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. 2020 Sep 3;9:e56571. doi: 10.7554/eLife.56571

Figure 6. Δmla with obgE* is synthetically sensitive to chemical modulation of stringent response or fatty acid biosynthesis.

(A) Normalized growth of cultures treated with 100 µg/mL serine hydroxamate which induces stringent response. Graphs represent normalized OD600 readings of treated vs untreated. Dashed line at 1.0 indicates no difference in growth of treated culture. Values < 1.0 mean growth was decreased after treatment. Bars are colored by genotype with individual circles representing biological replicates. The mla and obgE alleles are indicated below for ease of comparison. Lettering above denotes significantly different clusters as determined by a one-way ANOVA. (B) Normalized growth of cultures treated with 100 µg/mL cerulenin which inhibits fatty acid biosynthesis. Graphs are displayed identically as panel (A).

Figure 6.

Figure 6—figure supplement 1. Multiple sequence alignment of obgE across A. baumannii.

Figure 6—figure supplement 1.

Portion displayed is from amino acid positions 210 to 269, with position 258 annotated by an arrow. Asterisks represent conservation across all samples. Top 10 rows are strains utilized during this study. Any strains derived from the UW WT are denoted. Bottom 14 rows are from published A. baumannii genomes. I258 is boxed in red.
Figure 6—figure supplement 2. Quantification of (p)ppGpp levels after serine hydroxamate treatment at 100 µg/mL.

Figure 6—figure supplement 2.

Cultures were treated identically to that of Figure 6A except with the addition of 32P. Cultures were normalized by OD600 and equivalent OD units were harvested for each sample. Nucleotides were extracted and separated by TLC as described in the methods. Due to an overall lower OD600, UW ΔmlaF strains were loaded equivalently but less than other genotypes tested. (A) Densitometry was normalized by comparing treated versus untreated for both pppGpp and ppGpp and is biological triplicates. (B) Representative TLC image.
Figure 6—figure supplement 3. Increased stringent response inhibits de novo GPL biosynthesis.

Figure 6—figure supplement 3.

(A) Schematic detailing serine hydroxamate (SHX) treatment. Cultures were grown for 2 hr and pulsed with 10 µCi/mL. This labeled culture was immediately split in half, and half was treated with 1 mg/mL SHX. Following an hour of growth, cells were pelleted and total GPLs extracted via Bligh-Dyer. (B) Isolated GPLs were quantified via LSC. Counts of the treated culture were normalized to that of untreated. A value = 1 indicates no change (dashed line) in GPL content and a value <1 indicates a decrease in GPLs under SHX treatment. Individual circles represent biological replicates.