
Author(s):  
Kenji SAKAGUCHI ◽  
Shozo KOTANI ◽  
Hidekazu SUGINAKA ◽  
Yoshiyuki HIRACHI ◽  
Shuzo KASHIBA ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lytic Enzymes ◽  
Cell Wall Lytic Enzymes

Production of Yeast Cell-wall Lytic Enzymes on a Semi-defined Medium by a Streptomyces

Nature ◽  
1966 ◽  
Vol 210 (5034) ◽  
pp. 442-443 ◽  
Author(s):  
MARIA VICTORIA ELORZA ◽  
E. MUÑOZ RUIZ ◽  
JULIO R. VILLANUEVA
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Defined Medium ◽  
Yeast Cell Wall ◽  
Lytic Enzymes ◽  
Cell Wall Lytic Enzymes
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