A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli

The suf and isc operons of Escherichia coli have been implicated in Fe-S cluster assembly. However, it has been unclear why E. coli has two systems for Fe-S cluster biosynthesis. We have examined the regulatory characteristics and mutant phenotypes of both operons to discern if the two operons have redundant functions or if their cellular roles are divergent. Both operons are similarly induced by hydrogen peroxide and the iron chelator 2,2'-dipyridyl, although by different mechanisms. Regulation of the isc operon is mediated by IscR, whereas the suf operon requires OxyR and IHF for the response to oxidative stress and Fur for induction by iron starvation. Simultaneous deletion of iscS and most suf genes is synthetically lethal. However, although the suf and isc operons have overlapping functions, they act as distinct complexes because the SufS desulphurase alone cannot substitute for the IscS enzyme. In addition, suf deletion mutants are more sensitive to iron starvation than isc mutants, and the activity of the Fe-S enzyme gluconate dehydratase is diminished in the suf mutant during iron starvation. These findings are consistent with the model that the isc operon encodes the housekeeping Fe-S cluster assembly system in E. coli, whereas the suf operon is specifically adapted to synthesize Fe-S clusters when iron or sulphur metabolism is disrupted by iron starvation or oxidative stress.     

Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen.     

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis

Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.     

Interchangeability and distinct properties of bacterial Fe-S cluster assembly systems: functional replacement of the isc and suf operons in Escherichia coli with the nifSU-like operon from Helicobacter pylori

The assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational maturation of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUF encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nifSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-component systems, and to determine how ISC, SUF and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUF systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

Purification and biophysical characterization of a new [2Fe-2S] ferredoxin from Azotobacter vinelandii, a putative [Fe-S] cluster assembly/repair protein.

During the purification of site-directed mutant variants of Azotobacter vinelandii ferredoxin I (FdI), a pink protein, which was not observed in native FdI preparations, appeared to associate specifically with variants that had mutations in ligands to FdI [Fe-S] clusters. That protein, which we designate FdIV, has now been purified. NH(2)-terminal sequence analysis revealed that the protein is the product of a previously described gene, herein designated fdxD, that is in the A. vinelandii iscSUA operon that encodes proteins involved in iron-sulfur cluster assembly or repair. An apoprotein molecular mass of 12,434.03 +/- 0.21 Da was determined by mass spectrometry consistent with the known gene sequence. The monomeric protein was shown to contain a single [2Fe-2S](2+/+) cluster by UV/visible, CD, and EPR spectroscopies with a reduction potential of -344 mV versus the standard hydrogen electrode. When overexpressed in Escherichia coli, recombinant FdIV holoprotein was successfully assembled. However, the polypeptide of the recombinant protein was modified in some way such that the apoprotein molecular mass increased by 52 Da. Antibodies raised against FdIV and EPR spectroscopy were used to examine the relative levels of FdIV and FdI in various A. vinelandii strains leading to the conclusion that FdIV levels appear to be specifically increased under conditions where another protein, NADPH:ferredoxin reductase is also up-regulated. In that case, the fpr gene is known to be activated in response to oxidative stress. This suggests that the fdxD gene and other genes in the iron-sulfur cluster assembly or repair operon might be similarly up-regulated in response to oxidative stress.