Molecular underpinnings and biogeochemical consequences of enhanced diatom growth in a warming Southern Ocean.

The Southern Ocean (SO) harbors some of the most intense phytoplankton blooms on Earth. Changes in temperature and iron availability are expected to alter the intensity of SO phytoplankton blooms, but little is known about how these changes will influence community composition and downstream biogeochemical processes. We performed light-saturated experimental manipulations on surface ocean microbial communities from McMurdo Sound in the Ross Sea to examine the effects of increased iron availability (+2 nM) and warming (+3 and +6 °C) on nutrient uptake, as well as the growth and transcriptional responses of two dominant diatoms, Fragilariopsis and Pseudo-nitzschia We found that community nutrient uptake and primary productivity were elevated under both warming conditions without iron addition (relative to ambient -0.5 °C). This effect was greater than additive under concurrent iron addition and warming. Pseudo-nitzschia became more abundant under warming without added iron (especially at 6 °C), while Fragilariopsis only became more abundant under warming in the iron-added treatments. We attribute the apparent advantage Pseudo-nitzschia shows under warming to up-regulation of iron-conserving photosynthetic processes, utilization of iron-economic nitrogen assimilation mechanisms, and increased iron uptake and storage. These data identify important molecular and physiological differences between dominant diatom groups and add to the growing body of evidence for Pseudo-nitzschia's increasingly important role in warming SO ecosystems. This study also suggests that temperature-driven shifts in SO phytoplankton assemblages may increase utilization of the vast pool of excess nutrients in iron-limited SO surface waters and thereby influence global nutrient distribution and carbon cycling.

Diversity and Evolution of Iron Uptake Pathways in Marine Cyanobacteria from the Perspective of the Coastal Strain Synechococcus sp. Strain PCC 7002.

Marine cyanobacteria contribute to approximately half of the ocean primary production, and their biomass is limited by low iron (Fe) bioavailability in many regions of the open seas. The mechanisms by which marine cyanobacteria overcome Fe limitation remain unclear. In this study, multiple Fe uptake pathways have been identified in a coastal strain of Synechococcus sp. strain PCC 7002. A total of 49 mutants were obtained by gene knockout methods, and 10 mutants were found to have significantly decreased growth rates compared to the wild type (WT). The genes related to active Fe transport pathways such as TonB-dependent transporters and the synthesis and secretion of siderophores are found to be essential for the adaptation of Fe limitation in Synechococcus sp. PCC 7002. By comparing the Fe uptake pathways of this coastal strain with other open-ocean cyanobacterial strains, it can be concluded that the Fe uptake strategies from different cyanobacteria have a strong relationship with the Fe bioavailability in their habitats. The evolution and adaptation of cyanobacterial iron acquisition strategies with the change of iron environments from ancient oceans to modern oceans are discussed. This study provides new insights into the diversified strategies of marine cyanobacteria in different habitats from temporal and spatial scales. IMPORTANCE Iron (Fe) is an important limiting factor of marine primary productivity. Cyanobacteria, the oldest photosynthetic oxygen-evolving organisms on the earth, play crucial roles in marine primary productivity, especially in the oligotrophic ocean. How they overcome Fe limitation during the long-term evolution process has not been fully revealed. Fe uptake mechanisms of cyanobacteria have been partially studied in freshwater cyanobacteria but are largely unknown in marine cyanobacterial species. In this paper, the characteristics of Fe uptake mechanisms in a coastal model cyanobacterium, Synechococcus sp. PCC 7002, were studied. Furthermore, the relationship between Fe uptake strategies and Fe environments of cyanobacterial habitats has been revealed from temporal and spatial scales, which provides a good case for marine microorganisms adapting to changes in the marine environment.

Utilization of delactosed whey permeate for the synthesis of ethyl acetate with Kluyveromyces marxianus.

Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soil Pseudomonas species.

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis-anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.

Proteomic analysis reveals Renibacterium salmoninarum grown under iron-limited conditions induces iron uptake mechanisms and overproduction of the 57-kDa protein.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.

