More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash

Abstract

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 10²³ phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.

Keywords: Mycobacterium; bacteriophage; viral defense.

FIG 1

Viral defense systems in mycobacteriophages Sbash and Che9c. (A) The genome maps of phages Che9c and Sbash are shown with shared nucleotide sequence similarity represented as spectrum-colored shading between the genomes, with violet representing the most similar and red the least similar above a BLASTN E value threshold of 10⁻⁴. Genes are shown as colored boxes above and below genome rulers. An expanded view of the centers of the Che9c and Sbash genomes with putative gene functions indicated is shown at the bottom. Gene numbers are shown within the gene boxes, and the assigned Pham numbers are shown above the boxes, with the number of Pham members indicated in parentheses. (B) Plating efficiencies of mycobacteriophages on lysogens of phages Che9c and Sbash. A total of 108 mycobacteriophages were serially diluted and plated onto lawns of M. smegmatis mc²155, mc²155(Che9c), and mc²155(Sbash). The plating efficiencies on each of the lysogens are shown relative to plating on the nonlysogen. The cluster assignment is shown in parentheses following each phage name, and phages are sorted according to their cluster designation; Giles and Crossroads are highlighted in red type. Plating efficiencies between 1 and 10⁻², between 10⁻³ and 10⁻⁷, and at 10⁻⁸ or below are highlighted in green, yellow, and red, respectively. (C) Sbash-mediated defense against Typha, Crossroads and Giles is not repressor mediated. Ten-fold serial dilutions of phages Typha, Giles, Crossroads, Sbash, and Pipsqueaks were plated onto lawns of mc²155, mc²155(Sbash), and mc²155pGG07, which carries the Sbash repressor (see Fig. 3). Like strain mc²155(Sbash), strain mc²155pGG07 confers immunity to Sbash but does not inhibit infection by Typha, Crossroads or Giles. Phage Pipsqueaks is a control that is not affected by Sbash defenses. The pGH1000 vector control is shown in Fig. 3.

FIG 2

Sbash genes required for viral defense. (A) Lysogenic expression of Sbash prophage genes. RNAseq reads are mapped to the Sbash prophage, shown in its integrated orientation from attL to attR, with the relevant part of the genome map shown at the bottom. Putative lysogenically expressed operons are represented by arrows. The RNAseq reads are strand specific, and those mapping to forward and reverse DNA orientations are indicated. Results of RNAseq analysis of the whole prophage are shown in Fig. S1. (B) Sbash deletion derivatives. The seven deletion mutants are depicted, showing the region removed in each mutant. Genomes are aligned with the map shown at the top. (C) Plating efficiencies of phages on mutant Sbash lysogens. Ten-fold serial dilutions of phages Giles, Crossroads, Sbash, and Pipsqueaks were plated onto lawns of M. smegmatis mc²155 and mc²155(Sbash) and of mutant derivatives in which individual Sbash genes were knocked out. Sbash 30 and 31 are both required for defense against Crossroads; none of the deleted genes are required for defense against Giles infection.

FIG 3

Sbash 30 and 31 are necessary and sufficient to defend against Crossroads. (A) Recombinant clones tested for defense against phage Crossroads and Giles infection. A map of the central portion of the Sbash genome is shown with the insertions in plasmids indicated by horizontal lines and plasmid names. Plasmid vectors are all integration-proficient plasmids based on pGH1000 (red), pJV39 (black), or pCCK39 (green). Phages plate similarly on pJV39 and pGH1000 strains (data not shown). (B) Plating efficiencies of phages Giles, Crossroads, Sbash, and Pipsqueaks on recombinant strains. Ten-fold serial dilutions of each phage were spotted onto lawns of each strain, as indicated. All four phages infected mc²155pGH1000 and mc²155pJV39 (not shown) vector strains similarly.

