1.E.36 The Mycobacterial 2 TMS Phage Holin (M2 Hol) Family The Mycobactrerial 2 TMS holins have been identified and recognized by Catalao et al (2012). The Mycobacterium phage D29 gp11 protein (1.E.36.1.7) is a holin that, upon expression, rapidly kills both E. coli and Mycobacterium smegmatis. Shortening gp11 from its C-terminus resulted in diminished cytotoxicity and smaller holes. The two TMSs at the N-terminus alone do not integrate into the cytoplasmic membrane and do not show toxicity. Fusion of the two TMSs and a small C-terminal coiled-coil region resulted in restoration of cell killing. The second TMS is dispensable for toxicity, but the gp11 C-terminal region is necessary but not sufficient for toxicity.
This family belongs to the Holin VII Superfamily .
References:
Catalao MJ., Gil F., Moniz-Pereira J., Sao-Jose C. and Pimentel M. (2013). Diversity in bacterial lysis systems: bacteriophages show the way. FEMS Microbiol Rev. 37(4):554-71.
Delisle, A.L., G.J. Barcak, and M. Guo. (2006). Isolation and expression of the lysis genes of Actinomyces naeslundii phage Av-1. Appl. Environ. Microbiol. 72: 1110-1117.
Gan, Y., P. Liu, T. Wu, and S. Guo. (2016). Different characteristics between mycobacteriophage Chy1 and D29, which were classified as cluster A2 mycobacteriophages. Indian J. Med. Microbiol. 34: 186-192.
Kamilla, S. and V. Jain. (2015). Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death. FEBS J. [Epub: Ahead of Print]
Lella, M. and R. Mahalakshmi. (2013). Pro-Gly mediated conformational switch of mycobacteriophage D29 holin transmembrane domain I is lipid concentration driven. Chem Commun (Camb) 49: 9594-9596.
Lella, M. and R. Mahalakshmi. (2016). Engineering a Transmembrane Nanopore Ion Channel from a Membrane Breaker Peptide. J Phys Chem Lett 7: 2298-2303.
Lella, M. and R. Mahalakshmi. (2016). Solvation Driven Conformational Transitions in the Second Transmembrane Domain of Mycobacteriophage Holin. Biopolymers. [Epub: Ahead of Print]
Leprince, A., M. Nuytten, E. July, C. Tesseur, and J. Mahillon. (2022). Getting Outside the Cell: Versatile Holin Strategies Used by Distinct Phages to Leave Their Bacillus thuringiensis Host. J. Virol. 96: e0069622.
Examples:
TC#	Name	Organismal Type	Example
1.E.36.1.1	Putative holin, Gp33 of 127 aas and 2 TMSs	Mycobacterial phage	Gp33 of Mycobacterial phage Cjw1

1.E.36.1.10	Uncharacterized protein of 145 aas and 4 TMSs.		UP of Demequina flava

1.E.36.1.2	*Putative holin Gp36 of 84 aas and 2 TMSs (Catalão et al.* 2012).**	Mycobacterial phages	Gp36 of mycobacterial phage PBI1

1.E.36.1.3	Putative holin Gp10 of 137 aas and 2 or 3 TMSs	Mycobacterial phage	Gp10 of mycobacterial phage Timshel

1.E.36.1.4	Putative holin, Gp29 of 134 aas and 2 TMSs	Mycobacterial phage	Gp29 of mycobacterial phage Charlie

1.E.36.1.5	Putative holin Gp14 of 144 aas and 2 TMSs	Mycobacterial phage	Gp14 of mycobacterial phage EricB

1.E.36.1.6	Putative holin of 146 aas and 2 TMSs	Actinobacteria	Putative holin of Mycobacterium massiliense

1.E.36.1.7	Mycobacterial phage D29 holin of 141 aas, Gp11. Gp11 shows a lipid concentration-dependent conformational switch from an α-helix to a β-sheet structure (Lella and Mahalakshmi 2013). Toxicity has been studied showing that TMS 1 (but not TMS 2) and a C-terminal coiled-coil region are essential for activity because the latter is necessary for holin aggregation, insertion into the membrane and bacterial cell death (Kamilla and Jain 2015). A role for TMS 2 in pore formation has been proposed (Lella and Mahalakshmi 2016). The first TMS has been engineered to form a nanopore (Lella and Mahalakshmi 2016). The D29 and Chy1 phage holins are identical in amino acid sequence (Gan et al. 2016).	Virus	Holin, gp11 of Mycobacterial phage D29.

1.E.36.1.8	Putative holin of 138 aas and 2 TMSs	Actinobacteria	Putative holin of Mycobacterium abscessus

1.E.36.1.9	Holin of 136 aas and 2 TMSs, gp255.		gp255 of Mycobacterium phage Pleione

Examples:
TC#	Name	Organismal Type	Example
1.E.36.2.1	Putative holin of 69 aas and 2 TMSs	Actinobacteria	Putative holin of Actinomyces neuii

1.E.36.2.2	Putative holin of 67 aas and 2 TMSs, Gp31 of mycobacterial phage Brujita	Mycobacterial phage	Gp31 of mycobacterial phage Brujita

1.E.36.2.3	Putative holin of 67 aas and 2 TMSs	Mycobacterial phage	Gp27 of mycobacterial phage Che9c

1.E.36.2.4	Putative holin of 68 aas and 2 TMSs	Actinobacteria	Putative holin of Mobiluncus mulieris

Examples:
TC#	Name	Organismal Type	Example
1.E.36.3.1	*Holin of 64 aas and 2 TMSs, HolB (Delisle et al.* 2006; Leprince et al. 2022).**	Actinobacterial phage	HolB of phage Ar-1 or Vp4 (deep blue)

1.E.36.3.2	TMEM125 family member of 65 aas and 2 TMSs.		TMEM125 protein of Podoviridae sp.

Examples:
TC#	Name	Organismal Type	Example
1.E.36.4.1	Putative holin of 178 aas and 2 TMSs, Gp48	Mycobacterial phage	Gp48 and mycobacterial phage Ares

1.E.36.4.2	Holin of 173 aas and 2 TMSs.		*Holin of Mycobacterium* phage Jolie1**

Examples:
TC#	Name	Organismal Type	Example
1.E.36.5.1	*Putative holin of 106 aas and 2 TMSs, Gp54 (Catalão et al.* 2012).**	Mycobacterial phage	Gp54 of Mycobacterial phage Omega

1.E.36.5.2	Putative holin of 98 aas and 2 TMSs, Gp71	Mycobacterial phage	Gp71 of mycobacterial phage Corndog

Examples:
TC#	Name	Organismal Type	Example
1.E.36.6.1	Putative holin, Gp17 of 144 aas and 4 TMSs	Actinobacterial phage	Gp17 of Mycobacterial phage Daisy

1.E.36.6.2	Putative holin of 113 aas and 4 TMSs	Actinobacteria	Putative holin of Nocardia farcinica

1.E.36.6.3	Putative holin, Gp16 of 135 aas and 4 TMSs	Actinobacterial phage	Gp16 of mycobacterial phage Acadian

1.E.36.6.4	Putative holin, Gp32 of 150 aas and 4 TMSs	Actinobacterial phage	Gp32 of Mycobacterial phage Larva

1.E.36.6.5	Putative holin, Gp31 of 128 aas and ~3 TMSs	Actinobacterial phage	Gp31 of Mycobacterial phage TM4

Examples:
TC#	Name	Organismal Type	Example