Mutational and biochemical analyses of the endolysin LysgaY encoded by the Lactobacillus gasseri JCM 1131T phage φgaY

Sugahara, Kazuki N.; Yokoi, Kohei; Nakamura, Yuuko; Nishino, Taito; Yamakawa, Ayanori; Taketo, Akira; Kodaira, Ken-Ichi

doi:10.1016/j.gene.2007.08.023

Cited by 14 publications

(15 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Considering that Zn 2+ obviously assumes a crucial role in catalytic function of these enzymes, it was surprising to find that Ca 2+ and Mn 2+ were more effective in restoring lytic activity after . Similar observations regarding the effects of divalent metals on activity of cell wall hydrolases were reported earlier (Vasala et al 1995;Shida et al 2001;Pritchard et al 2004;Donovan et al 2006;Sugahara et al 2007;Mikoulinskaia et al 2009;Garcia et al 2010). For aminopeptidase A, a zinc metalloendopeptidase that can be reactivated by Ca 2+ or Mn 2+ after exposure to chelating agents (Danielsen et al 1980), it was recently shown that the Ca 2+ ion increases substrate affinity by binding to two aspartate residues of the enzyme and interacting with an acidic side chain of the substrate (Claperon et al 2008).…”

Section: Discussionsupporting

confidence: 75%

“…In general, pH optima above 8.0 seem uncommon for cell wall hydrolases. In fact, most other phage and bacterial lysins studied require neutral or slightly acidic conditions, with activity rapidly decreasing above pH 7.5 or 8 (Vasala et al 1995;Morita et al 2001;Loeffler et al 2003;Pritchard et al 2004;Yoong et al 2004;Cheng and Fischetti 2006;Donovan et al 2006;Fukushima et al 2007;Sugahara et al 2007). However, another L-alanoyl-D-glutamate peptidase from E. coli phage T5 revealed a pH profile similar to the enzymes reported here, with highest activity at pH 8.5 (Mikoulinskaia et al 2009).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution

Schmelcher

Waldherr

Loessner

2011

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118, HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH values at around pH 8-9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent metal cations, indicating their role in catalytic function. While substitution of the native Zn 2+ by Ca 2+ or Mn 2+ was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co 2+ and Mn 2+ resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent metals for activity.The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35% activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures is reversible and may be followed by rapid refolding into a functional state.

show abstract

Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution

Schmelcher

Waldherr

Loessner

2011

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Ctp1l has the conserved Glu and Asn residues proposed to be involved in catalytic activity (29,30) within the proposed N-terminal GH25 domain. Examination of other endolysins of the GH25 type has shown a modular structure with a catalytic domain at the N terminus linked to a C-terminal cell wall binding domain (29,30,34,38). Sheehan et al (36) demonstrated catalytic activity of the Nterminal domain of a GH25 endolysin from the lactococcal bacteriophage Tuc2009 fused to the C-terminal domain from a pneumococcal autolysin, with the resulting hybrid having a novel activity on pneumococcal cell walls.…”

Section: Discussionmentioning

confidence: 99%

“…A number of publications have demonstrated the ability of selected endolysins to retain lytic activity upon truncation (8,23,28). However, this has not been demonstrated for GH25-type endolysins, with removal of the C-terminal domain either reducing (34,38) or removing (30) activity. We also found that truncation of Ctp1l to the proposed catalytic domain alone (amino acids 1 to 197) abolished lytic activity, indicating that the C-terminal region is required for full activity, either by virtue of the presence of as-yet-unidentified cell wall binding motifs or to create the required protein structure.…”

Section: Discussionmentioning

confidence: 99%

Genomic Sequence and Characterization of the Virulent Bacteriophage φCTP1 from Clostridium tyrobutyricum and Heterologous Expression of Its Endolysin

Mayer

Payne

Gasson

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

The growth of Clostridium tyrobutyricum in developing cheese leads to spoilage and cheese blowing. Bacteriophages or their specific lytic enzymes may provide a biological control method for eliminating such undesirable organisms without affecting other microflora. We isolated the virulent bacteriophage CTP1 belonging to the Siphoviridae and have shown that it is effective in causing lysis of sensitive strains. The double-stranded DNA genome of CTP1 is 59,199 bp, and sequence analysis indicated that it has 86 open reading frames. orf29 was identified as the gene coding for the phage endolysin responsible for cell wall degradation prior to virion release. We cloned and expressed the ctp1l gene in E. coli and demonstrated that the partially purified protein induced lysis of C. tyrobutyricum cells and reduced viable counts both in buffer and in milk. The endolysin was inactive against a range of clostridial species but did show lysis of Clostridium sporogenes, another potential spoilage organism. Removal of the C-terminal portion of the endolysin completely abolished lytic activity.Clostridium tyrobutyricum has been identified as a major species associated with late blowing of hard and semihard cheeses (12,15). Outgrowth of C. tyrobutyricum during cheese manufacture affects both flavor, by the production of butyric acid, and cheese structure, as gas formation produces cracks and cavities. C. tyrobutyricum has also been isolated from spoiled processed cheese (24) and spoiled fruit pulp (5). The bacteria are also present in nonfood environments, such as soil, forage, silage, hay, and milk (14, 39). The heat-resistant spores survive the treatments involved in pasteurization, processing, and canning, and even low numbers of spores can cause food spoilage (37). Outgrowth of the clostridial spores in cheese can be controlled by the use of nitrate or hen egg white lysozyme in countries where these additions are permitted (13,17,40). Other potential methods of reducing gas formation in cheese include reducing spore numbers and/or germination using centrifugation, increased salt concentration, or low-temperature ripening (37). The use of bacteriocins has also shown promise (31), and polyphosphates have been shown to effect control in processed cheese spreads (22). The use of bacteriophages to control the growth of food spoilage bacteria has been well documented (10, 28). However, the use of phages for biocontrol has limitations due to narrow host specificities and the potential development of phage resistance. Endolysins are expressed by bacteriophages to effect the release of new virus particles from an infected cell by attacking the peptidoglycan wall (7, 20). They commonly contain both a catalytic activity, engendered by one or two of a number of peptidoglycan hydrolases, and a cell wall binding domain. The latter endows the enzyme with high specificity, usually for a single species or a group of closely related species. For Gram-positive bacteria, endolysins can also be effective when applied exogenously, establishing th...

show abstract

“…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.…”

Section: Additional Lysinsmentioning

confidence: 99%

Recombinant bacteriophage lysins as antibacterials

et al. 2010

View full text Add to dashboard Cite

Mutational and biochemical analyses of the endolysin LysgaY encoded by the Lactobacillus gasseri JCM 1131T phage φgaY

Cited by 14 publications

References 30 publications

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution

Genomic Sequence and Characterization of the Virulent Bacteriophage φCTP1 from Clostridium tyrobutyricum and Heterologous Expression of Its Endolysin

Recombinant bacteriophage lysins as antibacterials

Contact Info

Product

Resources

About