Bacterial Strains and Growth Conditions
Bacterial strains used in this study are listed in Table 1. E. coli strains were grown in Luria–Bertani (LB) medium (liquid or agar) overnight at 37°C.
Bdellovibrio bacteriovorus HD100-S1 was cultivated either in liquid cultures with E. coli prey or on double layer agar plates. In the latter case prey bacteria were resuspended within the top layer . Stationary phase prey bacteria (E. coli DSM 423 or E. coli JC3272) were harvested by centrifugation and dissolved in the same volume of Bdellovibrio buffer (3 mM ammonium acetate, 3 mM CaCl2, and 3 mM MgCl2, pH 7.5). For liquid cultures this suspension was directly inoculated with B. bacteriovorus HD100-S1 and shaken at 28°C until complete lysis of the prey bacteria (depending on the amount of predators 1–3 days).
Mutant B. bacteriovorus M1 is a spontaneous mutant which grows and replicates on heat killed prey bacteria. For this purpose, stationary phase prey bacteria were harvested by centrifugation, dissolved in the same volume of Bdellovibrio buffer and autoclaved for 20 min at 121°C.
The axenic B. bacteriovorus mutant M1.1 is a derivative of mutant M1 and was grown in standard bacteriological medium (PYE; liquid or agar ).
For synchronized cultivation of B. bacteriovorus, two cultivation steps were necessary. In the first step the prey suspension containing E. coli JC3272 was infected with B. bacteriovorus HD100-S1 from stock cultures and shaken at 28°C. After lysis cell debris and remaining prey bacteria were removed by centrifugation (10 min, 2,600×g at RT). 10 ml supernatant containing attack phase predators was added to 30 ml of freshly prepared prey bacteria to give a predator to prey ratio of approximately 1:3. Mixing was set as starting point of the synchronized cultivation, which was shaken at 28°C.
Bdellovibrio strains were stored at −80°C in either prey suspension or PYE supplemented with 30% glycerol.
Plasmid and Chromosomal DNA Preparations
Plasmids used in this work are listed in Table 1. Vector pSUP202 (Fig. 1) contains single restriction sites for inactivation of ampicillin resistance (ApR) gene, tetracycline resistance (TcR) gene, and chloramphenicol resistance (CmR) gene. The replicon is derived from pMB1 and contains the RP4 mob region (Fig. 1). The complete sequence of pSUP202 is available in the NCBI database (accession AY428809).
Plasmid preparations of B. bacteriovorus and E .coli were carried out using the Gene JetTM Plasmid Miniprep Kit (Fermentas, St. Leon-Rot, Germany).
Chromosomal DNA of B. bacteriovorus was prepared from purified predators cells by the Cetyltrimetylammoniumbromide (CTAB) extraction procedure . Briefly, cells were harvested by centrifugation and inoculated with sodiumdodecylsulfate (0.5% final concentration), RNase (170 μg/ml), and Proteinase K (100 μg/ml) for 1 h. Sodium chloride to a concentration of 0.8 M and 1% CTAB were added and the solution was incubated for 10 min at 65°C. An equal volume of chloroform was added and the mixture was centrifuged at 14,900×g for 10 min at RT. The upper phase was transferred to a clean tube and an equal volume of phenol/chloroform/isoamylalcohol (25/24/1, v/v/v, premixed) was added. The mixture was centrifuged at RT for 10 min at 14,900×g. The supernatant was transferred to a clean tube and DNA was precipitated by addition of 0.6 volume of isopropanol. The precipitate was washed with 70% ethanol, dried in a vacuum concentrator and redissolved in distilled water.
Amplification of DNA by Polymerase Chain Reaction
For polymerase chain reaction (PCR), 0.5 μl Taq polymerase (5 U/μL), 5 μl dNTPs (2.5 mM each), 3 μl magnesium chloride (50 mM), 5 μl reaction buffer, 5 μl forward and reverse primer (5 μM each), and 1 μl Template DNA (0.1–1.0 ng) in 50 μl reaction volume were used. All chemicals were obtained from Bioline (Luckenwalde, Germany). The PCRs were performed with an initial denaturation step of 95°C for 5 min and 35 cycles (denaturation at 95°C for 30 s, annealing for 30 s and elongation at 72°C). Amplified PCR fragments were purified for either cloning or sequencing using the MSB®Spin PCRapace Kit from Invitek (Berlin, Germany). Oligonucleotide primers were designed using the Accelrys (DS-) gene program 2.5 (2006, Accelrys, Inc., San Diego, USA) (Table 2). Oligonucleotides were supplied by Metabion (Martinsried, Germany).
