Adaptation of Dinoroseobacter shibae to oxidative stress and the specific role of RirA.

Dinoroseobacter shibae living in the photic zone of marine ecosystems is frequently exposed to oxygen that forms highly reactive species. Here, we analysed the adaptation of D. shibae to different kinds of oxidative stress using a GeLC-MS/MS approach. D. shibae was grown in artificial seawater medium in the dark with succinate as sole carbon source and exposed to hydrogen peroxide, paraquat or diamide. We quantified 2580 D. shibae proteins. 75 proteins changed significantly in response to peroxide stress, while 220 and 207 proteins were differently regulated by superoxide stress and thiol stress. As expected, proteins like thioredoxin and peroxiredoxin were among these proteins. In addition, proteins involved in bacteriochlophyll biosynthesis were repressed under disulfide and superoxide stress but not under peroxide stress. In contrast, proteins associated with iron transport accumulated in response to peroxide and superoxide stress. Interestingly, the iron-responsive regulator RirA in D. shibae was downregulated by all stressors. A rirA deletion mutant showed an improved adaptation to peroxide stress suggesting that RirA dependent proteins are associated with oxidative stress resistance. Altogether, 139 proteins were upregulated in the mutant strain. Among them are proteins associated with protection and repair of DNA and proteins (e. g. ClpB, Hsp20, RecA, and a thioredoxin like protein). Strikingly, most of the proteins involved in iron metabolism such as iron binding proteins and transporters were not part of the upregulated proteins. In fact, rirA deficient cells were lacking a peroxide dependent induction of these proteins that may also contribute to a higher cell viability under these conditions.

RirA of Dinoroseobacter shibae senses iron via a [3Fe-4S]1+ cluster co-ordinated by three cysteine residues.

In the marine bacterium, Dinoroseobacter shibae the transcription factor rhizobial iron regulator A (RirA) is involved in the adaptation to iron-limited growth conditions. In vitro iron and sulfide content determinations in combination with UV/Vis and electron paramagnetic resonance (EPR) spectroscopic analyses using anaerobically purified, recombinant RirA protein suggested a [3Fe-4S]1+ cluster as a cofactor. In vivo Mössbauer spectroscopy also corroborated the presence of a [3Fe-4S]1+ cluster in RirA. Moreover, the cluster was found to be redox stable. Three out of four highly conserved cysteine residues of RirA (Cys 91, Cys 99, Cys 105) were found essential for the [3Fe-4S]1+ cluster coordination. The dimeric structure of the RirA protein was independent of the presence of the [3Fe-4S]1+ cluster. Electro mobility shift assays demonstrated the essential role of an intact [3Fe-4S]1+ cluster for promoter binding by RirA. The DNA binding site was identified by DNase I footprinting. Mutagenesis studies in combination with DNA binding assays confirmed the promoter binding site as 3'-TTAAN10AATT-5'. This work describes a novel mechanism for the direct sensing of cellular iron levels in bacteria by an iron-responsive transcriptional regulator using the integrity of a redox-inactive [3Fe-4S]1+ cluster, and further contributes to the general understanding of iron regulation in marine bacteria.

Functional definition of the two effector binding sites, the oligomerization and DNA binding domains of the Bacillus subtilis LysR-type transcriptional regulator AlsR.

The LysR-type transcriptional regulator (LTTR) AlsR from Bacillus subtilis activates the transcription of the alsSD operon encoding enzymes for acetoin formation in response to the presence of acetate. The structural basis for effector binding, oligomerization, DNA binding, higher ordered complex formation, DNA bending and transcriptional control by B. subtilis AlsR was functionally characterized. The binding of two molecules of acetate per molecule AlsR was determined. Acetate-dependent transcription complex formation was observed. A structural model of AlsR was used to identify the amino acid residues V98, S100, H147 of the binding site 1, which were experimentally verified. The second binding site formed by T193, V194, A196, T201 and L202 mediated high acetate responsive induction. Residues L124, E225 Q74, I79 and R111 contributed to dimerization of AlsR. A22, Q29, P30, S33, K37, L39, E46, R50 and R53 of the winged helix-turn-helix motif were important for promoter recognition. The DNA binding domain alone dimerized and effectively bound the promoter. The LTTR promoter elements RBS and ABS had to be localized on the same site of the DNA. Higher ordered complex formation resulted in bending of promoter DNA and transcriptional activation.