FIG 4.
Induction of prophage λSo in biofilms is independent of hydrogen peroxide and superoxide. (A) Planktonic growth under aerobic conditions in LB medium containing 10 mM H2O2 of the S. oneidensis MR-1 wild type, the ΔoxyR deletion mutant, and mutant strains that harbor single amino acid substitution T104N or L197P in OxyR (OxyRT104N and OxyRL197P). As a reference, the growth curve of the wild type in the absence of H2O2 is also presented. Growth curves are derived from a representative experiment conducted in triplicate. Error bars represent standard deviations. (B) Expression, relative to the wild type, of katB (SO_1070), dps (SO_1158), ahpC (SO_0958), katG1 (SO_0725), and tonB (SO_3670) in the ΔoxyR and the OxyRT104N and OxyRL197P mutant strains, determined by quantitative real-time RT-PCR. Bars represent the mean values of two independent experiments, each normalized to the 16S rRNA and recA housekeeping genes. Standard deviations are displayed as error bars. (C) Left three lanes (+ H2O2), immunoblot analysis of phage λSo production in planktonic cells of the wild type and the OxyRT104N and OxyRL197P mutants. Cells were cultivated in LB medium until mid-exponential phase and subjected to 2 mM H2O2 for 2 h. Right three lanes (Biofilm), immunoblot analysis of phage λSo production in biofilm cells of the wild type and the two OxyR mutants. Cells were harvested from 24-h-old biofilms formed on glass beads under hydrodynamic conditions. Sample normalization was achieved by adjusting cell suspensions to the same OD600 and analysis of stained SDS-PAGE gels (see Fig. S2 in the supplemental material). Representative immunoblot patterns from at least two independent experiments are presented. (D) Immunoblot analysis of phage λSo production in wild-type biofilm cells harboring plasmid pBBR1-TT-Ptac-MSC5-sodB for constitutive overexpression of superoxide dismutase gene sodB. Cells were harvested from 24-h-old biofilms formed on glass beads under hydrodynamic conditions. Sample normalization was achieved by adjusting cell suspensions to the same OD600 and analysis of stained SDS-PAGE gels (see Fig. S2 in the supplemental material). Representative immunoblot patterns from at least two independent experiments are presented. (E) Immunoblot analysis of phage λSo production in wild-type cells harvested from biofilms formed under oxic conditions (+ O2) on glass beads (constant medium flow) and cells harvested from biofilms formed under anoxic conditions (− O2; N2 headspace) on glass beads (static conditions). Sample normalization was achieved by adjusting cell suspensions to the same OD600 and analysis of stained SDS-PAGE gels (see Fig. S2 in the supplemental material). Representative immunoblot patterns from at least two independent experiments are presented.