Role of the irr protein in the regulation of iron metabolism in Rhodobacter sphaeroides

In Rhizobia the Irr protein is an important regulator for iron-dependent gene expression. We studied the role of the Irr homolog RSP_3179 in the photosynthetic alpha-proteobacterium Rhodobacter sphaeroides. While Irr had little effect on growth under iron-limiting or non-limiting conditions its deletion resulted in increased resistance to hydrogen peroxide and singlet oxygen. This correlates with an elevated expression of katE for catalase in the Irr mutant compared to the wild type under non-stress conditions. Transcriptome studies revealed that Irr affects the expression of genes for iron metabolism, but also has some influence on genes involved in stress response, citric acid cycle, oxidative phosphorylation, transport, and photosynthesis. Most genes showed higher expression levels in the wild type than in the mutant under normal growth conditions indicating an activator function of Irr. Irr was however not required to activate genes of the iron metabolism in response to iron limitation, which showed even stronger induction in the absence of Irr. This was also true for genes mbfA and ccpA, which were verified as direct targets for Irr. Our results suggest that in R. sphaeroides Irr diminishes the strong induction of genes for iron metabolism under iron starvation

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

Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5’ UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5’ UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5’ UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5’ UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae

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

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 alpha-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

Growth curves of the <i>R. sphaeroides</i> wild type (black) and the 2.4.1Δ<i>irr</i> mutant (gray) under normal iron (continuous line) and under iron limitation (dashed line) conditions are shown.

<p>The optical density at 660 nm (OD₆₆₀) of microaerobically grown <i>R. sphaeroides</i> cultures was determined over time. The data represent the mean of at least three independent experiments and error bars indicate standard error of the mean.</p

Relative expression of <i>katE</i> (RSP_2779) in <i>R. sphaeroides</i> wild type and 2.4.1Δ<i>irr</i> mutant.

<p>(A) Real-time RT-PCR was used to investigate the relative expression of <i>katE</i> in 2.4.1Δ<i>irr</i> mutant 0 min (light gray bar) and 20 min (dark gray bar) after exposure to 1 mM H₂O₂ compared to the wild type. (B) Relative <i>katE</i> expression 20 min of 1 mM H₂O₂ in the <i>R. sphaeroides</i> wild type (white bar) and the <i>irr</i> deletion mutant (black bar). Values were normalized to <i>rpoZ</i> and to the control at time point 0. The data represent the mean of three independent experiments and error bars indicate standard deviation. Levels of significance are indicated as follows: *<i>P</i>≤0.01; **<i>P</i>≤0.05.</p

Validation of microarray data by real-time RT-PCR.

<p>Relative gene expression (A) in 2.4.1Δ<i>irr</i> under normal iron conditions compared to the wild type under normal iron conditions and (B) in 2.4.1Δ<i>irr</i> under iron limitation compared to normal iron conditions (light gray bars) and in wild type under iron limitation compared to normal iron conditions (dark gray bars). Values were normalized to <i>rpoZ</i> and to the respective control treatment. The data represent the mean of at least three independent experiments and error bars indicate standard deviation. Numbers in parentheses show the fold change of the respective genes as determined by microarray analysis.</p

Determination of 5′ ends of <i>mbfA</i> (RSP_0850) (A) and <i>ccpA</i> (RSP_2395) (B) mRNA by 5′ rapid amplification of cDNA ends (RACE).

<p>Separation of 5′-RACE products <i>mbfA</i> and <i>ccpA</i> obtained from RNA extracts of the wild type strain under normal iron conditions. PCR products obtained after second PCR (nested) were separated on a 10% polyacrylamid gel and stained with ethidium bromide. Determined 5′ ends are indicated by an arrow. The putative translational start is indicated by an asterisk. The Irr-box (ICE, iron control element) is marked by a frame.</p

Selection of iron-responsive genes in <i>R. sphaeroides.</i>

a<p>Significant changes are in bold. Numbers in parentheses failed to meet the set <i>A</i> value criteria, while plain numbers show a lower fold change than ≥1.75 or ≤0.57. Selected genes that missed the cut-offs are included in this table to fully represent functional groups discussed.</p>b<p>Values are taken from Peuser and colleagues (2011).</p

Binding of purified Irr to the promoter of <i>mbfA</i> and <i>ccpA</i> as determined by Electrophoretic Mobility Shift Assays.

<p>(A) Binding of Irr to the promoter region of <i>mbfA</i> (180 bp). All reactions contain the same amount of ³²P end-labeled DNA fragment (3.08 fmol/lane) comprising the promoter sequence. Lanes 1–4 contain no Irr; lanes 3 and 4 contain 0.6 µg BSA; lanes 5 and 7 contain 0.1 µg Irr; lane 6 and 11–13 contain 0.6 µg Irr; lane 8 contains 0.2 µg Irr; lanes 9 and 14–16 contain 0.3 µg Irr; lane 10 contains 0.4 µg. Reactions contain 1 mM MnCl₂ as indicated. Lanes 14–16 contain non-labeled DNA fragment <i>mbfA</i> in excess amount as cold competitor. Lanes 12 and 13 contain radioactively labeled <i>sitA</i> DNA fragment (180 bp) as unspecific DNA. (B) Binding of Irr to the promoter region of <i>ccpA</i> (168 bp). All reactions contain the same amount of ³²P end-labeled DNA fragment (3.68 fmol/lane) comprising the promoter sequence. Lanes 1–3 contain no Irr; lane 3 contains 0.6 µg BSA; lane 4 contains 0.1 µg Irr; lane 5 contains 0.2 µg Irr; lane 6 contains 0.4 µg Irr; lane 7 contains 0.6 µg Irr. Reactions contain 1 mM MnCl₂ as indicated. All reactions contain 1 µg of salmon sperm DNA as unspecific competitor. The asterisks and arrows show the location of free and Irr-bound ³²P end-labeled DNA fragments, respectively.</p