Effect of the Interaction between Elevated Carbon Dioxide and Iron Limitation on Proteomic Profiling of Soybean.

Elevated atmospheric CO2 (eCO2) and iron (Fe) availability are important factors affecting plant growth that may impact the proteomic profile of crop plants. In this study, soybean plants treated under Fe-limited (0.5 mM) and Fe-sufficient (20 mM) conditions were grown at ambient (400 µmol mol-1) and eCO2 (800 µmol mol-1) in hydroponic solutions. Elevated CO2 increased biomass from 2.14 to 3.14 g plant-1 and from 1.18 to 2.91 g plant-1 under Fe-sufficient and Fe-limited conditions, respectively, but did not affect leaf photosynthesis. Sugar concentration increased from 10.92 to 26.17 µmol g FW-1 in roots of Fe-sufficient plants and from 8.75 to 19.89 µmol g FW-1 of Fe-limited plants after exposure to eCO2. In leaves, sugar concentration increased from 33.62 to 52.22 µmol g FW-1 and from 34.80 to 46.70 µmol g FW-1 in Fe-sufficient and Fe-limited conditions, respectively, under eCO2. However, Fe-limitation decreases photosynthesis and biomass. Pathway enrichment analysis showed that cell wall organization, glutathione metabolism, photosynthesis, stress-related proteins, and biosynthesis of secondary compounds changed in root tissues to cope with Fe-stress. Moreover, under eCO2, at sufficient or limited Fe supply, it was shown an increase in the abundance of proteins involved in glycolysis, starch and sucrose metabolism, biosynthesis of plant hormones gibberellins, and decreased levels of protein biosynthesis. Our results revealed that proteins and metabolic pathways related to Fe-limitation changed the effects of eCO2 and negatively impacted soybean production.

Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

Identification of the iron-limitation stimulon in Staphylococcus lugdunensis.

During the infectious process, pathogens such as Staphylococcus lugdunensis have to cope with the condition of host-induced iron-limitation. Using the RNAseq approach, we performed the first global transcriptomic analysis of S. lugdunensis cells incubated in the absence and presence of iron chelator. One hundred and seventy-five genes were identified as members of the iron-limitation stimulon (127 up- and 48 downregulated). Six gene clusters known or likely required for the acquisition of iron have been identified. Among them, a novel Energy-Coupling Factor type transporter (ECF), homologous to the lhaSTA operon, has been found into a 13-gene putative operon and strongly overexpressed under iron-limitation condition. Moreover, the transcription of genes involved in resistance to oxidative stress (including catalase), virulence, transcriptional regulation, and hemin detoxification were also modified. These data provide some answers on the cellular response to the iron-limitation stress that is important for the opportunistic behavior of this pathogen.

Different iron storage strategies among bloom-forming diatoms.

Diatoms are prominent eukaryotic phytoplankton despite being limited by the micronutrient iron in vast expanses of the ocean. As iron inputs are often sporadic, diatoms have evolved mechanisms such as the ability to store iron that enable them to bloom when iron is resupplied and then persist when low iron levels are reinstated. Two iron storage mechanisms have been previously described: the protein ferritin and vacuolar storage. To investigate the ecological role of these mechanisms among diatoms, iron addition and removal incubations were conducted using natural phytoplankton communities from varying iron environments. We show that among the predominant diatoms, Pseudo-nitzschia were favored by iron removal and displayed unique ferritin expression consistent with a long-term storage function. Meanwhile, Chaetoceros and Thalassiosira gene expression aligned with vacuolar storage mechanisms. Pseudo-nitzschia also showed exceptionally high iron storage under steady-state high and low iron conditions, as well as following iron resupply to iron-limited cells. We propose that bloom-forming diatoms use different iron storage mechanisms and that ferritin utilization may provide an advantage in areas of prolonged iron limitation with pulsed iron inputs. As iron distributions and availability change, this speculated ferritin-linked advantage may result in shifts in diatom community composition that can alter marine ecosystems and biogeochemical cycles.

Phenotypic and proteomic approaches of the response to iron-limited condition in Staphylococcus lugdunensis.

