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

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.

Figure 1. LysA expression in E. coli.

A. Purified LysA-His₆ fractions after SDS-PAGE analysis and Coomassie Blue staining. LysA-His₆ was produced from pMJC41 in E. coli BL21 (DE3) after isopropyl β-D-1-thiogalactopyranoside induction. B. Expression of LysA-His₆ from pMJC41: detection of C-terminal His₆tag LysA shows the production of Lysin₃₈₄-His₆ and Lysin₂₄₁-His₆. C. LysA-His₆ synthesis from pMJC41 over the time is not always followed by Lysin₂₄₁-His₆ production. D. Synthesis of Lysin₂₄₁-His₆ from pMJC43. Removal of pET29b and LysA (Lysin₃₈₄-His₆) translational signals does not hinder Lysin₂₄₁-His₆ synthesis. The molecular masses in kDa of Lysin₃₈₄ and Lysin₂₄₁ are indicated on the left; positions of both proteins are indicated by an arrow on the right. Lysin₃₈₄ and Lysin₂₄₁ were detected by Western blotting with an anti-His₆ antibody, except for panel A.

Figure 2. Time course of Lysin₃₈₄ and Lysin₂₄₁ synthesis during Ms6 infection of M. smegmatis.

Lysin production in M. smegmatis was analysed after infection with Ms6-LysAHis₆ or Ms6_Δgp1-LysAHis₆ at an MOI of 10. Extracts were prepared from samples taken at 30-min intervals as described in Material and Methods. Samples were analysed by Western blotting and Lysin₃₈₄ and Lysin₂₄₁ synthesis was detected with an anti-His₆ monoclonal antibody. A. Lysin₃₈₄ and Lysin₂₄₁ synthesis is first detected 90 minutes postinfection both in Ms6-LysAHis₆ (upper panel) and Ms6_Δgp1-LysAHis₆ (lower panel) mutant phages. Only the results for 90 to 300 min postinfection are shown. B. Lysin₃₈₄ is synthesized to near undetectable levels during Ms6_Δgp1-LysAHis₆ infection of M. smegmatis (lower panel) whereas Lysin₂₄₁ production is comparable to the wild-type phage. The molecular masses in kDa of Lysin₃₈₄ and Lysin₂₄₁ are indicated on the left; positions of both proteins are indicated by an arrow on the right.

Figure 3. Construction of Ms6 lysA mutants.

Two complementary oligonucleotides that modify lysA ₂₄₁ 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 lysA ₃₈₄, 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.

Figure 4. Time course of endolysin synthesis during Ms6-LysAHis₆, Ms6-Lysin₃₈₄His₆ or Ms6-Lysin₂₄₁His₆ infection of M. smegmatis.

Lysin production in M. smegmatis was analysed after infection at an MOI of 10. Extracts were prepared from samples taken at 30-min intervals as described in Material and Methods. Samples were analysed by Western blotting and Lysin₃₈₄ and Lysin₂₄₁ synthesis was detected as already described. Both Lysin₃₈₄ and Lysin₂₄₁ synthesis could be detected beginning 90 minutes postinfection in Ms6-LysAHis₆ (upper panel). In Ms6-Lysin₃₈₄His₆ and Ms6-Lysin₂₄₁His₆, (lower panels) only Lysin₃₈₄ or Lysin₂₄₁ synthesis could be detected also beginning 90 min postinfection, respectively. Only the results for 90 to 270 min postinfection are shown. The molecular masses in kDa of Lysin₃₈₄ and Lysin₂₄₁ are indicated on the left; positions of both proteins are indicated by an arrow on the right.

Figure 5. Both Lysin₃₈₄ and Lysin₂₄₁ are required for efficient host cell lysis.

A. One step growth curves of Ms6 and lysin-mutant derivatives. For each curve the titers measured were divided by the titer at t = 0 for normalization (titer/titer t₀). B. Halo formation by mycobacteriophage Ms6 and lysin-mutant derivatives. Serial dilutions of the bacteriophages stocks containing ∼10¹⁰ particles ml⁻¹ were prepared and 100 µl of the 10⁻⁸ dilution was plated with 200 µl of an exponential growing culture of M. smegmatis as top agar lawns. Incubation was continued for 3 days at 37°C. Mycobacteriophage D29 was used as a negative control of halo formation . Scale bar represents 1 cm.

Figure 6. 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.