Physiological characterization of single-gene lysis proteins.

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.

Physiological characterization of single gene lysis proteins.

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: Qß 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.