BACKGROUND: Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2'-dipyridyl (DIP). RESULTS: No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. CONCLUSIONS: This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

Photosynthetic adaptation to light availability shapes the ecological success of bloom-forming cyanobacterium Pseudanabaena to iron limitation.

The poorly understood filamentous cyanobacterium Pseudanabaena is commonly epiphytic on Microcystis colonies and their abundances are often highly correlated during blooms. The response and adaptation of Microcystis to iron limitation have been extensively studied, but the strategies Pseudanabaena uses to respond to iron limitation are largely unknown. Here, physiological responses to iron limitation were compared between one Pseudanabaena and two Microcystis strains grown under different light intensities. The results showed that low-intensity light exacerbated, but high-intensity light alleviated, the negative effect of iron limitation on Pseudanabaena growth relative to two Microcystis strains. It was found that robust light-harvesting and photosynthetic efficiency allowed adaptation of Pseudanabaena to low light availability relative to two Microcystis strains only during iron sufficiency. The results also indicated that a larger investment in the photosynthetic antenna probably contributed to light/iron co-limitation of Pseudanabaena relative to two Microcystis strains under both light and iron limitation. Furthermore, the lower antenna pigments/chlorophyll a ratio and photosynthetic efficiency, and higher nonphotochemical quenching and saturation irradiance provided Pseudanabaena photoadaptation and photoprotection advantages over the two Microcystis strains under the high-light condition. The lower investment in antenna pigments of Pseudanabaena than the two Microcystis strains under high-light intensity is likely an efficient strategy for both saving iron quotas and decreasing photosensitivity. Therefore, when compared with Microcystis, the high plasticity of antenna pigments, along with the excellent photoadaptation and photoprotection ability of Pseudanabaena, probably ensures its ecological success under iron limitation when light is sufficient.

Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis.

Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe2+ efflux transporter from Listeria monocytogenes, as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron (efeUOB), ferric citrate (fecCDEF), and petrobactin (fpbNOPQ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin (dhbACEBF) and turns on bacillibactin (feuABC) and hydroxamate siderophore (fhuBCGD) uptake systems to scavenge iron from the environment and flavodoxins (ykuNOP) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response (fsrA, fbpAB, and fbpC) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.

Regulation of Sulfur Homeostasis in Mycorrhizal Maize Plants Grown in a Fe-Limited Environment.

Sulfur is an essential macronutrient for growth of higher plants. The entry of the sulfate anion into the plant, its importation into the plastids for assimilation, its long-distance transport through the vasculature, and its storage in the vacuoles require specific sulfate transporter proteins. In this study, mycorrhizal and non-mycorrhizal maize plants were grown for 60 days in an S-deprived substrate, whilst iron was provided to the plants in the sparingly soluble form of FePO4. On day 60, sulfate was provided to the plants. The gene expression patterns of a number of sulfate transporters as well as sulfate assimilation enzymes were studied in leaves and roots of maize plants, both before as well as after sulfate supply. Prolonged sulfur deprivation resulted in a more or less uniform response of the genes' expressions in the roots of non-mycorrhizal and mycorrhizal plants. This was not the case neither in the roots and leaves after the supply of sulfur, nor in the leaves of the plants during the S-deprived period of time. It is concluded that mycorrhizal symbiosis modified plant demands for reduced sulfur, regulating accordingly the uptake, distribution, and assimilation of the sulfate anion.

Proteomic responses to ocean acidification of the marine diazotroph Trichodesmium under iron-replete and iron-limited conditions.

