Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches

Abstract

Genetic switches are critical components of developmental circuits. Because temperate bacteriophages are vastly abundant and greatly diverse, they are rich resources for understanding the mechanisms and evolution of switches and the molecular control of genetic circuitry. Here, we describe a new class of small, compact, and simple switches that use site-specific recombination as the key decision point. The phage attachment site attP is located within the phage repressor gene such that chromosomal integration results in removal of a C-terminal tag that destabilizes the virally encoded form of the repressor. Integration thus not only confers prophage stability but also is a requirement for lysogenic establishment. The variety of these self-contained integration-dependent immunity systems in different genomic contexts suggests that these represent ancestral states in switch evolution from which more-complex switches have evolved. They also provide a powerful toolkit for building synthetic biological circuits.

Figure 1. Mycobacteriophages with integration-dependent immunity systemst

(A) Genome segments are aligned by regions encoding Int, Rep and Cro. Genes are shown as boxes above (transcribed rightwards) or below (transcribed leftwards) each genome, and are colored according to Phamily assignments using Phamerator (Cresawn et al., 2011); Phamily number is shown above or below each gene with the number of phamily members (i.e. homologues) in parentheses. Pairwise nucleotide sequence similarity is represented by spectrum-coloring between adjacent genomes, with violet being the most similar and red the least. The genomes are sorted into the clusters according to overall genome similarity: BPs and Halo, Cluster G; Island 3, Babsiella, and Brujita, Subcluster I1; BigNuz, Cluster P; Charlie and Redi, Cluster N (Hatfull, 2012). (B) Integration results in reconfiguration of phage repressor genes. The attP common core (green box) lies within the repressor (rep) genes such that strand exchange results in a truncated version of the repressor adjacent to attL, and the 3’ end of the gene (arrowhead) next to attR. (See Figures S1, S2, Tables S1, S2)

Figure 2. Viral and prophage repressor activities

(A) Serial dilutions of phages D29 (control), BPs, Brujita, and Charlie were spotted onto BPs, Brujita, and Charlie lysogens (first column) and onto strains carrying integrated plasmids (columns 2–4) expressing different forms of the BPs, Brujita, and Charlie repressors in rows 1 – 3 respectively. Plasmids in columns 2 express prophage repressors (Rep^pro) from their normal integration site, and in columns 3 and 4 express viral repressors (Rep^vir) or Rep^pro integrated at attB -L5. Plasmid pGWB63 has a stop codon such that the viral gene expresses the prophage repressor; pGWB66 expresses the viral repressor form from the hsp60 promoter and replicates extrachromosomally. M. smegmatis mc²155 is the control strain. Note that the Charlie lysogen confers partial but non-reciprocal immunity to Brujita, and is not repressor-mediated. (B) Gain of function mutants (GoF) of the BPs viral repressor. Mutant derivatives of pGWB43 (see panel A) that have gained immunity were isolated, plasmids recovered, and retransformed into M. smegmatis. The full gain of immunity by mutants GoF2 and GoF3, and the partial recovery of immunity in GoF1 is shown. (C) GoF2-7 contain frameshift mutations near the 3’ end of the viral repressor and generate altered C-termini (yellow). GoF1 has a single amino acid substitution at the penultimate alanine residue (arrow). Protein lengths (in amino acids) are shown. (D) Alignment of the ssrA proteolytic tag and the last 11 amino acids of the repressor proteins of phages from Clusters G (BPs), I1 (Brujita, Babsiella and Island3), N (Charlie and Redi), and P (Bignuz) phages. Arrow indicates the highly conserved penultimate alanine residue. (E) The C-terminal 13 resides of BPs Rep tagged to the C-terminus of GFP lead to a loss of fluorescence upon induction. An A135E substitution at the penultimate residue restores GFP activity. Data are represented as mean +/− SD. (F) Western blot of uninduced (U) and induced (I) M. smegmatis expressing no GFP (control, top panel), untagged GFP (second panel), GFP-13aa Rep (third panel), GFP-13aa A135E (bottom panel) with anti-GFP. (G) Serial dilutions of BPs, mutants with a deletion of the helix-turn-helix motif in the repressor (RepΔHTH), with the stabilizing A135E substitution, or with a frameshift mutation in the repressor (GoF2) were spotted onto lawns of M. smegmatis mc²155. Lysogeny frequencies were determined as described in Experimental Procedures.

