Figure 1.
Venn diagram depicting conservation of putative drug efflux transporters encoded within representative sequenced strains of Acinetobacter.
Each strain is represented by a colored oval, A. baumannii SDF (blue), AYE (red) and ATCC 17978 (yellow) and A. baylyi ADP1 (green). The number of efflux systems was determined using TransAAP. Numbers in brackets after strain names are the number of putative efflux systems encoded in that strain. Numbers in overlapping regions are the number of systems shared by those strains, as determined by comparative BlastP searches.
Table 1.
Bacterial strains, plasmids and primers.
Figure 2.
Growth characteristics of wild-type A. baylyi ADP1 or A. baylyi ADP1 single-step mutants over a time-course of 15 hours.
Saturated overnight cultures of A. baylyi ADP1 strains were inoculated in fresh MH media to an OD600 of 0.01 in a volume of 200 µl prior to incubation at 37°C with shaking. Growth assays were conducted in duplicate using three biological replicates. Standard deviation is shown.
Table 2.
Growth characteristics of wild-type and mutant A. baylyi strains.
Table 3.
MIC of compounds tested against A. baylyi ADPI strains.
Figure 3.
Efflux of ethidium bromide by A. baylyi ADP1 cells.
Wild-type and mutant strains were loaded with 15 µM ethidium bromide in the presence of the protonophore CCCP. Cells were energised using 0.4% glucose at the point indicated by an arrow to initiate efflux. Efflux was monitored fluorometrically using an excitation wavelength of 530 nm and emission wavelength of 610 nm. Assays were conducted in duplicate, and a representative experiment is shown.
Table 4.
Fold changes in expression of multidrug efflux systems in the chloramphenicol resistant mutant strains as determined by qRT-PCR.
Figure 4.
Bayesian tree depicting phylogenetic relationships of putative drug exporting RND superfamily transporters identified within the genomes of sequenced Acinetobacter spp.
(as determined using TransAAP). Also included are previously characterized transporters classified within the Hydrophobe/Amphiphile Efflux-1 family of the RND superfamily, as listed in the Transporter Classification Database (www.tcdb.org). The protein sequence of the A. baumannii ATCC 17978 RND efflux transporter from the Heavy Metal Efflux family, CzcA (A1S_2932), was used as an out-group. Branch lengths are proportional to phylogenetic distance and the confidence of each node is indicated by posterior probabilities. Proteins othologous to the major RND drug efflux systems encoded in Acinetobacter, AdeB, AdeJ and AdeG are indicated. Gray shaded boxes denote clades that include orthologs from both A. baumannii and A. baylyi.
Figure 5.
Stability of craA mRNA transcript in wild-type and Cm201 backgrounds.
Wild-type and Cm201 cells were grown to mid-logarithmic phase before transcription was arrested with the addition of 200 µg/ml rifampicin. Culture samples were harvested at time points T = 0, 5, 10 and 20, before RNA was extracted and cDNA was synthesised. qRT-PCR was conducted in duplicate using two biological replicates, and the abundance of the craA transcript relative to the rpoB transcript in transcriptionally arrested cells was determined. R2 values and standard deviation is shown.