FIG 4

Crossroads is the only subcluster L2 phage targeted by Sbash gp30/gp31 defense. (A) Genome maps of subcluster L2 phages, Crossroads, Kahlid, LilDestine, Wilder, Gardann, MkaliMitinis3, Faith1, BigCheese, and Rumpelstiltskin (top to bottom). Genomes are represented as described for Fig. 1. (B) Expanded view of the right genome ends of phages BigCheese, Crossroads, Wilder, and Rumpelstiltskin. Crossroads and Wilder were very similar in this region, whereas BigCheese and Rumpelstiltskin have 3-kbp to 5-kbp deletions. (C) Plating efficiencies of subcluster L2 phages on mc²155 and a Sbash lysogen. Ten-fold serial dilutions of each phage were plated as indicated.

FIG 5

Characterization of Crossroads defense escape mutants. (A) Plating of Crossroads defense escape mutants (DEMs) on host strains. Crossroads mutants were isolated as escapees on lawns of either a Sbash lysogen or a pGG05 recombinant strain, purified, and shown to escape Sbash prophage defense. Ten-fold serial dilutions of DEMs (phgg409, phgg411, phgg399, phgg407) were plated on bacterial lawns as indicated. (B) Organization at the extreme right end of the Crossroads genome showing genes 121 to 145. The genome is displayed as described for Fig. 1. (C) Mapping of Crossroads DEM mutations. Defense escape mutants of Crossroads were purified and sequenced, and the locations of the mutations were mapped to gene 132 and 141 and the 141 to 142 intergenic region. The location of the AAA ATPase domain in gp141 (residues 186 to 323) predicted by pfam is shown. (D) DEM mapping in the Crossroads 141-to-142 intergenic region. The DNA sequence is shown for the beginning of the leftward-transcribed gene 141 and its amino acid sequence, together with the sequences immediately upstream. The ATG translation start codon for gene 141 is underlined, and the ribosome binding site (RBS) is indicated. Four DEM mutations are shown: a single base substitution at coordinate 74,303, 26-bp and 9-bp duplications, and a 99-bp deletion. (E) Crossroads 132 or 139 to 141 is required for targeting by Sbash 30/31. Serial dilutions of a Crossroads mutant in which gene 132 is deleted (C’roadsΔ132) or genes 139 through 141 are deleted (C’roadsΔ139–141) together with Crossroads (C’roads), Sbash, and Pipsqueaks were plated onto lawns of M. smegmatis mc²155, a Sbash lysogen strain, and a pGG05 recombinant strain.

FIG 6

Expression of Crossroads gp132 and gp141. (A) Strains were constructed carrying extrachromosomal expression vector pCCK38 or plasmid pKSW06, in which Crossroads 132 expression can be induced by addition of ATc, in either M. smegmatis mc²155 (top row) or a Sbash lysogen (bottom row). Lawns were prepared on solid media with (Induced) or without (Uninduced) ATc, and 10-fold serial dilutions (from left to right) of phages were spotted. Phages tested were Crossroads, Crossroads Δ132, DEM mutants phgg356 and phgg408, Sbash, and Pipsqueaks. Expression of gp132 in mc²155(Sbash) results in an approximately 100-fold reduction in EOP of strain Δ132 and the DEMs relative to the uninduced control. It is unclear why some of these phages have reduced plating efficiencies on M. smegmatis (Sbash) pCCK38 relative to M. smegmatis mc²155pCCK38. (B) Strains containing integration-proficient vector pCCK39 or plasmid pGG38 in which Crossroads 141 can be induced with ATc were plated as described in the panel A legend, except the ATc concentration was reduced to 5 ng/ml and the plates were incubated for 3 days (all others were incubated for 1.5 days). Phages Crossroads, phgg348, phgg364, phgg399, Sbash, and Pipsqueaks were spotted as described in the panel A legend. Expression of gene 141 substantially reduced the plating efficiencies of DEM mutants in a Sbash lysogen. (C) Liquid cultures of strains containing the plasmids indicated on the left were grown to an OD₆₀₀ of 0.5, serially diluted 10-fold, and spotted onto solid media with (Induced) or without (Uninduced) ATc. The genes/mutants in each plasmid are noted at the right. Plasmids pGG37 and pKSW06 are integrating and extrachromosomal plasmids, respectively, containing Crossroads 132, and neither displayed toxicity in either a Sbash lysogen or a nonlysogen.

