Figure 1.
Modification of the EZ-Tn5-encoding vector for random mutagenesis in C. perfringens.
To allow selection of C. perfringens transformants after electroporation with the EZ-Tn5 transposon, a C. perfringens erythromycin resistance determinant (erm) was cloned into the multiple cloning site in the Epicentre EZ-TN5-encoding pMOD-2 vector creating pJVTN5. This plasmid also contains PvuII-recognized sequences flanking the mosaic end (ME) sites, which are specifically recognized by the EZ-Tn5 transposase.
Figure 2.
Southern blot analyses of C. perfringens random mutants obtained after electroporation with EZ-Tn5 transposomes.
After selection on BHI plates containing Erm (40 µg/ml), DNA was extracted from strain 13 transformants. Following digestion with EcoRI (A) or XbaI (B), the digested DNA was electrophoresed and blotted to a nylon membrane. DNA on the membranes was then hybridized with a Dig-labeled erm probe, as found in the C. perfringens-modified EZ-Tn5, and blots were developed as described in the Materials and Methods. Size of DNA fragments, in kilobases (kb), is shown at left.
Table 1.
Target gene of the EZ-Tn5 based transposon in C. perfringens strain 13.
Table 2.
Primers used in this study.
Figure 3.
Generation of a C. perfringens agrB mutant and complementing strains.
A) Southern blot analyses, as described in Fig. 2, using EcoRI-digested DNA from CPJV501 and a Dig-labeled probe that detected a single copy of the erm gene. Size of DNA fragments, in kilobases (kb) is shown at left. B) PCR was performed with DNA extracted from the indicated strain and the following pair of primers, agrBFwd and agrBRev in reactions containing DNA from strain 13 (S13), CPJV501 and CPJVp1; agrBFwd and argDR for CPJVp2 and agrF1 and agrD100R for CPJVp3. DNA ladders (100 bp or 1 kb) were included in the first and last lane of the gel. Asterisks show the expected PCR product when the primers amplified the Tn5-disprupted agrB gene. C) Genes cloned in the E. coli-C. perfringens shuttle plasmid pJIR750 to complement the agrB transposon mutant. As shown, P1 encodes the agrB gene alone, P2 the agrB and agrD genes and P3 encodes two-genes (CPE1562 and CPE1563) upstream the agrB gene (CPE1561) and agrB and agrD.
Figure 4.
The C. perfringens agrB locus regulates PFO production.
A and B) Hemoglobin (Hb) release assay. Culture supernatants obtained, at the indicated time point, from strain 13 (S13), S13 pfoA-null mutant (S13ΔpfoA), CPJV501 (ΔagrB), CPJVp1 (ΔagrB/P1), CPJVp2 (ΔagrB/P2) or CPJVp3 (ΔagrB/P3), were incubated (1∶1) with a 1% suspension of horse red blood cells for 30 min at 37°C. Non-inoculated TGY or 0.1% saponin (Saponin) was included as negative or positive control, respectively. PFO-induced Hb release was detected by obtaining the absorbance at 570 (A570). C) For each time point, the OD600 of the cultures is shown. For all panels, error bars represent the standard error of the mean calculated using data from three independent experiments.
Figure 5.
Early transcription of pfoA and plc genes is regulated by the C. perfringens agr locus.
Total RNA was extracted from a 2 h TGY culture of the wt strain 13 (S13), CPJV501 (ΔagrB), CPJVp1, CPJVp2 or CPJVp3. Quantitative RT-PCR was then performed with 20 ng of each RNA and primers that amplified the (A) pfoA gen (pfoAF1 and pfoAR1) or the (B) plc gene (cpaF and cpaR). Average CT values were normalized to the polC gene and the fold differences were calculated using the comparative CT method (2−ΔΔCT) [44]. Values below each bar indicate the calculated fold change relative to the wt strain 13. Panels shown are representative of three independent experiments.
Figure 6.
A C. perfringens secreted factor(s) regulates PFO production.
