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.

Keywords: bacteriophage evolution; bacteriophage lysis; bacteriophages.

Fig 1

Peptidoglycan biosynthesis pathway and Sgl inhibition (A). Genomes of ssDNA (ϕX174) and ssRNA (Qβ, M, PP7, MS2, KU1, Hgal, and PRR1) phages. Adapted from reference (9). SsRNA phages have three core conserved genes in the same order: mat (orange), coat (black), and rep (green). These phages also encode a lysis gene (blue) that is embedded in an alternative reading frame in one of the three core genes or intergenic spaces. In Qβ, the maturation gene has a dual function, serving as both the maturation protein and the Sgl. (B). Sgls of ssRNA and ssDNA phages such as ϕx174, Qβ, and M inhibit different enzymatic steps in the PG biosynthesis pathway.

Fig 2

Sgl lysis profiles in defined minimal media. Lysis profiles of Sgls in minimal media supplemented with 16 amino acids, 0.01% thiamine, and 0.2% glycerol as the carbon source. When the cultures reached an OD of ~0.2, they were induced with 0.4% L-arabinose. From top to bottom: pBAD24 vector with no gl (closed circles), E (closed downward triangle), A₂ (open circles), Sgl^M (closed diamonds), Sgl^PP7 (open squares), L (closed squares), Sgl^KU1 (closed upward triangles), and Sgl^PRR1 (open triangles).

Fig 3

³[H]mDAP labeling of peptidoglycan. Growth curve of E. coli is shown as open circle (O), and the amount of ³[H]mDAP counted at different time points is shown as closed squares (◼). The first arrow indicates when ³[H]mDAP was added to the bacterial culture, and the second arrow indicates the induction of the plasmid. The dual-sided arrows indicate the time between cessation of ³[H]mDAP incorporation and lysis. Each construct has a minimum of one repeat, and the representative one is shown in each case. For type I Sgls, after induction, each time point was filtered twice (see Materials and Methods). The average is plotted, and the error bars represent the variation.

Fig 4

Type I and type II lysis morphology. Shown are phase contrast images of E. coli after induction of different lysis genes. (A) Vector only. (B–D) Type I Sgls: E, A₂, and Sgl^M. (E–H) Type II Sgls: L, Sgl^KU1, Sgl^Hgal, and Sgl^PRR1. For panels (A–G), cultures were grown at 37°C to an optical density of ~0.2 and induced with 0.4% L-arabinose. For panel (H), cultures were induced with 0.075 mM crystal violet. Ten minutes prior to lysis, 1.5-μL samples were taken from culture flasks, put onto glass slides, covered with a cover slip, and imaged using the 100× objective lens. Collages were made using Inkscape, and each represents the population of cells seen in triplicate imaging experiments. Furthermore, to minimize bias in the selection of cells with blebs for the collages, all cells showing blebs on an image were incorporated into collages.

Fig 5

Analysis of lesions caused by Sgls. The y-axis represents the location of the bleb along the cell length with 0.0 representing the poles of the cell and 0.5 representing middle of the cell. The x-axis indicates the type of Sgl for each set of measurements, including type I (E, A₂, Sgl^M, and Sg^PP7) and type II (L, Sgl^KU1, Sgl^Hgal, and Sgl^PRR1) Sgls. Measurements of cells (n = 25 for each Sgl) were done using the measurement tool from ImageJ. The box plot was created using GraphPad Prism 9. To minimize bias in the selection of cells with blebs for the analysis, all cells showing blebs in an image were measured. A comparison between type I Sgls and type II Sgls was conducted using a Levene and Fligner–Killeen test, both of which yielded a P-value less than 0.001.

Fig 6

Sgl^PP7 portrays type I lysis morphology and isotope labeling. (A) Microscopy images of bleb formation after the induction of the Sgl^PP7. (B) Isotope labeling of the peptidoglycan layer using ³[H]mDAP. The open circles represent the optical density of the culture, and the closed squares represent the counts per minute of ³[H]mDAP incorporated into insoluble material. The solid arrow on the left indicates the time at which ³[H]mDAP was added to the growing culture. The middle solid arrow shows when the culture was induced with 0.4% L-arabinose. The dual-sided arrow indicates the time between cessation of ³[H]mDAP incorporation and lysis.