FIG. 2.

H₂O₂ production (A) and biofilm formation (B) by ΔackA suppressor mutants. (A) H₂O₂ production was determined relative to the parent strain (IU1781; bar 1) in limited aeration (static) cultures as described in Materials and Methods, and standard errors from multiple independent determinations are indicated. Bars 2 to 4, constructed unencapsulated mutants; bars 5 and 6, mucoid large colony suppressors; bars 7 and 10, medium-size rough colony suppressors; bars 8, 9, 11, and 12, medium-sized rough colony suppressors in which the ΔackA and cps2E (ΔA) mutations were repaired to the ackA⁺ and cps2E⁺ alleles. Relevant genotypes are shown and are also listed in Table S1 of the supplemental material. Bar 2, strain IU1945; bar 3, IU3286; bar 4, IU3309; bar 5, IU3254; bar 6, IU3255; bar 7, IU3250; bar 8, IU3252; bar 9, IU3311; bar 10, IU3251; bar 11, IU3253; bar 12, IU3313. (B) Biofilms formed by overnight cultures in polystyrene tubes were stained with crystal violet and quantitated based on the A₅₅₀ as described in Materials and Methods. Panels from left: 1, parent encapsulated strain IU1781; 2, unencapsulated strain IU3250, a rough, medium-sized colony ΔackA suppressor containing the spxB (Trp339STOP) and cps2E (ΔA) mutations; 3, unencapsulated strain IU3252, medium-sized rough colony suppressor IU3250 repaired back to ackA⁺; 4, unencapsulated strain IU3309, an ackA⁺ spxB⁺ strain containing the constructed cps2E (ΔA) allele; 5, unencapsulated strain IU1945, an ackA⁺ spxB⁺ strain containing the large Δcps2A′-Δcps2H′ deletion. See Table S1 for full genotypes. A₅₅₀ values after crystal violet staining were normalized to the density (OD₆₂₀, ≈0.5 to 0.6) of the culture supernatants containing unattached cells. Averages are based on three or more independent determinations, and standard errors are indicated.