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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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Construction of Ms6 lysA mutants.Two complementary oligonucleotides that modify lysA241 GTG start codon (valine) to TGG (tryptophan) and introduce an MscI restriction site, or two complementary oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, were co-transformed with Ms6-LysAHis6 genomic DNA; primary plaques were recovered and screened by PCR and MscI or HindIII digestion to identify a mixed plaque containing wild-type and mutant phages DNA. The mixed primary plaque was diluted and plated; the lysate was screened to check for phage viability, and purified secondary plaques were screened to identify pure mutant phages of Ms6-Lysin384His6 and Ms6-Lysin241His6, expressing only Lysin384 or Lysin241, respectively.
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pone-0020515-g003: Construction of Ms6 lysA mutants.Two complementary oligonucleotides that modify lysA241 GTG start codon (valine) to TGG (tryptophan) and introduce an MscI restriction site, or two complementary oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, were co-transformed with Ms6-LysAHis6 genomic DNA; primary plaques were recovered and screened by PCR and MscI or HindIII digestion to identify a mixed plaque containing wild-type and mutant phages DNA. The mixed primary plaque was diluted and plated; the lysate was screened to check for phage viability, and purified secondary plaques were screened to identify pure mutant phages of Ms6-Lysin384His6 and Ms6-Lysin241His6, expressing only Lysin384 or Lysin241, respectively.

Mentions: As a result of synthesis of Lysin241 and Lysin384 during phage infection, we next investigated the influence of each endolysin form in phage growth parameters. Once more, we took advantage on the fact that the BRED recombineering strategy has already been described to efficiently introduce base changes that confer an amino acid substitution [27]. To eliminate synthesis of Lysin384 or Lysin241 we designed oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, or two complementary oligonucleotides that modify the lysA241 GTG start codon to TGG (tryptophan), and introduce an MscI restriction site, respectively. Both mutant phages Ms6-Lysin384His6 (producing Lysin384) and Ms6-Lysin241His6 (producing Lysin241) were readily isolated, demonstrating that for plaque formation only one of the two LysA forms, Lysin384 or Lysin241 is required (Fig. 3). Nevertheless, we considered whether the absence of one of the two endolysin forms during M. smegmatis phage infection (Fig. 4) could confer an altered lysis phenotype. To address this question one step growth curves and determination of phage growth parameters (latent period, rise period and burst size) were carried out to compare the phages infection cycle. The one step growth experiment (Fig. 5A) shows that in a phage Ms6-Lysin241His6 infection, the latent period is prolonged 30 min in comparison with the Ms6wt infection and a decrease in the burst size was observed which means that a delay exists in the detection of phage release from cells infected with Ms6-Lysin241His6. In agreement, absence of Lysin384 leads to smaller size phage plaques (Fig. 5B), meaning that Lysin384 is important for infective particles release. On the other hand, Ms6-Lysin384His6 phage release starts 90 min later than with Ms6wt. This indicates that similarly to Lysin384, Lysin241 has an obvious function in completion of lysis, although it does not have a significant effect in the number of phage particles released. Examination of Ms6-Lysin384His6 phage plaques shows that although not larger in size, plaques are more turbid than wild-type Ms6 probably due to a partial host cell lysis (Fig. 5B). These results strongly suggest that even though only one of the two LysA forms, Lysin384 or Lysin241, is required to accomplish host cell lysis, both enzymes are necessary for complete and efficient lysis of M. smegmatis.


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)

Construction of Ms6 lysA mutants.Two complementary oligonucleotides that modify lysA241 GTG start codon (valine) to TGG (tryptophan) and introduce an MscI restriction site, or two complementary oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, were co-transformed with Ms6-LysAHis6 genomic DNA; primary plaques were recovered and screened by PCR and MscI or HindIII digestion to identify a mixed plaque containing wild-type and mutant phages DNA. The mixed primary plaque was diluted and plated; the lysate was screened to check for phage viability, and purified secondary plaques were screened to identify pure mutant phages of Ms6-Lysin384His6 and Ms6-Lysin241His6, expressing only Lysin384 or Lysin241, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111421&req=5

pone-0020515-g003: Construction of Ms6 lysA mutants.Two complementary oligonucleotides that modify lysA241 GTG start codon (valine) to TGG (tryptophan) and introduce an MscI restriction site, or two complementary oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, were co-transformed with Ms6-LysAHis6 genomic DNA; primary plaques were recovered and screened by PCR and MscI or HindIII digestion to identify a mixed plaque containing wild-type and mutant phages DNA. The mixed primary plaque was diluted and plated; the lysate was screened to check for phage viability, and purified secondary plaques were screened to identify pure mutant phages of Ms6-Lysin384His6 and Ms6-Lysin241His6, expressing only Lysin384 or Lysin241, respectively.
Mentions: As a result of synthesis of Lysin241 and Lysin384 during phage infection, we next investigated the influence of each endolysin form in phage growth parameters. Once more, we took advantage on the fact that the BRED recombineering strategy has already been described to efficiently introduce base changes that confer an amino acid substitution [27]. To eliminate synthesis of Lysin384 or Lysin241 we designed oligonucleotides that introduce a stop codon and a HindIII restriction site downstream of the start codon of lysA384, or two complementary oligonucleotides that modify the lysA241 GTG start codon to TGG (tryptophan), and introduce an MscI restriction site, respectively. Both mutant phages Ms6-Lysin384His6 (producing Lysin384) and Ms6-Lysin241His6 (producing Lysin241) were readily isolated, demonstrating that for plaque formation only one of the two LysA forms, Lysin384 or Lysin241 is required (Fig. 3). Nevertheless, we considered whether the absence of one of the two endolysin forms during M. smegmatis phage infection (Fig. 4) could confer an altered lysis phenotype. To address this question one step growth curves and determination of phage growth parameters (latent period, rise period and burst size) were carried out to compare the phages infection cycle. The one step growth experiment (Fig. 5A) shows that in a phage Ms6-Lysin241His6 infection, the latent period is prolonged 30 min in comparison with the Ms6wt infection and a decrease in the burst size was observed which means that a delay exists in the detection of phage release from cells infected with Ms6-Lysin241His6. In agreement, absence of Lysin384 leads to smaller size phage plaques (Fig. 5B), meaning that Lysin384 is important for infective particles release. On the other hand, Ms6-Lysin384His6 phage release starts 90 min later than with Ms6wt. This indicates that similarly to Lysin384, Lysin241 has an obvious function in completion of lysis, although it does not have a significant effect in the number of phage particles released. Examination of Ms6-Lysin384His6 phage plaques shows that although not larger in size, plaques are more turbid than wild-type Ms6 probably due to a partial host cell lysis (Fig. 5B). These results strongly suggest that even though only one of the two LysA forms, Lysin384 or Lysin241, is required to accomplish host cell lysis, both enzymes are necessary for complete and efficient lysis of M. smegmatis.

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