Figure 3. Divergent transcription in BPs

(A) Organization of the rep-cro intergenic region showing the P_R and P_Rep promoters, the O_R operator, and the locations of repressor-insensitive mutations within O_R (see Figure S3). (B) The promoters P_R and P⁶ (located between genes 5 and 6) were fused to mCherry and fluorescence units determined in M. smegmatis mc²155, a BPs lysogen [mc²155(BPs)], or strains expressing Rep¹³⁶ , Rep¹⁰³ or RepA135E. The P_R promoter activities of two repressor insensitive mutants (A29486C, T29489C) are also shown. Data are represented as mean +/− SD. (C) The trace derived by sequencing PCR products of ligated BPs gp34 (cro) mRNA shows that transcription starts at the first codon; cro coding sequence is shown in green. Cro is expressed from a leaderless mRNA. (D) Dilutions of either BPs or a D29 control phage were spotted onto lawns of strains carrying plasmids pGB109 or pGB110. The sequence below shows the mutations and substitutions (red) at each of the two rep plausible start sites in each plasmid. Loss of the upstream start codon (in pGB109) destroys immunity. (E) BPs Rep¹⁰³-DNA complexes were formed and separated by native gel electrophoresis. DNA substrates are: 366 bp with the entire 33-34 intergenic region, 139 bp containing O_R, similar fragments containing either mut 1 or mut 2 in O_R (A29486C and T29489C respectively), or a 110 bp DNA containing the 26-27 intergenic region (O₂₇). BPs Rep¹⁰³ concentrations are: none, 0.16 µM, 0.54 µM, 1.6 µM, 5.4 µM, 16 µM, and 54 µM. Migration of DNA and complexes (cmplx) are indicated, and the multiple complexes formed with the 33-34 substrate are labeled C1 – C4. Controls with non-specific DNA and competition with O_R-specific oligonucleotides are shown in Figure S4. (F) Promoter activity of P_Rep and a P_Rep T29336C mutation (see Fig. 6) is shown, as in panel B. Data are represented as mean +/− SD. (G) Determination of the P_Rep transcription start site, as in panel C. The coding sequence of gene 33 (rep) is shown in green. Rep also is expressed from a leaderless mRNA.

