Determination of plasmid copy number.

The plasmid copy number (PCN) of pLPV plasmids in A. baumannii ATCC 19606^T and E. coli DH5α was determined by real-time quantitative PCR (RT-qPCR), as previously described (22). All of the following experimental procedures were applied also to determine the PCN of pVRL1, employed as a reference plasmid in which the PCN has previously been determined (22). pLPV2Z was chosen as a representative pLPV plasmid for PCN determination because it was comparable in size to pVRL1 (7,545 bp versus 7,276 bp, respectively). Three primer pairs for the amplification of the Gm resistance gene (aacC1) and of the d-1-deoxyxylulose 5-phosphate synthase gene (dxs) of A. baumannii ATCC 19606^T (locus tag HMPREF0010_02600) or E. coli DH5α (EcoCyc database accession number G6237) were used (Table 1). The aacC1 gene is present in single copy in the pLPV and pVRL1 vectors, while it is absent in A. baumannii ATCC 19606^T and E. coli DH5α chromosomes, and dxs is a single-copy gene in both A. baumannii ATCC 19606^T and E. coli DH5α. Therefore, RT-qPCR quantification of aacC1 and dxs in samples containing both plasmid and A. baumannii ATCC 19606^T or E. coli DH5α chromosomes, relative to samples containing known amounts of plasmid or A. baumannii ATCC 19606^T or E. coli DH5α chromosomal DNA alone, makes it possible to extrapolate the PCN of pLPV vectors in these bacteria. RT-qPCR was performed using an AriaMX real-time PCR system (Agilent) with software (version 1.1). Reactions were performed in a 20-μl total volume, containing 1× iTaq Universal SYBR Green Supermix (Bio-Rad), 0.4 μM each 10 μM concentrated primer, and 2 μl of 2-fold-diluted template DNA (final DNA amount ranging from 0.05 ng to 50 ng per sample). Separate reaction mixtures were prepared for detection of chromosomal or plasmid-specific amplicons for each template DNA concentration. The thermal cycling protocol was as follows: initial denaturation of 4 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/extension for 45 s at 60°C. Cycle threshold (C_T) values were determined after automatic fine-tuning of the baseline and manual adjustment of the fluorescence threshold. Standard curves (R² ≥ 0.99) from five independent samples containing A. baumannii ATCC 19606^T or E. coli DH5α chromosomal DNA (chromosome) or containing plasmid alone were generated, placing the log value of the amount of template DNA (determined according to dilution) on the x axis and the average C_T value on the y axis. Standard curves were used to extrapolate the copy number of pLPV vectors in samples containing both chromosomal and plasmid DNA.