1. Volume 49, Issue 2, Pages 237-248 (January 2013)
Integration-Dependent Bacteriophage Immunity Provides Insights into the Evolution of Genetic Switches 
Gregory W. Broussard, Lauren M. Oldfield, Valerie M. Villanueva, Bryce L. Lunt, Emilee E. Shine, Graham F. Hatfull 
Molecular Cell 
Volume 49, Issue 2, Pages (January 2013)
DOI: /j.molcel
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2. Figure 1
Mycobacteriophages with Integration-Dependent Immunity Systems
(A) Genome segments are aligned by regions encoding Int, Rep, and Cro. Genes are shown as boxes above (transcribed rightward) or below (transcribed leftward) 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., homologs) 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 Figure S1, Figure S2, Table S1, and Table S2.)
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3. 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 column 2 express prophage repressors (Reppro) from their normal integration site and in columns 3 and 4 express viral repressors (Repvir) or Reppro 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 nonreciprocal immunity to Brujita and is not repressor mediated.
(B) Gain-of-function mutants (GoF) of the BPs viral repressor. Mutant derivatives of pGWB43 (see A) that have gained immunity were isolated, and 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–Gof7 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 the Experimental Procedures.
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4. Figure 3 Divergent Transcription in BPs
(A) Organization of the rep-cro intergenic region showing the PR and PRep promoters, the OR operator, and the locations of repressor-insensitive mutations within OR (see Figure S3).
(B) The promoters PR 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 PR 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 as follows: 366 bp with the entire 33–34 intergenic region, 139 bp containing OR, similar fragments containing either mut 1 or mut 2 in OR (A29486C and T29489C, respectively), or a 110 bp DNA containing the 26–27 intergenic region (O27). BPs Rep103 concentrations are as follows: none, 0.16 μM, 0.54 μM, 1.6 μM, 5.4 μM, 16 μM, and 54 μM. Migration of DNA and complexes (cmplx) is indicated, and the multiple complexes formed with the 33–34 substrate are labeled C1–C4. Controls with nonspecific DNA and competition with OR-specific oligonucleotides are shown in Figure S4.
(F) Promoter activity of PRep and a PRepT29336C mutation (see Figure 6) is shown, as in (B). Data are represented as mean ± SD.
(G) Determination of the PRep transcription start site, as in (C). The coding sequence of gene 33 (rep) is shown in green. Rep also is expressed from a leaderless mRNA.
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5. Figure 4 The Role of Integrase in the Establishment of Lysogeny
(A) Organization of noncanonical 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 cotransformation of attP plasmids with int-expressing plasmid pBL03 (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 × 103cfu/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 the Experimental Procedures and reported as percentage 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 (KanR) 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 D).
(F) Addition of five 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 D) were grown either with or without selection for the specified number of generations and the proportion of colonies retaining the plasmid determined.
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6. 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 PR (pGWB76) or the repressor-insensitive mutant PRT29489C (pGWB78). Lysogenization frequencies of phages on M. smegmatis pGWB78 were determined and can be compared with the parent strain in Figure 2G and Figure 4C.
(B) The activities of PR and PRep promoter reporter plasmids were measured in a strain expressing Cro from PR (red bars) or from the depressed PR (green bars), both on a lysogen and a nonlysogen. Data are represented as mean ± SD.
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7. 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 PR activity. Data are represented as mean ± SD.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 A Model for Integration-Dependent Bacteriophage Immunity
During infection by phages using integration-dependent immunity systems, the promoters PR and PRep 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 ssrA-like 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 and antagonizes repressor action, and lytic growth ensues. A putative function located between the rep and int genes (inhib?) may also downregulate 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 OR and downregulates PR, and cro expression ceases. Lysogeny is thus established. Repressor is made constitutively from PRep, which is not autoregulated, and binds with relatively low affinity. Spontaneous induction may occur in conditions in which 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).
Molecular Cell  , DOI: ( /j.molcel )
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