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. 2022 Jan-Mar;14(1):46-53.
doi: 10.18502/ajmb.v14i1.8169.

Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function

Morteza Miri  1 , Sepideh Yazdianpour  1 , Shamsozoha Abolmaali  2 , Shakiba Darvish Alipour Astaneh  1
Affiliations

Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function

Morteza Miri et al. Avicenna J Med Biotechnol. 2022 Jan-Mar.

Abstract

Background: To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a Bacillus subtilis-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli).

Methods: The sequences of MurNAc-LAA genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in E. coli. Furthermore, the structure and function of LysM2 was evaluated in silico. The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied.

Results: LysM2, on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli. In silico analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, and Proteus vulgaris in agar diffusion assays.

Conclusion: Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.

Keywords: Antibacterial activity; Bacteriophage SPP1; Endolysin; Siphoviridae.

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Conflict of interest statement

Conflict of Interest The authors declare that they have no competing interest.

Figures

Figure 1.
Figure 1.
A) The phylogenetic tree for the optimized LysM2, based on the Neighbor-Joining method. B) In silico investigation of the catalytic domain of LysM2
Figure 2.
Figure 2.
A, B) Secondary structure prediction for LysM2 protein. C) Frequency of positively charged amino acids is higher in the N-terminal protein. D) The three-dimensional structure of LysM2 protein. Purple, red, and blue colors indicate extended strand, coil, and helix, respectively in both figures, A and D.
Figure 3.
Figure 3.
A) Evaluation of LysM2 protein stability based on Ramachandran plot showed 2.2% of the residues were in the outlier region; 5.6% in the allowed area, and 92.1% of amino acids in the favored region. B) The results of analyzing LysM2 protein with the ProSA web server; Z-score plot, NMR spectroscopy (dark blue), and X-ray crystallography (light blue); the plot showed local model quality using plotting energies as a function of amino acids position in the sequence.
Figure 4.
Figure 4.
The photogram of LysM2 protein on 12% SDS-PAGE. The relevant 33 kDa band is indicated in the Lanes 1, 2: LysM2 protein, Lane 3: Control, Lanes 4, 5: Purified protein, Lane 6: Ladder protein 10-250 kDa, Lane 7: Purified control without IPTG induction, Lane 8: E. coli BL21DE3, Lane 9: Pre induction of pET28a without the insert.
Figure 5.
Figure 5.
A) Agar diffusion of LysM2 against Proteus (sp). B) The lytic activity of LysM2 against Proteus (sp).
See this image and copyright information in PMC

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