Figure 4. The role of integrase in the establishment of lysogeny

(A) Organization of non-canonical mycobacteriophage integrases. Each contains a proposed catalytic domain (red) and a core-type DNA binding domain (blue), but lack the arm-type DNA binding domain of λ Int (green). However, they contain C-terminal extensions (yellow) (see Figure S5). (B)attP requirements for integration were determined by co-transformation of attP plasmids with int-expressing plasmid pLB03 (Table S4) and selection for the attP plasmid. Transformation frequencies for plasmids pBL06, pBL08, pBL10, pBL14, pBL26, pBL15, pBL23, pBL24, pBL39 (top to bottom with BPs coordinates shown) are expressed as percentages of pBL06 (6.7 × 10³ cfu/µg DNA). Deletion beyond the right side of the attP core (at 29,211) is predicted to interfere with tRNA function. Competency of cells using plasmid pMH94 was 2 × 10⁵ transformants/µg DNA. (C) Serial dilutions of BPs and BPs mutants were spotted on a lawn of M. smegmatis mc²155. Lysogeny frequencies were determined as described in Experimental Procedures and reported as % recovery. The mutant (Δint) that removes sequences downstream of the repressor yields 100% recovery of colonies – reflecting the plaque turbidity – but does not form recoverable stable lysogens (Figure S6). (D) Integration-proficient plasmids carrying a selectable marker (Kan^R) and phage cassette including attP and int stably transform by chromosomal integration. The transformation frequencies (transⁿ/µg DNA) of various plasmids are shown. Substitutions stabilizing either Brujita or Charlie Int (in pGWB87 and pES14 respectively) substantially increase transformation efficiency. (E) Int C-termini are aligned with the M. smegmatis ssrA tag, showing their spacing from the catalytic tyrosine. Arrow indicates the penultimate residue where substitutions stabilize Int. The BPs C-terminus is atypically longer and does not have a canonical ssrA tag. Charlie Int has a valine residue at the penultimate position which likely accounts for the 50-fold higher transformation efficiency of pGWB82 relative to pGWB81 (see panel D). (F) Addition of 5 C-terminal residues of Brujita Int destablizes GFP and the A296E substitution restores activity. Western blots show that activities reflect GFP protein levels (data not shown), and addition of the C-terminal 60 aa of BPs Int to GFP has the same effect (data not shown). Data are represented as mean +/− SD. (G) Brujita mutants with deletions of either int(Δint) or rep (Δrep) make clear plaques and do not form lysogens. Both a stabilized form of Int and a catalytically defective Int fail to form lysogens (see Figure S7). (H) Transformants of pGWB81 (wt Int) or pGWB87 (IntA296E; see panel D) were grown either with or without selection for the specified number of generations and the proportion of colonies retaining the plasmid determined.

Figure 5. BPs Cro antagonizes lysogeny

(A) Serial dilutions of BPs and BPs mutants were spotted onto M. smegmatis mc²155 and strains expressing BPs cro from the wild type promoter P_R (pGWB76) or the repressor insensitive mutant P_R T29489C (pGWB78). Lysogenization frequencies of phages on M. smegmatis pGWB78 were determined and can be compared with the parent strain in Figures 2G and 4C. (B) The activities of P_R and P_Rep promoter reporter plasmids were measured in a strain expressing Cro from P_R (red bars) or from the depressed P_R (green bars), both on a lysogen and non-lysogen. Data are represented as mean +/− SD.

Figure 6. All clear-plaque mutations map within the BPs 32–34 locus

(A) Independent clear plaque mutants were isolated using three mutant parent phages that have increased turbidity, RepA135E (red), RepGoF2 (green) and Δint (blue), and the mutations mapped. The locations of mutations or amino acid substitutions in repressor are shown. (B) Effects of clear plaque mutations on P_R activity. Data are represented as mean +/− SD.

Figure 7. A model for integration-dependent bacteriophage immunity

During infection by phages using integration-dependent immunity systems, the promoters P_R and P_Rep are both active, leading to synthesis of Cro, Repressor (Rep) and Integrase (Int). The outcome of infection is decided by the stability of Int determined through its C-terminal ssrAlike tag. If Int fails to accumulate due to proteolysis, then only the viral form of Rep is made which in turn is degraded through its C-terminal ssrA-like tag; Cro accumulates, antagonizes repressor action, and lytic growth ensues. A putative function located between the rep and int genes (inhib?) may also down-regulate repressor synthesis from the viral genome. If Int escapes proteolysis (either at high moi or when cellular proteases are at low levels) then integration occurs, the active truncated form of the repressor is made which binds to O_R and downregulates P_R and cro-expression ceases. Lysogeny is thus established. Repressor is made constitutively from P_Rep, which is not autoregulated, and binds with relatively low affinity. Spontaneous induction may occur in conditions where Rep falls below critical levels, or by expression of Int, perhaps from the M. smegmatis genes adjacent to attR, Msmeg_6348, Msmeg_6151, or Msmeg_5759 for the three attB sites described here (See Figure S1).