Tables listing genomic and trasncriptomic data, antibiotic resistance of all tested isolates, and oligonucleotide sequences used in this study.
(a) Findings from sequence analysis of mutated genes in the genomes of PMRLow and PMRHigh isolates. Mutations were identified using the MUMmer program. Additional PCR reactions using Pfu DNA polymerase were performed to confirm mutational positions. All position numbers are based on the genes of A. baumannii ATCC 17978. (b) Whole-genome sequence analyses of Lab-WT, PMRLow, and PMRHigh isolates using the PacBio sequencing technique. The genome sequences were downloaded from EzGenome and then their chromosomes were analyzed using the CLgenomics program. (c) Lists of deleted regions in the chromosomes of PMRLow and PMRHigh. The genome sequences were compared to the reference genome, A. baumannii ATCC 17978, and whole genomes were aligned using Snapgene. (d) Identification of antibiotic-resistance genes (ARGs) in Lab-WT, PMRLow, and PMRHigh strains. Totally 20 ARGs were detected using the CLC Genomics Workbench v.10.0.1 (QIAGEN, Germany). (e) Identification of insertion sequence (IS) elements in the Lab-WT, PMRLow, and PMRHigh. All parameters were set as default. IS elements were analyzed using the IS finder (https://isfinder.biotoul.fr/blast.php). (f) All information on the acquisition of RNA-seq data in this study. Total RNAs were extracted from exponentially grown Lab-WT, Lab-WT strain + polymyxin B (PMB), PMRHigh and PMRHigh strain + PMB. RPKM represents reads per kilobase of transcript per million mapped sequence reads. (g) List of genes that were up- or downregulated in PMRHigh, Lab-WT + PMB, and PMRHigh + PMB in comparison with control strains. Up- (fold change > 1.4, RPKM > 50) or downregulated (fold change < 0.7, RPKM > 50) genes were indicated. The intersection area and numbers are described in Figure 3—figure supplement 1. All locus tags are based on the genes of A. baumannii ATCC 17978. *TM = transmembrane region, ND = not detected. (h) Relative abundance of proteins in three conditions that increased outer membrane vesicles (OMVs): Lab-WT + PMB, PMRHigh + PMB, and PMRHigh strains compared to Lab-WT and PMRHigh. The quantity of protein in each spot was normalized to the total valid spot intensity. By comparing each gel image of proteins in strains under overproduction of OMVs, significantly changed spots were selected based on up- (fold change > 2, intensity > 1000) or down-expressed (fold change < 0.5, intensity > 1000) protein. The total number of final selected proteins for MALDI-TOF analysis was 43 spots. *ND = not detected. (i) Minimum inhibitory concentrations (MICs) of antibiotics used in this study. (j) Bacterial strains, plasmids, and primers used in this study. (k) Primers used in this study. Additional PCR reactions using Pfu DNA polymerase were performed for confirming mutational positions listed in (a) (18 genes).