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A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6.

Catalão MJ, Milho C, Gil F, Moniz-Pereira J, Pimentel M - PLoS ONE (2011)

Bottom Line: Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts.Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site.In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis.

View Article: PubMed Central - PubMed

Affiliation: Centro de Patogénese Molecular, Unidade dos Retrovírus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.

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Peptidoglycan hydrolysis by E. coli-produced Lysin384 and Lysin241 in M. luteus cells.A. Lytic activity of lysin extracts was assessed by in situ renaturation after SDS-PAGE using a gel matrix containing M. luteus cells as substrate (upper panel). Peptidoglycan hydrolysis by renatured proteins within the gel produces clear zones that no longer stain with methylene blue. Lysozyme and bovine serum albumin (BSA) represent positive and negative controls, respectively. A cell-free control gel was run in parallel and stained with Coomassie blue (lower panel). The molecular masses in kDa of BSA, lysozyme, Lysin384 and Lysin241 are indicated on the left; positions of proteins are indicated by an arrow on the right. B. Effect of Lysin384 or Lysin241 activity on lawns of B. subtilis (upper panel) and M. smegmatis (lower panel). 20 µl of E. coli:pMJC41 or E. coli:pMJC42 extracts containing Lysin384 or Lysin241 were spotted onto the bacterial lawn of the test strain and incubated overnight at 37°C. After overnight incubation, the presence of a clear zone was examined. E. coli:pET29b induced extract was used as a negative control.
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pone-0020515-g006: Peptidoglycan hydrolysis by E. coli-produced Lysin384 and Lysin241 in M. luteus cells.A. Lytic activity of lysin extracts was assessed by in situ renaturation after SDS-PAGE using a gel matrix containing M. luteus cells as substrate (upper panel). Peptidoglycan hydrolysis by renatured proteins within the gel produces clear zones that no longer stain with methylene blue. Lysozyme and bovine serum albumin (BSA) represent positive and negative controls, respectively. A cell-free control gel was run in parallel and stained with Coomassie blue (lower panel). The molecular masses in kDa of BSA, lysozyme, Lysin384 and Lysin241 are indicated on the left; positions of proteins are indicated by an arrow on the right. B. Effect of Lysin384 or Lysin241 activity on lawns of B. subtilis (upper panel) and M. smegmatis (lower panel). 20 µl of E. coli:pMJC41 or E. coli:pMJC42 extracts containing Lysin384 or Lysin241 were spotted onto the bacterial lawn of the test strain and incubated overnight at 37°C. After overnight incubation, the presence of a clear zone was examined. E. coli:pET29b induced extract was used as a negative control.

Mentions: LysA has been previously described as not affecting E. coli growth rate unless permeabilization of the plasma membrane by chloroform addition which results in immediate lysis [18]. To follow growth and viability of E. coli strains expressing Lysin241, the lysA DNA fragment corresponding to lysA241 was cloned into pQE30 vector allowing expression of Lysin241 under the control of the regulated T5 bacteriophage promoter. Although induction of Lysin241 did not result in E. coli lysis unless chloroform was added, growth seems to be halted over the induction period (data not shown). Ms6 LysA holds a PGRP domain and its hydrolase activity was already demonstrated; the protein was shown to cleave the bond between L-Ala and D-muramic acid and to release up to 70% of the diaminopimelic acid present in isolated mycobacterial cell walls which confirmed the amidase activity of the enzyme (unpublished results). To more directly assess the enzymatic activity of Lysin384 and Lysin241 we tested their ability to generate a zone of clearing in a zymogram assay. Lytic activity in lysin-producing E. coli extracts was checked by in situ protein renaturation after SDS-PAGE, using gel-incorporated autoclaved M. luteus cells as the substrate. As shown in Fig. 6A both Lysin384 and Lysin241 have hydrolase activity. Peptidoglycan hydrolysitic activity in zymograms was already demonstrated for other three mycobacteriophage LysA proteins (Che8 Gp32, Bxz1 Gp236 and Corndog Gp69) [22] and TM4 Gp29 [38].


A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6.

