FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae

Abstract

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

Heme and nitric oxide binding by the transcriptional regulator DnrF from the marine bacterium Dinoroseobacter shibae increases napD promoter affinity

“…2). This finding is in accordance with previous observations (28). This repression is mediated via the palindromic sequence 5Ј-TTGATN 4 ATCAA-3Ј, which is located 96.5 bp upstream of the napD translational start site (Fig.…”

“…1B). This NO-independent increase of nirS-lacZ expression is due to an anaerobic induction mediated by FnrL (28). Addition of 50 nM NO led to ␤-galactosidase activities of 16 Miller units after 15 min, 93 Miller units after 30 min, and 119 Miller units after 60 min.…”

“…RNA preparation following DNA array analysis was performed as described elsewhere (28,32). Generated data have been deposited in the NCBI Gene Expression Omnibus (47) and are accessible by Geo Series accession number GSE95560.…”

“…The marine bacterium Dinoroseobacter shibae DFL12 T (DSM 16493), as one representative of the highly abundant Roseobacter group of marine bacteria, possesses a fine-tuned hierarchical network for the adaptation to anaerobic conditions (28). One FnrL and three Dnr regulators (DnrD, DnrE, and DnrF) mediate the transition from aerobic to anaerobic conditions by activating denitrification genes in the presence of nitrate (28).…”

“…In a previous study, we determined the role of FnrL and the three Dnr regulators (DnrD, DnrE, and DnrF) for their response to anaerobiosis in the presence of nitrate (28). FnrL and DnrD were identified as key players for the regulation of the transition process.…”