FIG 7

RNAseq analysis of Crossroads and DEM mutants. (A) RNA was isolated at early (30 min) and late (150 min) time points after infection of M. smegmatis with wild-type Crossroads (green) or DEMs phgg348 (aqua) and phgg417 (red); reads mapping to forward and reverse strands are shown as indicated. Arrows at the bottom indicate the early and late transcribed regions, with gene numbers denoting the positions. (B) Expanded view of RNAseq reads mapping to the reverse strands at the right end of the genome. DEM mutants phgg419 (blue) and phgg413 (magenta) are shown in addition to those shown in panel A and are similarly colored. Arrows indicate leftward transcription of the two genes, 132 and 141, in which DEM mutants mapped. Reads are shown for the reverse strand only.

FIG 8

Mechanism of Sbash 30-to-31-mediated defense against Crossroads. (A) RNAseq analysis of Crossroads and DEM mutant infection of M. smegmatis mc²155pGG05. RNA was isolated at early (30 min) and late (150 min) time points after infection of M. smegmatis mc²155pGG05 with wild-type Crossroads (green) or DEMs phgg417 (red) and phgg348 (aqua); reads mapping to forward and reverse strands are shown as indicated. Scales were adjusted to reflect approximately similar levels of early gene expression. (B) Comparison of Sbash and lambda genomes. Segments of the two phage genomes were aligned to show similarities between Sbash defense genes 30 and 31 and lambda exclusion genes rexA and rexB. The positions of promoters expressing Sbash 30/31 and lambda cI/rexAB are shown, and the locations of a DUF4747 conserved domain in Sbash gp30 and lambda RexA are also indicated. The function of Sbash gp29 is not known, but it likely a virion tail gene; the lambda gene orientation has been reversed for illustration purposes. (C) TMHMM prediction of membrane topology of Sbash gp31 and RexB. Probabilities of amino acids being located within the membrane and cytoplasm and externally are shown in red, purple, and blue, respectively. The positions of two separate amino acid substitutions in Sbash gp31 that inactivate defense are shown (see Fig. S2B). (D) A model for Sbash 30/31-mediated defense. Sbash gp31 is proposed to be membrane located but inactive as an ion channel until infection with phage Crossroads. During early lytic growth of phage Crossroads, gp132 and gp141 act either directly or indirectly through Sbash gp30 to activate the gp31 ion channel, leading to loss of membrane potential and of intracellular ATP, interruption of macromolecular synthesis, and loss of cell viability. It is not known if Sbash gp30 interacts directly with gp31 in either the inactive or the activated state, but substitutions in the central cytoplasmic loop of Sbash gp31 are inactive for defense, consistent with an interaction between Sbash gp30 and gp31.

FIG 9

CarolAnn genes 43 and 44 confer defense against Crossroads. (A) Genome comparison of mycobacteriophage Sbash and Gordonia phage CarolAnn. Shading between the genomes reflects nucleotide sequence similarities, although there are only two short regions of weak similarity at the extremities of Sbash 30 and CarolAnn 43. Sbash gp30/CarolAnn gp44 and Sbash gp31/CarolAnn gp43 homologous pairs share 50% and 43% amino acid identity, respectively. Sbash 30 and 31 are transcribed rightward and are displaced from the repressor gene (gene 43), whereas CarolAnn 43 and 44 are coexpressed with the repressor (gene 45). (B) Serial dilutions of Sbash, Crossroads, Pipsqueaks, and 12 Crossroads DEM mutants (labeled phggXXX) were plated onto lawns of M. smegmatis mc²155pMH94 (vector) and M. smegmatis mc²155pMM16, which carries CarolAnn genes 43 and 44.