Dataset Information

Physiological characterization of single-gene lysis proteins.

ABSTRACT: Single-strand RNA (ssRNA) and single-strand DNA phages elicit host lysis using a single gene, in each case designated as sgl. Of the 11 identified Sgls, three have been shown to be specific inhibitors of different steps in the pathway that supplies lipid II to the peptidoglycan (PG) biosynthesis machinery. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here, we designate these as type I Sgls. In this formalism, the other eight Sgls are assigned to type II, the best-studied of which is protein L of the paradigm F-specific ssRNA phage MS2. Comparisons have suggested that type II Sgls have four sequence elements distinguished by hydrophobic and polar character. Environmental metatranscriptomics has revealed thousands of new ssRNA phage genomes, each of which presumably has an Sgl. Here, we describe methods to distinguish type I and type II Sgls. Using phase contrast microscopy, we show that both classes of Sgls cause the formation of blebs prior to lysis, but the location of the blebs differs significantly. In addition, we show that L and other type II Sgls do not inhibit the net synthesis of PG, as measured by radio-labeling of PG. Finally, we provide direct evidence that the Sgl from Pseudomonas phage PP7 is a type I Sgl, in support of a recent report based on a genetic selection. This shows that the putative four-element sequence structure suggested for L is not a reliable discriminator for the operational characterization of Sgls.

Importance

The ssRNA phage world has recently undergone a metagenomic expansion upward of a thousandfold. Each genome likely carries at least one single-gene lysis (sgl) cistron encoding a protein that single-handedly induces host autolysis. Here, we initiate an approach to segregate the Sgls into operational types based on physiological analysis, as a first step toward the alluring goal of finding many new ways to induce bacterial death and the attendant expectations for new antibiotic development.

SUBMITTER: Antillon SF

PROVIDER: S-EPMC10955853 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Publications

Physiological characterization of single-gene lysis proteins.

Antillon S Francesca SF Bernhardt Thomas G TG Chamakura Karthik K Young Ry R

Journal of bacteriology 20240301 3

Single-strand RNA (ssRNA) and single-strand DNA phages elicit host lysis using a single gene, in each case designated as <i>sgl</i>. Of the 11 identified Sgls, three have been shown to be specific inhibitors of different steps in the pathway that supplies lipid II to the peptidoglycan (PG) biosynthesis machinery. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here, we designate these a ...[more]

PMID: 38426721

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Project description:Until recently only 11 distinct Sgls (single gene lysis proteins) have been experimentally identified. Of these, three have been shown to be specific inhibitors of different steps in the pathway that supplies Lipid II to the peptidoglycan (PG) biosynthesis machinery: Qβ A2 inhibits MurA, ϕX174 E inhibits MraY, and Lys from coliphage M inhibits MurJ. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here we propose to designate these as members of type I Sgls, to distinguish them from another Sgl, the L protein of the paradigm ssRNA phage MS2. Although none of the other distinct Sgls have significant sequence similarity to L, alignments suggested the presence of four domains distinguished by hydrophobic and polar character. The simplest notion is that these other Sgls have the same autolytic mechanism and, based on this, constitute type II. Although the number of experimentally confirmed Sgls has not changed, recent environmental metagenomes and metatranscriptomes have revealed thousands of new ssRNA phage genomes, each of which presumably has at least one Sgl gene. Here we report on methods to distinguish type I and type II Sgls. Using phase-contrast microscopy, we show that both classes of Sgls cause the formation of blebs prior to lysis, but the location of the blebs differs significantly. In addition, we show that L and other type II Sgls do not inhibit net synthesis of PG, as measured by incorporation of 3[H]-diaminopimelic acid. Finally, we provide support for the unexpected finding by Adler and colleagues that the Sgl from Pseudomonas phage PP7 is a type I Sgl, as determined by the two methods. This shows that the sharing the putative 4-domain structure suggested for L is not a reliable discriminator for operational characterization of Sgls. Overall, this study establishes new ways to rapidly classify novel Sgls and thus may facilitate the identification of new cell envelope targets that will help generate new antibiotics.

Project description: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.

Dataset Information

Physiological characterization of single-gene lysis proteins.

Importance

Publications

Physiological characterization of single-gene lysis proteins.

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