*1.E.18 The Lactococcus lactis* Phage r1t Holin (r1t Holin) Family** The L. lactis phage r1t genome includes two adjacent genes, Orf48 and Orf49, which encode a holin and a lysin, probably an N-acetyl-muramoyl-L-alanine amidase, respectively. Orf48 exhibits 2 putative hydrophobic TMSs separated by a short β-turn region. It also has a hydrophobic N-terminus and a highly charged C-terminus. Thus, Orf48/Orf49 constitute the phage r1t lysis cassette. An essential role of Orf49 in cell lysis by Orf48 has been demonstrated (van Sinderen et al., 1996). Orf48 is homologous to the gp4 holin of Mycobacterium phage Ms6 (Garcia et al., 2002). Like most double-stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin-endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. The lysis function of Gp1, encoded by the gp1 gene that lies immediately upstream of lysA, was revealed (Catalão et al. 2010). Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of the Ms6 holin. Gp1 does not belong to the holin class of proteins, but it shares several characteristics with molecular chaperones. Gp1 interacts with LysA, and this interaction is necessary for LysA delivery to its target. PhoA fusions showed that in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1 which is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. Catalao et al. proposed that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone-like protein, in a holin-independent manner. The transport reaction catalyzed by phage r1t Orf48 is: lysin (in) → lysin (out)
References:
Catalão, M.J., F. Gil, J. Moniz-Pereira, and M. Pimentel. (2010). The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan. Mol. Microbiol. 77: 672-686.
Catalão, M.J., F. Gil, J. Moniz-Pereira, and M. Pimentel. (2011). Functional analysis of the holin-like proteins of mycobacteriophage Ms6. J. Bacteriol. 193: 2793-2803.
Garcia, M., M. Pimentel, and J. Moniz-Pereira. (2002). Expression of Mycobacteriophage Ms6 lysis genes is driven by two σ⁷⁰-like promoters and is dependent on a transcription termination signal present in the leader RNA. J. Bacteriol. 184: 3034-3043.
Prabhu, A., J. Zaugg, C.X. Chan, S.J. McIlroy, and C. Rinke. (2025). Insights Into Phylogeny, Diversity and Functional Potential of Poseidoniales Viruses. Environ Microbiol 27: e70017.
van Sinderen, D., H. Karsens, J. Kok, P. Terpstra, M.H.J. Ruiters, G. Venema and A. Nauta (1996). Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t. Mol. Microbiol. 19: 1343-1355.
Examples:
TC#	Name	Organismal Type	Example
1.E.18.1.1	*The L. lactis* phage r1t holin, Orf49**	Firmicute phage	*Orf49 holin of L. lactis* phage r1t (spQ38134)**

1.E.18.1.10	Putative holin, GP2, of 135 aas and 4 TMSs		*Putative holin of Rhodococcus* phage E3**

1.E.18.1.11	Putative holin of 80 aas and 2 TMSs		Holin of Agrococcus pavilionesis

1.E.18.1.12	Holin of 78 aas and 2 TMSs.		Holin of uncultured Methanobrevibacter sp.

1.E.18.1.2	*The Mycobacteriophage Ms6 hol* gene product, Gp4. May be an antiholin, counteracting the effect of the holin Gp5 (Catalão et al. 2011).**	Actinobacterial phage	gp4 of Mycobacteriophage Ms6

1.E.18.1.3	Protein of unknown function	Firmicutes	*Uncharacterized protein of Anaerostipes caccae* (67 aas)**

1.E.18.1.4	Holin	Actinobacteria	Holin of Corynebacterium ulcerans

1.E.18.1.5	Putative holin, gp37	Actinobacterial phage	gp37 of mycobacterial phage Che9d

1.E.18.1.6	Putative holin	Actinobacteria	Putative holin of Propionibacterium acnes

1.E.18.1.7	Putative holin	Actinobacteria	Putative holin of Renibacterium salmoninarium

1.E.18.1.8	Putative holin	Firmicutes	Putative holin of Eubacterium saphenum

1.E.18.1.9	Uncharacterized 4 TMS holin of 168 aas with two 2 TMS repeats.		UP of Rhodococcus opacus

Examples:
TC#	Name	Organismal Type	Example