Growth and dinitrogen (N2) fixation of the globally important diazotrophic cyanobacteria Trichodesmium are often limited by iron (Fe) availability in surface seawaters. To systematically examine the combined effects of Fe limitation and ocean acidification (OA), T. erythraeum strain IMS101 was acclimated to both Fe-replete and Fe-limited concentrations under ambient and acidified conditions. Proteomic analysis showed that OA affected a wider range of proteins under Fe-limited conditions compared to Fe-replete conditions. OA also led to an intensification of Fe deficiency in key cellular processes (e.g., photosystem I and chlorophyll a synthesis) in already Fe-limited T. erythraeum. This is a result of reallocating Fe from these processes to Fe-rich nitrogenase to compensate for the suppressed N2 fixation. To alleviate the Fe shortage, the diazotroph adopts a series of Fe-based economic strategies (e.g., upregulating Fe acquisition systems for organically complexed Fe and particulate Fe, replacing ferredoxin by flavodoxin, and using alternative electron flow pathways to produce ATP). This was more pronounced under Fe-limited-OA conditions than under Fe limitation only. Consequently, OA resulted in a further decrease of N2- and carbon-fixation rates in Fe-limited T. erythraeum. In contrast, Fe-replete T. erythraeum induced photosystem I (PSI) expression to potentially enhance the PSI cyclic flow for ATP production to meet the higher demand for energy to cope with the stress caused by OA. Our study provides mechanistic insight into the holistic response of the globally important N2-fixing marine cyanobacteria Trichodesmium to acidified and Fe-limited conditions of future oceans.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

Elemental Stoichiometry and Photophysiology Regulation of Synechococcus sp. PCC7002 Under Increasing Severity of Chronic Iron Limitation.

Iron (Fe) is an essential cofactor for many metabolic enzymes of photoautotrophs. Although Fe limits phytoplankton productivity in broad areas of the ocean, phytoplankton have adapted their metabolism and growth to survive in these conditions. Using the euryhaline cyanobacterium Synechococcus sp. PCC7002, we investigated the physiological responses to long-term acclimation to four levels of Fe availability representative of the contemporary ocean (36.7, 3.83, 0.47 and 0.047 pM Fe'). With increasing severity of Fe limitation, Synechococcus sp. cells gradually decreased their volume and growth while increasing their energy allocation into organic carbon and nitrogen cellular pools. Furthermore, the total cellular content of pigments decreased. Additionally, with increasing severity of Fe limitation, intertwined responses of PSII functional cross-section (σPSII), re-oxidation time of the plastoquinone primary acceptor QA (τ) and non-photochemical quenching revealed a shift in the photophysiological response between mild to strong Fe limitation compared with severe limitation. Under mild and strong Fe limitation, there was a decrease in linear electron transport accompanied by progressive loss of state transitions. Under severe Fe limitation, state transitions seemed to be largely supplanted by alternative electron pathways. In addition, mechanisms to dissipate energy excess and minimize oxidative stress associated with high irradiances increased with increasing severity of Fe limitation. Overall, our results establish the sequence of physiological strategies adopted by the cells under increasing severity of chronic Fe limitation, within a range of Fe concentrations relevant to modern ocean biogeochemistry.

Proteomic response of Streptococcus pneumoniae to iron limitation.

Iron is an essential trace element and involved in various key metabolic pathways in bacterial lifestyle. Within the human host, iron is extremely limited. Hence, the ability of bacteria to acquire iron from the environment is critical for a successful infection. Streptococcus pneumoniae (the pneumococcus) is a human pathobiont colonizing symptomless the human respiratory tract, but can also cause various local and invasive infections. To survive and proliferate pneumococci have therefore to adapt their metabolism and virulence factor repertoire to different host compartments. In this study, the response of S. pneumoniae to iron limitation as infection-relevant condition was investigated on the proteome level. The iron limitation was induced by application of the iron chelator 2,2'-bipyridine (BIP) in two different media mimicking different physiological traits. Under these conditions, the influence of the initial iron concentration on pneumococcal protein expression in response to limited iron availability was analyzed. Interestingly, one major difference between these two iron limitation experiments is the regulation of proteins involved in pneumococcal pathogenesis. In iron-poor medium several proteins of this group were downregulated whereas these proteins are upregulated in iron-rich medium. However, iron limitation in both environments led to a strong upregulation of the iron uptake protein PiuA and the significant downregulation of the non-heme iron-containing ferritin Dpr. Based on the results, it is shown that the pneumococcal proteome response to iron limitation is strongly dependent on the initial iron concentration in the medium or the environment.

