Figure 1.
Variability of Gifsy phage repressors.
A. Comparison of the Gifsy-1 prophage genomes from Salmonella enterica serovar Typhimurium strains LT2 and ATCC14028. Diagrams were made from sequence data obtained in the course of this study, complemented with data from [21] and [12]. Percentages indicate DNA sequence identities. Green coloring shows a portion of LT2's Gifsy-1 prophage more than 99% identical to the corresponding region of Gifsy-2. Genes marked by an asterisk are named on the basis of their sequence similarity to known genes of other phages or bacteria. B. Alignment of the deduced amino acid sequences of the repressors of prophages Gifsy-1 (GfoR) Gifsy-3 (GfhR) and Gifsy-2 (GftR) from strain ATCC14028.
Figure 2.
Fate of phage repressors during induction and the role of LexA.
Strains harboring C-terminally 3xFLAG-tagged versions of the repressors of prophages Gifsy-1 (A) Gifsy-2 (B), Gifsy-3 (C) and Fels-1 (D) were exposed to Mitomycin C (1 µg mL−1) for the indicated times and processed for immunodetection of the 3xFLAG epitope as described [20]. Strains used were MA8407 (A), MA8259 (B), MA8408 (C) and MA8456 (D). E,F. Effect of the lexA3 mutation on induction of lacZ reporter fusions in the Gifsy-2 prophage (E) or in the Fels-1 prophage (F). Cultures were spread on LB X-gal indicator plates; filter paper disks were placed on the surface and soaked with 5 µL of 2 mg mL−1 Mitomycin C. The strains used were MA8756 (lexA+) and MA8757 (lexA3) in E and MA8410 (lexA+) and MA8573 (lexA3) in F. For complete strain genotypes, see Table 1.
Figure 3.
LexA-controlled prophage loci required for induction.
A. Alignment of DNA sequences preceding the dinI gene homologues of Gifsy-1, Gifsy-2 and Gifsy-3 prophages. The dinI translation initiation codon is underlined. The −35 and −10 motifs of putative promoters are highlighted in light green. A light brown box encompasses sequences matching the consensus for LexA binding. B. Effect of replacing the LexA box of the Gifsy-2 prophage with an araC-PBAD promoter module on induction of a Gifsy-2-borne recE-lacZ fusion (strain MA8357). C. Expression of Gifsy-2's gftA gene alone (strain MA8430) is sufficient to elicit induction of the prophage.
Figure 4.
A. Alignment of antirepressor sequences and amino acid changes in GftA mutants. B. Gene organization near the left end of the Fels-1 prophage. The diagram in B was drawn using information from Salmonella enterica serovar Typhimurium strain LT2 genome sequence [21]. Repressor and antirepressor genes (fsoR and fsoA, respectively) were identified in the course of this study. C. Induction of Gifsy-2-borne recE-lacZ fusion in strains carrying gftA or fsoA genes, or their 3xFLAG-tagged variants, fused to the chromosomal PBAD promoter. Arabinose (10 mM) was added at time zero. Cells collected at the indicated times were assayed for β-galactosidase as described [48].
Table 1.
Strains used in this work.
Figure 5.
Monitoring Gifsy phage repressors and antirepressors under inducing conditions.
Strains harboring 3xFLAG-tagged versions of both repressor and antirepressor genes in prophage Gifsy-1 (A; strain MA8729), Gifsy-2 (B; strain MA8728) or of the antirepressor gene in Fels-1 (C; MA8605) were exposed to Mitomycin C (1 µg mL−1) for 30 or 60 min. Chloramphenicol (10 µg mL−1) was added to samples subjected to the 60 min treatment and incubation continued for additional 30 min or 60 min. Bacteria were harvested and processed for immunodetection of epitope-tagged proteins as described [47].
Figure 6.
Repressor-antirepressor pulldown assays.
The strains used harbor 3xFLAG-tagged versions of antirepressor genes under the control of the chromosomal PBAD promoter and carry or lack plasmids expressing 6His-tagged versions of cognate repressors. Crude extracts from cells grown in the presence or absence of arabinose were incubated with nickel beads, and rinsed in low-concentration imidazole buffer (10 mM). The bound proteins were eluted from the column using high imidazole concentrations (250 mM). Eluates were applied in duplicates to 15% polyacrylamide gels and one was stained with Coomassie brilliant-blue (A and B) while the other was processed for immunodetection using anti-FLAG monoclonal antibodies (C and D). A,C. GtfA pulldown by GtgR (strain MA8567 plus or minus plasmid pSEB10; left three lanes contain samples prior to the nickel binding step); B,D. GfoA pulldown by GfoR (strain MA8731 plus or minus plasmid pSEB11). E. Far western detection of GtgR∶GftA interaction. A crude extract of strain MA8567 carrying gtgR plasmid pSEB10 was separated on a 15% gel and the gel blotted onto a PVDF membrane. The membrane was split into two halves; one was incubated with the extract of a strain expressing GftA-3xFLAG (1) while the other was left untreated (2). The two strips were processed for hybridization with anti-FLAG antibodies. For more details, see Materials and Methods.
Figure 7.
Gel exclusion chromatography of GfoR and GfoR∶GfoA complexes.
Proteins were purified from strain MA8731 containing the GfoR plasmid pSEB11 as described in Materials and Methods. The strain was grown without (A) or with arabinose (B). About 10 µg of each protein preparation were applied to an Amersham Sephadex G75 column. 0.5 mL fractions were collected after the passage of the void volume, dried, resuspended in protein loading buffer and separated on a 12% SDS-polyacrylamide gel. Band identification was confirmed by anti-FLAG Western blotting (data not shown). The column was size-calibrated using α-chymotrypsin, ovalbumin and bovine serum albumin from Sigma-Aldrich.
Figure 8.
Gel-shift assay with purified GftR and GftA proteins.
A radioactively labeled 267 bp DNA fragment (approximately 5 ng) was mixed with increasing amounts of purified GftR protein (∼0.25, 0.5, 1, 2, 4 or 8 pmol) in BB buffer (Tris-HCl pH 7.5 20 mM, NaCl 50 mM, EDTA 0.2 mM, MgCl2 1 mM, glycerol 5%, PMSF 10 mM, sonicated salmon sperm DNA 50 µg mL−1). After 15 min at room temperature, aliquots from the sample with the highest protein∶DNA ratio were mixed with increasing amounts of purified GftA protein (∼0.01, 0.05, 0.1, 0.5, 2 or 10 pmol) and incubation continued for all samples, for additional 30 min. Samples were loaded on a non-denaturing 5% polyacrylamide gel. The gel was fixed in an acid-ethanol bath, dried and radioactivity was detected and quantified by phosphorimaging.
Table 2.
DNA oligonucleotides used as PCR primers for plasmid construction and cloning.