Heme coordination and nitric oxide binding by Dnr regulators are essential for adaptation of Dinoroseobacter shibae to anaerobiosis

The adaptation strategies and regulatory networks behind the adaptation of D. shibae DFL12T to nitrosative stress under anaerobic starvation was investigated. Furthermore, the role of the oxygen sensing regulator FnrL and the heme coordinating regulators DnrD and DnrF were determined under nitrosative stress conditions. Therefore transcriptional analyses were performed using regulator knock out strains. Dinoroseobacter shibae DFL12T (DSM 16493T) and corresponding fnrL, dnrD and dnrF depletion strains (DS001 (DfnrL), DS002 (DdnrD) and DS004 (DdnrF)) were grown in artificial saltwater minimal medium (SWM) supplemented with 16.9 mM succinate in baffled flasks shaking at 200 rpm for aerobic growth and for anaerobic cultivation 25 mM pyruvate was added and incubation was performed in serum flasks with rubber stoppers shaking a 100 rpm. For nitrosative stress conditions 50 nM of NO saturated water was added. D. shibae wild type and mutant strains were grown under aerobic conditions up to the mid exponential growth phase (OD578 nM 0.5) and shifted to anaerobiosis to mimicke the physiologic conditions in the marine habitat. The samples were taken before (as reference) NO injection and after 30 minutes of nitrosative stress. Three biological replicas were analyzed. Comparison: Identification of genes induced or repressed in the Dinoroseobacter shibae DSM 16493T Crp/ Fnr regulator mutant strains and wild type strain the under nitrosative stress conditions.