Fig 6. Loss of MlaA results in a reduction in gonococcal colony size that is exacerbated in the presence of the antimicrobial peptide polymyxin B.
(A) Supernatants from mid-logarithmic cultures of WT and ΔmlaA bacteria were separated by low-speed centrifugation and filtration, treated with DNAseI, and precipitated with a pyrogallol red-molybdate-methanol procedure. Precipitated supernatants and whole cell lysates were standardized by the OD600 of the source culture, separated by SDS-PAGE, and probed with indicated antisera. (B) WT FA1090, isogenic knockout ΔmlaA, complementation strain ΔmlaA/Plac::mlaA, and PldA overexpression strain ΔmlaA/Plac::pldA were cultured aerobically in liquid medium for 3 h, back diluted to an OD600 of 0.1, cultured 2 h longer, serially diluted, and spotted onto GCB without (left column) or with (right column) polymyxin B (800 U/mL) and either without (top row) or with (bottom row) 0.5 mM IPTG. Dilution spots from each condition were imaged with a Bio-Rad ImageDoc system. (C) CFUs for permissive and restrictive conditions with or without IPTG were counted and relative viability was calculated. Experiment was performed on three separate occasions (mean ± SEM on graph; *p < 0.05), and typical plate images are presented. (D) Representative micrographs from 10−4 dilution taken with a Zeiss AxioObserver.D1 microscope at 10× magnification 0.25 Phase Contrast 1 of WT (Row 1), ΔmlaA (Row 2), and ΔmlaA/Plac::mlaA (Row 3). MlaA expression was induced by inclusion of 0.1 mM IPTG in the solid medium for the complementation strain. (E) Images of 10−4 dilution were also taken at 2.5× magnification 0.06 Phase Contrast 1 and colony diameters were measured with ImageJ software. Colonies were measured for each of two independent experiments for the − polymyxin B condition (WT, n = 548; ΔmlaA, n = 755; ΔmlaA/Plac::mlaA, n = 664) and for the + polymyxin B condition (WT, n = 836; ΔmlaA, n = 1197; ΔmlaA/Plac::mlaA, n = 1121; mean ± SEM on graphs; *p < 0.05). (F) Rapidly growing liquid cultures of WT, ΔmlaA, ΔmlaA/Plac::mlaA, and ΔmlaA/Plac::pldA incubated in the presence or absence of polymyxin B were lysed and treated with proteinase K to isolate LOS. Subsequently, LOS was separated by SDS-PAGE and visualized by silver staining. IPTG was added to ΔmlaA/Plac::mlaA and ΔmlaA/Plac::pldA cultures to 0.5 mM as indicated. DF, dilution factor; OD600, optical density at 600 nm; IPTG, isopropyl β-D-thiogalactopyranoside; LOS, lipooligosaccharide; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.