A and B) Physical complementation of the ΔagrB mutant by co-culture with a ΔpfoA mutant of strain 13 or CN3685. C. perfringens strain 13 (S13), CPJV501 (ΔagrB), S13 pfoA-null mutant (S13ΔpfoA), CPJV501 and S13 pfoA-null mutant (ΔagrB/S13ΔpfoA) or CPJV501 and CN3685 pfoA-null mutant (ΔagrB/CN3685ΔpfoA) were inoculated in TGY and incubated at 37°C for the indicated time. Culture supernatants obtained, at the indicated time point, were incubated (1∶1) with a 1% suspension of horse red blood cells for 30 min at 37°C. Non-inoculated TGY or 0.1% saponin was included as negative or positive control, respectively (not shown). PFO-induced Hb release was detected by obtaining the absorbance at 570 nm (A570). C) The physical complementation shown in panels A and B requires a secreted factor to regulate PFO production. Strain 13 (S13), CPJV501 (ΔagrB) or S13 pfoA-null mutant (ΔpfoA) was inoculated in 100 mm tissue culture dishes containing 25 ml of TGY. Another 100 mm tissue culture dish containing a transwell filter device (0.4 µm pore size) received 25 ml of TGY. Then, the S13 pfoA-null mutant was inoculated into the top chamber and CPJV501 was inoculated into the bottom chamber of the dish (bottom ΔagrB/Top S13ΔpfoA) and incubated for the indicated time. Culture supernatants obtained at the indicated time points were incubated (1∶1) with a 1% suspension of horse red blood cells for 30 min at 37°C. PFO-induced Hb release was detected by obtaining the absorbance at 570 nm (A570). For all panels, error bars represent the standard error of the mean calculated using data from three independent experiments.
Figure 7.
The C. perfringens agrB locus regulates CPA production.
A) ELISA analyses. Culture supernatants obtained, at the indicated time point, from strain 13 (S13), CPJV501 (ΔagrB), CPJVp1 (ΔagrB/P1), CPJVp2 (ΔagrB/P2), CPJVp3 (ΔagrB/P3) or purified CPA was used to coat a 96-well microplate overnight at 4°C. The wells were incubated with a mouse monoclonal anti-CPA antibody followed by a HRP-conjugated anti-mouse antibody. The bound antibody was detected with a TMB substrate solution and the color reaction stopped with sulphuric acid (0.18 M). A450 was determined using an ELISA reader. Error bars represent the standard error of the mean calculated using data from three independent experiments. B) Western blot showing the agr locus regulates production of CPA. Strain 13 (S13), CPJV501 (ΔagrB) or CPJVp3 (ΔagrB/P3) was inoculated in TGY and incubated at 37°C for 4 h. Bacteria were then pelleted by centrifugation, resuspended in lysis buffer and sonicated. Equal amount (25 µl) of bacterial lysates was run in a 12% SDS-PAGE, transferred to nitrocellulose membrane and western blotted with a monoclonal anti-CPA antibody. As a control, 25 µl of CPA-containing concentrated supernatant proteins was added to the gel. The expected molecular weight in kDa of CPA is shown at the left. Shown is a representative figure of three independent experiments.
Figure 8.
Organization and RT-PCR analysis of the agr operon.
A) RT-PCR reactions were performed with 50 ng of RNA extracted from an overnight TGY culture of the wt strain 13. RT-PCR reactions included (+) or not (−) retrotranscriptase (RT). The following pair of primers were used to detect mRNA transcripts from every two-adjacent ORF's, agr104L and agr103R (L4-R3, which should generate a 321 bp PCR product), agr103L and agr102R (L3-R2, which should generate a 315 bp PCR product), agr102L and agr101R (L2-R1, which should generate a 420 bp PCR product) or agr101L and agrDR (B–D, which should generate a 520 bp PCR product). A 100-bp DNA ladder is shown at left. B) Schematic representation of the agr locus showing primers used for RT-PCR reactions.