Use of a Bacteriophage Lysin to Identify a Novel Target for Antimicrobial Development
Figure 2
Identification and analysis of 2-epimerase in B. anthracis.
(A) sps loci of the B. cereus lineage. Islands of variable sps genes are connected by gray regions and denoted by different colors. Conserved flanking sequences are shown. Red shaded loci (not in Ames) are cell wall-biosynthetic genes similar to that encoded by Ames. Inverted arrows are repeat elements. Susceptibility to PlyG lysis and GFP-PlyGBD surface binding are shown. For PlyG lysis, the “+++” designation is based on an assay [8] for zones of complete bacteriolysis on agar plates overlaid with the indicated organism and treated with a 10 µl drop of PBS containing 1 µg of PlyG; “++” indicates a slight reduction in activity compared to complete bacteriolysis, and “-“ indicates an absence of bacteriolysis. For PlyGBD binding, designations are based on exposure times needed to clearly visualize binding of GFP-PlyGBD to target organisms by fluorescence microscopy; “+++” indicates a <5 second exposure, “+” indicates a 15–30 second exposure, and “-“ indicates no fluorescence is observed. Abbreviations: w/c, whole-cell binding; p/s, polar/septal binding. (B) Genetic representation of 2-epimerase double mutant, RS1205. (C) Growth of RS1205 (with indicated IPTG concentrations) compared to the parental wild-type strain ΔSterne. Mean averages are shown (n = 3) with standard deviations. (D) Morphological analysis of RS1205 after five hours of growth without IPTG. Phase contrast images and corresponding fluorescence fields are shown for GFP-PlyGBD-labeled RS1205 (5 second exposure) and B. anthracis ΔSterne (30 second exposure). For Deltavision images, NPS (red) was labeled with rhodamine-PlyGBD, division septa (green) were labeled with vancomycin BODIPY FL, and DNA (blue) was labeled with DAPI. TEM images are shown with scale bars (500 nm) and arrows denote some division septa. (E) Phase contrast microscopic images of RS1205 grown for 12 hours with and without IPTG (5 µM).