FIG 1.

Bacillus anthracis Sterne variants with mutations in gneY- and gneZ-encoded UDP-GlcNAc 2-epimerase activities. (A) Schematic representation of the B. anthracis Sterne chromosome organization surrounding the gneY (bas5047) and gneZ (bas5117) genes and of variants with a gneY deletion (ΔgneY; YW15) and insertion of lacI and P_spac upstream of gneY either without (P_spac-gneY; YW29) or with (P_spac-gneY; gneZ::spc; YW6) replacement of gneZ. The plasmid pJK4 provides for the constitutive expression of lacI and is selected by growing cells in the presence of kanamycin (neo, neomycin). (B) B. anthracis strains Sterne(pJK4), YW29(pJK4), and YW6(pJK4) were spread on BHI agar plates with 20 μg ml⁻¹ kanamycin for plasmid selection and incubated at 37°C for 16 h either without (−IPTG) or with (+IPTG) 1 mM isopropyl-β-d-thiogalactoside supplement. (C) B. anthracis Sterne(pJK4), YW29(pJK4), and YW6(pJK4) were cultured overnight in BHI with 20 μg ml⁻¹ kanamycin and 1 mM IPTG. Bacteria were centrifuged, washed, and suspended in fresh medium either without (−IPTG) or with (+IPTG) 1 mM IPTG supplement and incubated with rotation at 37°C to monitor vegetative growth. At timed intervals, culture aliquots were withdrawn and absorbance at 600 nm was recorded (A₆₀₀). Data are representative of three independent experimental determinations.