The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina.

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (-F6) isolated from iron-deficient culture contained Chl d-bound PSI-IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).

The long goodbye: the rise and fall of flavodoxin during plant evolution.

Ferredoxins are electron shuttles harbouring iron-sulfur clusters that connect multiple oxido-reductive pathways in organisms displaying different lifestyles. Some prokaryotes and algae express an isofunctional electron carrier, flavodoxin, which contains flavin mononucleotide as cofactor. Both proteins evolved in the anaerobic environment preceding the appearance of oxygenic photosynthesis. The advent of an oxygen-rich atmosphere proved detrimental to ferredoxin owing to iron limitation and oxidative damage to the iron-sulfur cluster, and many microorganisms induced flavodoxin expression to replace ferredoxin under stress conditions. Paradoxically, ferredoxin was maintained throughout the tree of life, whereas flavodoxin is absent from plants and animals. Of note is that flavodoxin expression in transgenic plants results in increased tolerance to multiple stresses and iron deficit, through mechanisms similar to those operating in microorganisms. Then, the question remains open as to why a trait that still confers plants such obvious adaptive benefits was not retained. We compare herein the properties of ferredoxin and flavodoxin, and their contrasting modes of expression in response to different environmental stimuli. Phylogenetic analyses suggest that the flavodoxin gene was already absent in the algal lineages immediately preceding land plants. Geographical distribution of phototrophs shows a bias against flavodoxin-containing organisms in iron-rich coastal/freshwater habitats. Based on these observations, we propose that plants evolved from freshwater macroalgae that already lacked flavodoxin because they thrived in an iron-rich habitat with no need to back up ferredoxin functions and therefore no selective pressure to keep the flavodoxin gene. Conversely, ferredoxin retention in the plant lineage is probably related to its higher efficiency as an electron carrier, compared with flavodoxin. Several lines of evidence supporting these contentions are presented and discussed.

Characterizing the flavodoxin landscape in Clostridioides difficile.

Clostridioides difficile infections have become a major challenge in medical facilities. The bacterium is capable of spore formation allowing the survival of antibiotic treatment. Therefore, research on the physiology of C. difficile is important for the development of alternative treatment strategies. In this study, we investigated eight putative flavodoxins of C. difficile 630. Flavodoxins are small electron transfer proteins of specifically low potential. The unusually high number of flavodoxins in C. difficile suggests that they are expressed under different conditions. We determined high transcription levels for several flavodoxins during the exponential growth phase, especially for floX. Since flavodoxins are capable of replacing ferredoxins under iron deficiency conditions in other bacteria, we also examined their expression in C. difficile under low iron and no iron levels. In particular, the amount of fldX increased with decreasing iron concentration and thus could possibly replace ferredoxins. Moreover, we demonstrated that fldX is increasingly expressed under different oxidative stress conditions and thus may play an important role in the oxidative stress response. While increased fldX expression was detectable at both RNA and protein level, CD2825 showed increased expression only at mRNA level under H2O2 stress with sufficient iron availability and may indicate hydroxyl radical-dependent transcription. Although the exact function of the individual flavodoxins in C. difficile needs to be further investigated, the present study shows that flavodoxins could play an important role in several physiological processes and under infection-relevant conditions. IMPORTANCE: The gram-positive, anaerobic, and spore-forming bacterium Clostridioides difficile has become a vast problem in human health care facilities. The antibiotic-associated infection with this intestinal pathogen causes serious and recurrent inflammation of the intestinal epithelium, in many cases with a severe course. To come up with novel targeted therapies against C. difficile infections, a more detailed knowledge on the pathogen's physiology is mandatory. Eight putative flavodoxins, an extraordinarily high copy number of this type of small electron transfer proteins, are annotated for C. difficile. Flavodoxins are known to be essential electron carriers in other bacteria, for instance, during infection-relevant conditions such as iron limitation and oxidative stress. This work is a first and comprehensive overview on characteristics and expression profiles of the putative flavodoxins in the pathogen C. difficile.