Transformation and mobilization of cloning vectors in Acinetobacter spp

Abstract

, and RK2-derived plasmids were introduced into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413 by transformation and conjugal mobilization. The transformation frequencies of BD413 were 4.2 x 106 to 6.3 x 106 transformants per ,ug of DNA per 109 recipient cells. Conjugal mobilization frequencies were 1.1 x 10-1 to 8.5 x 10-1 per recipient. An improved method for the transformation of A. calcoaceticus BD413 is reported.R300B-, R1162-, and RSF1010-based vectors have been used for gene cloning in Pseudomonas (2)(3)(4)(5)(18)(19)(20) and Methylophilus (23) spp. Plasmid pRK290 is a 20-kilobase (kb) broad-host-range RK2 derivative that can be mobilized into a number of gram-negative organisms, including Acinetobacter spp. (9). A previous report suggested that transformation might be used to introduce plasmid cloning vehicles into Acinetobacter strains (10). We examined plasmid transformation in Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413 by using a variety of broadhost-range vectors and transformation protocols, and we have optimized conditions for plasmid transformation. We examined conjugal mobilization as an alternative to transformation for the introduction of cloning vectors into Acinetobacter spp.Acinetobacter sp. strain HO1-N and A. calcoaceticus BD413 were previously described (8,12,21). Rifampicinresistant mutants of each strain were isolated after nitrosoguanidine mutagenesis (1). Escherichia coli HB101, HB1O1(pRK290), and HB101(pRK2013) were obtained from Donald R. Helinski (9, 11). Plasmids R300B and pTB107, an R300B derivative carrying a 1.5-kb kanamycin resistance determinant, were obtained from Peter T. Barth (5,6,14,23). Plasmids RSF1010, pKT230, pKT231, and pKT248 were obtained from K. N. Timmis (2-4).L broth (pH 7.2) consisted of 1% tryptone, 0.5% yeast extract, and 0.5% NaCl. Solid medium contained 1.6% agar. Antibiotics were added to L agar at the following final concentrations: rifampin, 100 ,ug/ml; kanamycin, 50 ,ug/ml; tetracycline, 15 ,ug/ml; streptomycin, 15 ,ug/ml; sulfadiazine, 250 ,ug/ml; and chloramphenicol, 50 ,ug/ml. Recipient Acinetobacter cell cultures were grown overnight in shaken, 50-ml L-broth cultures at 30°C. Cultures were diluted to approximately 107 cells per ml with L broth and grown for 2 to 6 h at 30°C, and samples of the cultures were used according to the specified transformation protocols.Plasmid DNA was isolated by an alkaline sodium dodecyl sulfate extraction procedure (17) or by the procedure of Clewell and Helinski (7), with 0.5% Triton X-100 substituted for Brij 58 and deoxycholate. Plasmids were purified by CsCl-ethidium bromide density gradient ultracentrifugation.The transformation procedures examined were those of * Corresponding author. Mandel and Higa (16), Lederberg and Cohen (15), Kushner (13), and Eichenlaub and Steinbach (10). Transformations were carried out for 1 h and were terminated by the addition of DNase to 5 ,ug/ml. Transformants and exconjugants carrying R300B, pTB107, and pRK290 were selected on L-agar...

Occurrence, Transfer and Mobilization in Epilithic Strains of Acinetobacter of Mercury-resistance Plasmids Capable of Transformation

“…The observation of highest transformation frequencies on media containing higher concentrations of organic nutrients (Fig. 3 c) was consistent with reports that rapid growth of recipient cells promotes transformation (Cruze et al, 1979;Singer et al, 1986). Transformation of A .…”

Cloning and expression of Acinetobacter calcoaceticus catechol 1,2-dioxygenase structural gene catA in Escherichia coli

“…In accord with the observations of Singer et al (24), no evidence of an A. calcoaceticus restriction system was observed when the transformation frequency with donor DNA purified from E. coli was compared with that with DNA purified from A. calcoaceticus. Transformation frequencies (per microgram of DNA) were lower by 1,000-fold for EcoRI-cut ADP1 chromosomal donor DNA than for EcoRI-cut pIB1362 donor DNA.…”

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

“…These IncQbased plasmids were mobilized by pRK2013 into E. tarda, P. aeruginosa, and V. anguillarum strains. While we were inter- (28) and other Pseudomonas species (12). There are several advantages of using the plasmids described above for studies of infectivity.…”