Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta

Abstract

Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some models, generate large “pores” in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive Gordonia rubripertincta NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1–4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered.

A genome-wide cytotoxicity screen of cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth

“…The high level of toxicity following the expression of these proteins is especially intriguing given the low sequence conservation of the three proteins: although Girr gp35 and Waterfoul gp32 are members of the same pham, they only share 35% amino acid identity, and Hammy gp32 is part of a separate pham with only ∼20% identity to the other two proteins. Although the single TMD proteins are not annotated as holins, a recent report on >80 actinobacteriophages that infect Gordonia rubripertincta ( Pollenz et al . 2022 ) shows that the lysis cassette may contain up to four different holin-like genes where the terminal gene typically encodes a protein with a single TMD.…”

“…2022 ), and TOPCONS ( Tsirigos et al 2015 ). TMD proteins were characterized based on agreement of all three programs as detailed in Pollenz et al . (2022) .…”

Unlocking prophage potential:In silicoand experimental analysis of a novelMycobacterium fortuitumLysinB containing a peptidoglycan-binding domain

“…The holin gene encodes for a protein of size 7.4 kDa, which shares 61.1% identity with the holin from cluster I2 mycobacteriophage Che9c. Comparable to other phage-encoded holin, this, too, consists of two transmembrane helices, which could destabilize the membrane proton motive force of the host in a time-dependent manner (Pollenz et al, 2022). phiE1336 demonstrates distinct PGBD and EAD.…”

Genome sequence of Soos: a siphovirus of the CP cluster infecting Gordonia rubripertincta

“…Soos shares 84.1% GCS with annotated phage Clawz (GenBank accession MT498058) , and 90.6% GCS with the draft genome of phage Sting ( https://phagesdb.org/phages/Sting ), the only other phages in cluster CP. Like Clawz, Soos includes a simple lysis cassette with a single, large endolysin gene preceding a holin gene ( 16 ). Nucleotide sequence comparison of Soos using BLASTn revealed that outside of cluster CP, Soos is most similar to GMA4 (GenBank accession NC_030939.1 ), a phage without sufficient GCS to be assigned to any cluster and with which Soos shares ~4% GCS.…”