Matings were performed with the donor strain E. coli S17.1λpir harboring the RP4 transfer region chromosomally integrated. 20 ml of B. bacteriovorus recipients from liquid cultures containing about 108
Bdellovibro bacteria were concentrated tenfold by centrifugation and resuspended in 2 ml Bdellovibrio buffer. Donors were grown to stationary phase and concentrated tenfold in the same buffer. 100 μl of each suspension were mixed and transferred onto nitrocellulose filters (0.22 μm pore size) which were placed on top of LB agar plates. Matings were incubated overnight at 28°C. The next day, the bacteria were resuspended in 2 ml Bdellovibrio buffer and spread on double layer agar plates with E. coli JC3272 [pBR29] under selective conditions (25 μg/ml streptomycin, 12.5 μg/ml chloramphenicol) to determine the transfer frequency. The cells were incubated at 28°C until plaques became visible in the lawn of prey cells.
In case of stability experiments the resuspended bacteria were added to a freshly prepared prey suspension of E. coli JC3272 (see Fig. 2). After lysis of this first culture aliquots of the predatory bacteria were used for inoculation of the following cultures: cultures with E. coli JC3272, cultures with E. coli JC3272 [pBR29] under selective conditions (25 μg/ml streptomycin, 12.5 μg/ml chloramphenicol, 12.5 μg/ml tetracycline) or E. coli JC3272 [pIV2] (100 μg/ml kanamycin). Complete lysis of prey cells in the liquid cultures takes place within 2 days and is visible by clearance of the prey culture and microscopic control (Nikon-Optiphot-2, phase-contrast 1000×).
Determination of Plasmid Copy Number Using Real-Time PCR
To estimate the copy number of the plasmid pSUP404.2 within B. bacteriovorus, the number of plasmids was compared to the number of chromosomal copies using Real-Time PCR. Total DNA was isolated from the strain B. bacteriovorus HD100-S1 containing the plasmid pSUP404.2 and analyzed with the ABI-7500 Real-Time PCR System (Applied Biosystems, Foster City, USA). Primers and probes were designed (Table 2) using the Primer Express software version 3.0 (Applied Biosystems). The NanoDrop Spectrophotometer ND-1000 (peQLab, Erlangen; Germany) was used to determine the DNA concentrations. Serial dilutions from 1.66 × 10−1 to 1.66 × 10−8 ng DNA (plasmid pSUP404.2) and 9.882 ng to 9.882 × 10−7 ng DNA (chromosome) were prepared for calibration. The number of plasmid molecules (size of 11,026 bp) contained in 1 μg was calculated to be 2.25 × 1010. In comparison 1 μg chromosomal DNA (3,782,950 bp) consists of approximately 2.41 × 108 molecules (, accession NC_005363).
Plasmid copy number was calculated by dividing the number of plasmids by the number of chromosomal molecules. All data were analyzed using the 7500 Software version 2.0.1 (2008, Applied Biosystems).
DNA Sequencing and Computer Analysis
DNA sequencing was done by primer walking. Sequencing of either the pSUP404.2 plasmid or the newly generated gfp-construct (pNR24) was done by QIAGEN sequencing services (Hilden, Germany). The obtained sequences were analyzed using the Lasergene programme “SeqMan” (DNASTAR, Inc., Madison, USA). The nucleotide sequence of pSUP404.2 was deposited at EMBL database (accession FN907923).
Fluorescent bacteria were diluted 1:2 with 0.8% agarose and spotted on glass slides. Bacteria were visualized with a Zeiss Axioskop microscope at a magnification of 1000× using a mercury lamp with a filter for blue excitation.