Construction of MAP deletion mutant by specialized transduction

If not stated otherwise, all protocols were performed according to Park and colleagues with slight modifications (BARDAROV et al., 2002, 1997; PARK et al., 2008).

2.6.1 Phage related protocols

The lytic mycobacteriophages, which were used in this thesis, are listed in table 3.

2.6.2 Determination of phage concentration

For the determination of mycobacteriophage concentration, M. smegmatis mc² 155 was grown to an OD600 of 3. Of each bacteria suspension, 100 µl were mixed with ten-fold serial dilutions of 50 µl phage lysate prepared in 450 µl mycobacteriophage (MP) buffer (10 mM Tris, pH 7.6, 100 mM NaCl, 10 mM MgSO4, 2 mM CaCl2). The samples were incubated at 30°C for 30 min. Then, 3 ml of the 55°C pre-warmed MB top agarose (0.5 g MB 7H9, 0.1 g NaCl, 0.75 g glucose, 0.5 g agarose in 100 ml ddH2O) were added to the cell-phage mixtures, mixed gently by vortexing, and poured onto Middlebrook phage (MBP) agar plates. Subsequently, the plates were incubated at 30°C for 5 days. The induced confluent lysis dilution was used to calculate the titer of the phage suspension.

2.6.3 Preparation of mycobacteriophage stocks from plate lysate

Ten aliquots of M. smegmatis grown in LB broth to an OD600 of 1.5 were infected with the induced confluent lysis dilution of mycobacteriophages as described above (see 2.6.2). As soon as confluent lysis was reached (3-5 days), the plates were incubated with 7 ml MP buffer at RT for 30 min. Subsequently, the top layer was scraped off with a sterile scalpel, transferred to a 50 ml reaction tube, and centrifuged at 41.800 x g and 4°C for 30 min. The supernatant containing the phages was filtered (0.45 µm) and stored at 4°C.

2.6.4 Isolation of phage DNA

The phage DNA was isolated in accordance to the protocol of SAMBROOK et al.

(1989). Five ml of a phage solution with a concentration of 10⁷ plaque forming units (pfu) per ml or higher were precipitated with 30 % [v/v] polyethylene glycol (PEG)/ NaCl (20 % [w/v] PEG 6.000, 2.5 M NaCl) in a 15 ml reaction tube at 4°C overnight. After centrifugation at 14.500 x g for 10 min, the phage pellet was resuspended in 100 µl TE buffer with 30 µl DNase (1 mg/ ml) and RNase A (10 mg/ ml, Boehringer Ingelheim Pharma GmbH, Ingelheim, Germany), and incubated at 37°C for 1 h to reduce a contamination with bacterial nucleic acids.

Following, a 0.5 volume of TE-equilibrated phenol (pH 8) was added and incubated at -80°C overnight. Then, the mixture was thawed, mixed with chloroform/ isoamyl alcohol (24:1 [v/v]) transferred in a 2 ml reaction tube and centrifuged at 10.000 x g and 4°C for 30 min for phase separation. The liquid phase was transferred in a new 2 ml reaction tube. The chloroform/ isoamyl alcohol extraction was performed and repeated twice. Finally, one-tenth volume of Na-Acetate (3 M, pH 5.2) and 2.5 volume of 96 % (v/v) ethanol were added for phage DNA precipitation. The samples were incubated on ice for 15 min and centrifuged at 10.000 x g and 4°C for 30 min. The DNA was spooled with a pipette tip, dried, and resuspended in DNase/ RNase free ddH2O. The phage DNA was stored at -20°C for further analysis.

2.6.5 Packaging of phage DNA in lambda phage heads

The packaging of phage DNA in lambda phage heads was performed with the Gigapack III Plus Packaging Extract from Agilent Technologies (Agilent Technologies, Life Sciences and Chemical Analysis, Waldbronn, Germany) in accordance to the manufacturer´s protocol.

2.6.6 Construction of the allelic exchange substrate cosmid for furA deletion

The up- and downstream elements were amplified by PCR using the MAPwt genome DNA as template. Oligonucleotides with AflII and XbaI restriction sites were used for the upstream (1022 bp) and oligonucleotides with XhoI and SpeI restriction sites were used for the downstream (1094 bp) element. The fragments were ligated in the TOPO vector and transformed in E. coli HB101. Positive transformants were picked and controlled by sequencing to check for the correct insert. Then, the plasmid was isolated, restricted with a suitable restriction enzyme as described above, purified by gel electrophoresis, and ligated one after another in the restricted, gel purified recombinant cosmid vector pYUB854. The new cosmid pMFur1510 was controlled for correct up- and downstream elements by sequencing and stored at -20°C for further analysis.

2.6.7 Construction of mycobacteriophage for furA deletion

For the construction of the new mycobacteriophage, a concatamer of purified phAE87 DNA was obtained by self-ligation. Further, both pYUB854 and phAE87 concatamers were restricted with PacI and ligated. The concatamer was packaged into λ phage heads using the Gigapack III Plus Packaging Extract and transduced in E. coli HB101 according to the manufacturer´s protocol. The cosmid integry of hygromycin resistant transformants was controlled by restriction digest and sequencing. The correct plasmid was isolated and electroporated in

M. smegmatis mc² 155. Aliquots of 100 µl electroporated cells were mixed with 200 µl of M. smegmatis mc² 155, grown in LB medium to an OD600 of 1, and 1.5 ml MB top agarose. These preparations were immediately incubated on MBP agar plates at 30°C for 5 days. Then, plaques were picked with a sterile Pasteur pipette and kept in MB buffer at 4°C overnight. Five µl of each phage solution were mixed with 200 µl M. smegmatis mc² 155, grown in LB medium to an OD600 of 1 and with 1.5 ml MB top agarose. The bacteria-phage mix was incubated in parallel on two plates at 37°C and 30°C to confirm the temperature-sensitivity of the new phage phAE151. The presence of the AES in the phAE151 was confirmed by sequencing the PCR fragments using the same oligonucleotide combinations as for the up- and downstream elements.

2.6.8 Specialized transduction of MAP

The specialized transduction was performed in accordance to PARK et al. (2008) with some modifications. MAP was grown to an OD600 of 1 in MB 7H9 containing OADC and Tween^® 80. The bacteria were singularized by vortexing with glass beads (2-3 mm diameter) for 15 min. Then, the culture was allowed to stand on ice for 30 min and the supernatant was transferred in a sterile 50 ml reaction tube. The bacteria were centrifuged at 7.200 x g at RT for 10 min and washed three times in 30 ml MP buffer. Afterwards, the pellet was resuspended in 5 ml MP buffer and kept on ice for 20 min to reduce clumps. Following, 1 ml of the supernatant were mixed with 1 ml of mycobacteriophage lysate and a MOI of 10:1 in a 12 ml tube and incubated for 4 h at 37°C. Subsequently, 2 ml MB 7H9 broth containing 5 % OADC were added and the samples were incubated at 100 rpm and 37°C for 24 h. Then, the culture was placed on ice for 30 min and 300 µl of the supernatant were plated on 37°C pre-warmed MB 7H10 agar plates containing OADC and hygromycin B (50 µg/ ml) as well as on one agar plate with hygromycin B (75 µg/ ml) only. The plates were incubated for 8 weeks at 37°C and hygromycin resistant clones were monitored for the successful deletion by PCR, qRT-PCR, and sequencing.