Figure 5. Programmed cell lysis and biofilm formation in fermenting cells are governed by the SrrAB two-component regulatory system.
Panel A; Fermentative biofilm formation is dependent upon SrrAB. Biofilm formation is displayed following aerobic or fermentative growth in the WT (JMB 1100) carrying pLL39 (pEV) or the ΔsrrAB (JMB 1467) strains carrying either pLL39 (pEV) or pLL39_srrAB (psrrAB). Panel B; A hemB mutant forms SrrAB-dependent biofilms aerobically. Biofilm formation following aerobic growth is displayed for the WT, ΔsrrAB, hemB::Tn (JMB 6037), and ΔsrrAB hemB::Tn (JMB 6039) strains. Panel C; Transcript levels corresponding to genes involved in programmed cell lysis and biofilm formation are altered in a ΔsrrAB strain. Biofilms of the WT and ΔsrrAB strains were cultured fermentatively, mRNA was extracted, and the abundances of the atlA, tarO, tarA, tarB, and tarH transcripts were quantified. Data were normalized to 16S rRNA levels, and thereafter, to levels observed in the WT. Panel D; The fermentative biofilm formation phenotypes associated with the ΔsrrAB and atlA::Tn mutations are not additive. Biofilm formation is displayed following fermentative growth for the WT, ΔsrrAB, atlA::Tn (JMB 6625), and ΔsrrAB atlA::Tn (JMB 6624) strains. Panel E; Autolysis of fermenting S. aureus is decreased in a strain lacking SrrAB. The WT, ΔsrrAB, and atlA::Tn strains were cultured fermentatively and autolysis was examined (pH of 5). Panel F; eDNA accumulation is decreased in a strain lacking SrrAB. Biofilms of the WT, ΔsrrAB, and atlA::Tn strains were cultured fermentatively and eDNA was quantified. The data were normalized to the viable cell count and thereafter to the levels in the WT. Panel G; atlA in multicopy partially suppresses the biofilm formation defect of the ΔsrrAB strain. Fermentative biofilm formation is displayed for the WT and ΔsrrAB strains carrying either patlAAM H263A or patlA. Panel H; Heat-killed cells of a ΔsrrAB strain are less amenable towards AtlA-dependent lysis. Murein-hydrolase activity for cell-wall associated proteins (CW-extracts) detached from a ΔatlA strain (KB 5000) carrying patlA and combined with fermentatively cultured and heat-killed WT or ΔsrrAB strains as substrates are displayed. Data presented represent the average value of eight wells (Panels A, B, D-G) or biological triplicates (Panel C and F). Data in Panels E and H represent the average value of technical duplicates from one set of autolysis assays or substrate preparations. The heat-killed substrates were prepared or autolysis assays were conducted on least three separate occasions and similar results were obtained. Error bars in all panels represent standard deviations. Error bars are displayed for all data, but might be too small to see on occasion. Statistical significance was calculated using a two-tail Student's t-test and p-values>0.05 were considered to be not significant while * indicates p-value of <0.05.
Figure 5—figure supplement 1. Biofilm formation of a ΔsrrAB strain is largely unaltered upon supplementing anaerobic biofilms with the alternate terminal electron acceptor nitrate.
