Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Abstract

BackgroundHigh intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.ResultsOne third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.ConclusionSome R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.

The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5’ Leader Featuring the sRNA UpsM

“…R . sphaeroides strains were cultivated at 32°C in malate minimal-salt medium [50]. To grow the cells aerobically, cultures were either gassed with air in Meplat bottles to attain a concentration of 160 to 180 μM of dissolved oxygen or by continuous shaking of Erlenmeyer flasks containing 20% culture by volume at 140 rpm.…”

“…For R . sphaeroides RNA Illumina cDNA libraries were prepared by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/) as described before without prior RNA fragmentation or size fractionation [50]. Illumina cDNA libraries resulting from TEX treated R .…”

An RpoHI-Dependent Response Promotes Outgrowth after Extended Stationary Phase in the Alphaproteobacterium Rhodobacter sphaeroides

“…The R. sphaeroides 2.4.1 wild-type strain (DSM 158) and the mutants 2.4.1 ΔrpoHI, 2.4.1 ΔrpoHII, 2.4.1 ΔrpoHI rpoHII, and 2.4.1 ΔrpoHI(pRKrpoHI) (see Table S2 in the supplemental material) were grown at 32°C in minimal salt medium containing malate (0.022 M) as the carbon source (38). Low-aeration growth conditions were established by continuous shaking of Erlenmeyer flasks at 140 rpm with a culture volume of 80%, leading to a dissolved oxygen concentration of approximately 25 to 30 M in exponential phase with strong changes throughout growth, as measured using the GMH 3610 (Greisinger) digital oxygen measuring device (Fig.…”

“…The transcripts were not fragmented in order to obtain mainly sequencing reads of the 5= end of the transcripts. Libraries for Illumina sequencing of cDNA were constructed by Vertis Biotechnology AG, Germany, as described previously (38). The resulting cDNA libraries were sequenced on an Illumina HiSeq 2000 machine in single-read mode running 100 cycles.…”

“…The fluorescence measurements were performed as described elsewhere (38). RNA isolation and quantification.…”