The diversity and applications of microbial iron metabolism and iron-containing proteins

This collection reflects research that will advance the understanding of hemoproteins, iron-sulfur cluster proteins and other iron-containing proteins, proteins involved in homeostasis of iron/heme and their regulation, or ecological impacts of iron/heme on interacting hosts and microbial communities.

Iron is essential to virtually all living organisms but can be toxic when present in excess; as a result, to maintain intracellular iron homeostasis, a variety of highly effective and tightly regulatory systems have evolved^1,2. Although a trace amount of iron may exist in the labile form, the element is mainly found in metalloproteins in one of three forms: in hemes, in iron-sulfur clusters or non-heme iron proteins³. In the cell, these iron-containing proteins perform a myriad of functions in diverse biological processes, such as oxygen transport, electron transfer, respiration, photosynthesis, oxidative stress response, signal sensing and transduction, and catalysis³. Given the importance of iron homeostasis and iron-dependent proteins, they represent novel and promising targets for therapeutics⁴.

Our Communications Biology collection of “Microbial iron metabolism: hemes, homeostasis and iron proteins” contains 12 original research articles at the time of writing this editorial. Two papers report molecular mechanisms by which two-component systems (TCS) regulate siderophore synthesis in Gram-negative bacteria. Siderophores are small, chemically diverse iron chelators with extremely high affinity and siderophore-dependent iron acquisition is the most efficient strategy for microorganisms to obtain iron from the low-iron environments⁵. On one hand, the canonical TCS BfmRS of Pseudomonas aeruginosa, which has been established to be critical regulator for biofilm formation and quorum sensing, was found to additionally regulate a number of genes that encode siderophore biosynthesis enzymes in response to high osmolarity⁶. On the other hand, Xie et al. discovered a novel response regulator SsoR, whose sensor kinase remains to be identified, to be constitutively active independent of phosphorylation because it is locked into the activating form in Shewanella oneidensis⁷. Importantly, both BfmRS and SsoR appear to be conserved in many bacteria, suggesting that the mechanisms unraveled may be widespread. A transcriptomic view on the metabolic response to iron availability in a pathogen, Pseudomonas aeruginosa is shown in a report from Leinweber et al.⁸ The twist here is the use of a competitor (Burkholderia cenocepacia) supernatant to expose the connection of microbial communication to the response to nutrient availability. The metabolic response to low iron is drastically different from that in a high iron environment when competitor molecules around, suggesting that competition is has a large influence on the bacterial response to iron limitation. For example, transcripts of the siderophore pyoverdine are more abundant in the presence of B. cenocepacia supernatant, while those of the siderophore pyochelin are less abundant—opposed to a higher abundance without supernatant.

Given that heme can serve as the primary iron source for many bacteria, particularly pathogens, heme consumption and degradation play an important role in iron homeostasis⁹. Among hemoproteins, those with heme(s) covalently attached are called cytochrome (cyt) c and the heme attachment process is called cyt c biosynthesis or maturation⁹. Yeasmin et al. carried out a comprehensive biochemical analysis of the cyt c biosynthesis system CcsBA from Campylobacterota (formerly ε-Proteobacteria), such as Helicobacter pylori and Campylobacter jejuni, and provided evidence to support that mechanisms of heme interaction are conserved during heme trafficking¹⁰. On the heme degradation side, Ran et al. identified a heme oxygenase in Cyanobacteria that breaks down heme(s) within cyt c, releasing biliverdin IXα and iron¹¹. Interestingly, while microorganisms generally exploit heme degradation as an iron source, biliverdin IXα produced by this heme oxygenase is more important for the physiology of cyanobacteria because it is essential for synthesis of phycobiliprotein, which is vital for growth of cyanobacterial cells during chlorosis and regreening through its nitrogen-storage and light-harvesting functions, respectively.

In addition to the biological relevance of heme cofactors, their intense color can be used as a bio-reporter. Kupke et al. developed a heme-binding reporter based on TNFα that can be used in a simple assay to study intramembrane-cleaving proteases (I-CLiPs¹²). I-CLiP-mediated proteolysis induces a color change between white and green that reflects on the activity of the protease. They used the assay based on the heme-binding reporter to study the bacterial intramembrane-cleaving zinc metalloprotease RseP inside cells. These findings expand the methodological repertoire for detecting protease activity inside cells and may open new routes to heme-based reporters or biosensing.

A research article by Gadar et al. reports a new strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections caused by carbapenem-resistant Acinetobacter baumannii¹³. They discovered that kaempferol, which compromises biofilm formation but not planktonic growth when used alone, promotes killing of colistin at sub-inhibitory concentrations by disrupting iron homeostasis. Importantly, this strategy works with clinical strains of A. baumannii and Escherichia coli.

