antibiotic susceptible a baumannii atcc 17978 strain background  (ATCC)

Journal: mBio

Article Title: Hfq orchestrates a robust RNA-RNA interaction network in Acinetobacter baumannii

doi: 10.1128/mbio.03231-25

Figure Lengend Snippet: Aar regulates expression of CarO and RsuA. ( A ) Circos plot of RIL-seq chimeras containing Aar. Circle represents the chromosome and three plasmids of AB5075-UW, and numbers indicate genome position (in Megabases). Aar location is indicated in black text, and gray text indicates RNA targets also identified by Hi-GRIL-seq . Links between Aar and other positions indicate chimeras detected in exponential phase (EP, blue) and stationary phase (SP, orange). ( B ) RIL-seq data for carO region. RIL-seq IP panel indicates read depth detected in a representative stationary phase IP sample. RIL-seq ctrl panel indicates read depth detected in a representative stationary phase control sample. Total RNA panel indicates read depth in a representative total RNAseq sample. Aar chimeras panel indicates position of chimeras detected containing Aar as RNA2. Yellow bars indicate annotations; the internal black arrows indicate the direction of transcription. For IP, control, and Total RNA tracks, only reads mapping to the forward strand are shown. ( C ) IntaRNA prediction of the interaction between carO and Aar. The carO start codon is indicated in green text. Locations of the Aar-M1 and carO -M1C mutant alleles are indicated. Numbers indicate the nucleotide position relative to the start codon of carO (above) and first nucleotide of Aar (below). The Aar “seed” sequence (CUCC) previously identified by Hamrock and colleagues is underlined. ( D ) β-galactosidase activity (in Miller Units) of AB5075 WT cells or its ∆ aar derivative containing a translational rsuA::lacZ reporter integrated at the Tn 7 attachment site in the presence of an empty vector (pEV) or Aar expression vector (pAar-FL). The β-galactosidase assay was repeated independently at least twice with biological triplicate cultures. Data from a single representative experiment are plotted as the mean activity with error bars representing one SD of the mean. Significance was assessed by a two-tailed t -test comparing the activity to that of WT cells with empty vector. Asterisks indicate significant differences with P -value ≤ 0.05 (*), P -value ≤ 0.001 (***). ( E–G ) Western blot analyses to detect CarO-V in cells grown to early stationary phase (6 h, OD 600 ≈ 2.0) in the presence or absence of the Aar plasmid and/or inducer. In panels E and F , cells encode an allele of carO with a C-terminal VSV-G epitope sequence at the chromosomal carO locus in wild-type AB5075 (WT) or an aar deletion mutant background (∆ aar ). In panel G , cells encode the carO M1C -V allele in the ∆ aar background. In panel H , cells encode an allele of carO with a C-terminal VSV-G epitope sequence in otherwise wild-type A. baumannii ATCC 17978. For Western blotting, whole-cell lysates prepared from cells of the indicated strains/plasmids were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by Western blot with antibodies recognizing the VSV-G epitope (VSV-G) or the RNA polymerase Alpha subunit (RpoA). Markers in gray text indicate the position of protein size standards (in kDa). The predicted molecular weight of CarO-V is 26 kDa, and RpoA is 38 kDa. Western blot experiments were completed with biological triplicate cultures and repeated independently at least twice. Data from a single experiment are shown. In panels E and G , samples in lanes with a dash (–) were collected from cells harboring an empty vector (pMJG598). Where indicated in panels F and H , cultures included 50 ng/mL anhydrotetracycline (aTc) as an inducer. pEV, empty vector pMJG598, pAar-FL, pMJG598 harboring Aar under control of its native promoter, pAar, pMJG598 with Aar under control of the P tetA promoter, pAar-M1, pMJG598 with Aar-M1 allele under control of the P tetA promoter.

Article Snippet: In the antibiotic-susceptible A. baumannii ATCC 17978 strain background, hfq is not essential.

Techniques: Expressing, Control, Mutagenesis, Sequencing, Activity Assay, Plasmid Preparation, Two Tailed Test, Western Blot, SDS Page, Molecular Weight