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

Abstract

Background: High 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.

Results: One 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.

Conclusion: Some 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.

Figure 1

Correlation between oxic and anoxic RNAseq analysis. The scatter-plot represents a comparison of log₂ fold changes between the oxic and anoxic RNAseq data sets. (A) Colour is used for the regulated genes (log₂ > 1 or < -1, green and red spots, respectively) to indicate whether changes are of similar magnitude under both conditions (log₂ ratio difference between approaches < 1, green spots) or are biased towards one condition (log₂ ratio difference > 1, red spots). Non-regulated genes (log₂ < 1 and > -1) are shown as grey spots. (B) Genes were grouped into six clusters according to their expression pattern as described in Results. For a complete list of genes and information on their functions, see Additional file 1: Table S1.

Figure 2

Growth curves and ROS level measurements. Characterisation of wild type R. sphaeroides (black) and the 2.4.1∆oxyR mutant (grey) was performed in oxic conditions in the presence (A) or absence (B) of iron. The optical density at 660 nm (OD₆₆₀) was determined over time, and growth is indicated as continuous line. The intracellular levels of ROS (squares) are presented in arbitrary units. Both data sets represent the mean of at least three independent experiments, and the error bars indicate the standard deviation.

Figure 3

Determination of intracellular levels of ROS in wild type R. sphaeroides and the 2.4.1∆ oxyR mutant. Cultures were grown under normal iron (black) and iron-limiting (white) conditions in oxic and anoxic (-O₂) environments. ROS generated by the cells were analysed after reaction with 10 mM 2,7-DCFH-DA. Cells incubated with 250 μM Paraquat (PQ) served as a positive control. The autofluorescence of cells without dye was subtracted from the measured values. The fluorescence intensity was normalised to the optical densities of the samples. The resulting values are presented in arbitrary units. The data represent the mean of three independent experiments, and the error bars indicate the standard deviation.