Catalão MJ, Milho C, Gil F, Moniz-Pereira J, Pimentel M - PLoS ONE (2011)

Peptidoglycan hydrolysis by E. coli-produced Lysin384 and Lysin241 in M. luteus cells.A. Lytic activity of lysin extracts was assessed by in situ renaturation after SDS-PAGE using a gel matrix containing M. luteus cells as substrate (upper panel). Peptidoglycan hydrolysis by renatured proteins within the gel produces clear zones that no longer stain with methylene blue. Lysozyme and bovine serum albumin (BSA) represent positive and negative controls, respectively. A cell-free control gel was run in parallel and stained with Coomassie blue (lower panel). The molecular masses in kDa of BSA, lysozyme, Lysin384 and Lysin241 are indicated on the left; positions of proteins are indicated by an arrow on the right. B. Effect of Lysin384 or Lysin241 activity on lawns of B. subtilis (upper panel) and M. smegmatis (lower panel). 20 µl of E. coli:pMJC41 or E. coli:pMJC42 extracts containing Lysin384 or Lysin241 were spotted onto the bacterial lawn of the test strain and incubated overnight at 37°C. After overnight incubation, the presence of a clear zone was examined. E. coli:pET29b induced extract was used as a negative control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111421&req=5

pone-0020515-g006: Peptidoglycan hydrolysis by E. coli-produced Lysin384 and Lysin241 in M. luteus cells.A. Lytic activity of lysin extracts was assessed by in situ renaturation after SDS-PAGE using a gel matrix containing M. luteus cells as substrate (upper panel). Peptidoglycan hydrolysis by renatured proteins within the gel produces clear zones that no longer stain with methylene blue. Lysozyme and bovine serum albumin (BSA) represent positive and negative controls, respectively. A cell-free control gel was run in parallel and stained with Coomassie blue (lower panel). The molecular masses in kDa of BSA, lysozyme, Lysin384 and Lysin241 are indicated on the left; positions of proteins are indicated by an arrow on the right. B. Effect of Lysin384 or Lysin241 activity on lawns of B. subtilis (upper panel) and M. smegmatis (lower panel). 20 µl of E. coli:pMJC41 or E. coli:pMJC42 extracts containing Lysin384 or Lysin241 were spotted onto the bacterial lawn of the test strain and incubated overnight at 37°C. After overnight incubation, the presence of a clear zone was examined. E. coli:pET29b induced extract was used as a negative control.
Mentions: LysA has been previously described as not affecting E. coli growth rate unless permeabilization of the plasma membrane by chloroform addition which results in immediate lysis [18]. To follow growth and viability of E. coli strains expressing Lysin241, the lysA DNA fragment corresponding to lysA241 was cloned into pQE30 vector allowing expression of Lysin241 under the control of the regulated T5 bacteriophage promoter. Although induction of Lysin241 did not result in E. coli lysis unless chloroform was added, growth seems to be halted over the induction period (data not shown). Ms6 LysA holds a PGRP domain and its hydrolase activity was already demonstrated; the protein was shown to cleave the bond between L-Ala and D-muramic acid and to release up to 70% of the diaminopimelic acid present in isolated mycobacterial cell walls which confirmed the amidase activity of the enzyme (unpublished results). To more directly assess the enzymatic activity of Lysin384 and Lysin241 we tested their ability to generate a zone of clearing in a zymogram assay. Lytic activity in lysin-producing E. coli extracts was checked by in situ protein renaturation after SDS-PAGE, using gel-incorporated autoclaved M. luteus cells as the substrate. As shown in Fig. 6A both Lysin384 and Lysin241 have hydrolase activity. Peptidoglycan hydrolysitic activity in zymograms was already demonstrated for other three mycobacteriophage LysA proteins (Che8 Gp32, Bxz1 Gp236 and Corndog Gp69) [22] and TM4 Gp29 [38].

Bottom Line: Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts.Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site.In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis.

View Article: PubMed Central - PubMed

Affiliation: Centro de Patogénese Molecular, Unidade dos Retrovírus e Infecções Associadas, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.

Show MeSH
Related in: MedlinePlus