Fe-S clusters are ubiquitous cofactors that are essential for many biological processes. The most abundant species in proteins are the rhombic [2Fe-2S] and cubic [4Fe-4S] forms. Although Fe-S clusters are relatively simple in structure and composition, their synthesis and assembly into apoproteins is a highly complex and coordinated process in living cells. In both bacteria and eukaryotes, different biogenesis machines have been discovered that support the maturation of Fe-S proteins according to uniform biosynthetic principles.

Three different systems have been identified for the biogenesis of Fe-S proteins in bacteria: the ISC assembly¹⁴ and SUF¹⁵ systems, for the maturation of housekeeping Fe-S proteins under normal and oxidative stress conditions, and the NIF system, for the specific maturation of nitrogenase in diazotrophic bacteria¹⁶. The SUF pathway is the sole machinery for Fe-S cluster biogenesis in archaea, cyanobacteria and many Gram-positive, thermophilic, and pathogenic bacteria¹⁷. The maturation of cytosolic and nuclear Fe-S proteins in eukaryotes is mediated by the cytosolic iron-sulfur cluster assembly (CIA) system¹⁸. The maturation of bacterial Fe–S proteins has been intensely studied, while the mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the CIA pathway is far less understood. Vasquez et al. report cryo-EM structures of a key component of the CIA targeting complex (CTC), the HEAT-repeat protein Met18 from Saccharomyces cerevisiae that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster¹⁹. Depending on the presence of Cia2, Met18 adopts different oligomeric states, which are responsible for recognizing client proteins of different sizes and shapes.

Iron-sulfur enzymes also play a crucial role in biological nitrogen fixation. The reduction of atmospheric nitrogen to ammonia is performed by nitrogenases, which are amongst the most complex representatives of iron-sulfur enzymes. Nitrogenases are divided into three different classes and differ in the composition of their active sites: molybdenum (Mo), vanadium (V) and iron (Fe) only forms.

The biochemical characteristics of two [4Fe-4S] cluster-containing desulfidases (TudS), one from an uncultivated Gammaproteobacterium and one from Pseudomonas putida, are reported in an exciting paper by Fuchs et al.²⁰ TudS is shown to be involved in recycling and detoxifying tRNA-derived nucleosides. The data presented clearly show that 4-thiouridine-5’-monophosphate is the preferred substrate of TudS, whose homologs are widely distributed. Interestingly, the desulfuration involves a [4Fe-5S] cluster intermediate.

Payne et al. investigate the synthesis of the metallocofactors of the molybdenum (Mo) nitrogenase, which is the most abundant and best-characterized form, from Methanococcus maripaludis²¹. Methanogens inhabit sulfide-rich or iron-rich environments that promote the formation of insoluble sulfides, which lead to low availability of metals or sulfur species. This nitrogenase can synthesize the nitrogenase cofactor from pyrite as the source of iron and sulfur, suggesting that iron-rich environments are most likely to be the origin of nitrogen fixation on early Earth. This study highlights the importance of the elements iron and sulfur in the origin of life.

Plants depend on nitrogen-fixing bacteria to provide essential nutrients that plants need for optimal growth and productivity. Our soils have limited quantities of nitrogen sources, which are not sufficient for providing crops with all the nitrogen they need. Modern agriculture relies on synthetic fertilizers to make up that nitrogen gap. The overuse of synthetic fertilizers and pesticides in agriculture leads to higher emissions of nitrous oxide, which is a greenhouse gas contributing to global warming. Therefore, a long-term goal of plant biotechnology is to engineer crops to express functional nitrogenase to reduce the need for synthetic fertilizers. However, the complexity of nitrogenase biosynthesis makes this a very challenging task. Baysal et al. generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH) together with NifM to assist in NifH polypeptide folding²². NifH from Hydrogenobacter thermophilus carries a [4Fe-4S] cluster in its active form and performs electron transfer to the MoFe protein nitrogenase component (NifDK), an essential step in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co).

It was shown that NifH revealed a low [4Fe-4S] cluster occupancy, which limited the enzyme activity. This highlights how essential [Fe-S] cluster biogenesis and transport are to enable the full functionality of enzymes.

Another addition to the collection deals with bacterial metals in general in a methodological context: The in vivo monitoring of e.g., urogenital microbiota via magnetic resonance imaging as presented by Donnelly et al. is highly dependent on the association of bacteria to metals²³. Especially the iron-to-manganese content is important, with Lactobacillus containing mainly manganese, while most other bacterial genera contain more iron than manganese. The specific differences in the metal content result in different MRI relaxation rates, which can be appointed with high accuracy to specific strains.

The wide range of contributions in this collection, including both basic and applied science, shows the importance and fascinating biochemistry and biophysics of iron in the microbial world and how it can be used to combat or detect pathogens.