Iron Stress Response Research Articles

Utilizing nutrient type compounds as anti-bacterial compounds: arginine and cysteine inhibit Salmonella survival in egg white.

Because of growing levels of antibiotic resistance, new methods to combat bacteria are needed. We hypothesized that because bacteria evolved to survive in specific environments, the addition of compounds, including nutrient type compounds, to an environment, might result in a modification of that environment that will disrupt bacterial growth or in maladaptive bacterial behavior, i.e., gene expression. As a proof of concept, we focused on the egg white environment and the pathogen Salmonella. Despite egg white's antibacterial nature, Salmonella is able to survive and grow in egg white, and this ability of Salmonella leads to infection of chicks and humans. Here, the 20L-amino-acids were screened for their ability to affect the growth of Salmonella in egg white. L-arginine and L-cysteine were found to reduce growth in egg white in physiologically relevant concentrations. To determine the mechanism behind L-arginine inhibition TnSeq was utilized. TnSeq identified many Salmonella genes required for survival in egg white including genes required for iron import, biotin synthesis, stress responses, cell integrity, and DNA repair. However, a comparison of Salmonella in egg white with and without L-arginine identified only a few differences in the frequency of transposon insertions, including the possible contribution of perturbations in the cell envelope to the inhibition mechanism. Finally, both D-arginine and D-cysteine were found to inhibit Salmonella in egg white. This implied that the effect of arginine and cysteine in egg white is chemical rather than biological, likely on the egg white environment or on the bacterial outer membrane. To conclude, these results show that this approach of addition of compounds, including nutrient type compounds, to an environment can be used to limit bacterial growth. Importantly, these compounds have no inherent anti-bacterial properties, are used as nutrients by animals and bacteria, and only become anti-bacterial in a specific environmental context. Future research screening for the effects of compounds in relevant environments might uncover new ways to reduce pathogen levels in the poultry industry and beyond.

Unveiling Agricultural Biotechnological Prospects: The Draft Genome Sequence of Stenotrophomonas geniculata LGMB417.

Stenotrophomonas species are recognized as rhizobacteria that play a pivotal role in promoting plant growth by making substantial contributions to enhanced soil fertility, nutrient recycling, and phytopathogen control. Employing them as bioinputs constitutes an environmentally sound strategy, particularly within the rhizospheric community. This study revealed the draft genome sequence of Stenotrophomonas geniculata LGMB417, which was originally isolated from root samples of maize (Zea mays L.). This research assessed the potential of a bacterial strain at the molecular level through genome mining, aiming to identify genes with biotechnological significance for promoting plant growth and protection. The assembly findings indicate that strain LGMB417 possesses a genome size of 4,654,011bp, with a G + C content of 66.50%. The draft genome sequence revealed the presence of gene clusters responsible for the synthesis of secondary metabolites and carbohydrate active enzymes (CAZymes), glycoside hydrolases (23), glycosyltransferases (18), carbohydrate esterases (5), polysaccharide lyases (2), carbohydrate-binding modules (2), and auxiliary activities (1). Several genes related to growth promotion were found in the genome, including those associated with phosphate transport and solubilization, nitrogen metabolism, siderophore production and iron transport, hormonal modulation, stress responses (such as to drought, temperature fluctuations, osmotic challenges, and oxidative conditions), and volatile organic compounds (VOCs). Subsequent phases will encompass investigations utilizing gene expression methodologies, with future explorations concentrating on facets pertinent to agricultural production, including comprehensive field studies.

GmGLU1 and GmRR4 contribute to iron deficiency tolerance in soybean.

Iron deficiency chlorosis (IDC) is a form of abiotic stress that negatively impacts soybean yield. In a previous study, we demonstrated that the historical IDC quantitative trait locus (QTL) on soybean chromosome Gm03 was composed of four distinct linkage blocks, each containing candidate genes for IDC tolerance. Here, we take advantage of virus-induced gene silencing (VIGS) to validate the function of three high-priority candidate genes, each corresponding to a different linkage block in the Gm03 IDC QTL. We built three single-gene constructs to target GmGLU1 (GLUTAMATE SYNTHASE 1, Glyma.03G128300), GmRR4 (RESPONSE REGULATOR 4, Glyma.03G130000), and GmbHLH38 (beta Helix Loop Helix 38, Glyma.03G130400 and Glyma.03G130600). Given the polygenic nature of the iron stress tolerance trait, we also silenced the genes in combination. We built two constructs targeting GmRR4+GmGLU1 and GmbHLH38+GmGLU1. All constructs were tested on the iron-efficient soybean genotype Clark grown in iron-sufficient conditions. We observed significant decreases in soil plant analysis development (SPAD) measurements using the GmGLU1 construct and both double constructs, with potential additive effects in the GmRR4+GmGLU1 construct. Whole genome expression analyses (RNA-seq) revealed a wide range of affected processes including known iron stress responses, defense and hormone signaling, photosynthesis, and cell wall structure. These findings highlight the importance of GmGLU1 in soybean iron stress responses and provide evidence that IDC is truly a polygenic trait, with multiple genes within the QTL contributing to IDC tolerance. Finally, we conducted BLAST analyses to demonstrate that the Gm03 IDC QTL is syntenic across a broad range of plant species.

The Distribution of Ferritins in Marine Copepods

Iron is an essential element for the functioning of cellular processes. Ferritins, the major intracellular iron storage proteins, convert the free Fe2+ into the nontoxic Fe3+ which can be stored and transported where needed. To date, little is known about the iron metabolism in copepods; however, in these crustaceans, ferritins have been used as biomarkers of stress and diapause. A limiting factor of these studies has been the use of a single ferritin transcript as a biomarker. In this paper, we in silico mined the publicly available copepod transcriptomes to characterize the multiplicity of the ferritin transcripts in different orders and families. We also examined the expression of ferritin in three ecologically important copepods—Calanus finmarchicus, C. helgolandicus and Temora stylifera—during development and under stress conditions. A full-length transcript encoding ferritin heavy chain has been identified in all 27 mined transcriptomes, with 50% of the species possessing multiple transcripts. Ferritin expression increased in C. finmarchicus during the early–late development transition, and in T. stylifera females exposed to oxylipins at sea. Overall, our results suggest that copepod ferritins can be involved in iron storage, larval development and stress response, thus representing potential biomarker genes for ocean health status monitoring.

Cinnamomum zeylanicum Blume Essential Oil Inhibits Metastatic Melanoma Cell Proliferation by Triggering an Incomplete Tumour Cell Stress Response.

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity.

Soybean GmVIT1 Gene Confers Plant Tolerance to Excess Fe/Mn Stress

Iron (Fe) and (Mn) are essential for the plant but are toxic when in excess. Vacuolar iron transporters (VITs) are involved in plant metal storage and detoxication. In this study, we screened two soybean cultivars (HN51 and SN37) with different responses to iron stress. From HN51 and SN37, we identified a new gene GmVIT1, for which expression is closely related to iron stress response by transcriptomic and quantitative analysis. We obtained GmVIT1 and GmVIT1 promoter from the iron deficiency-tolerant soybean variety Heinong51. Sequence analysis showed that GmVIT1 contained a conserved 170-residue VIT domain and localized at the tonoplast. Moreover, GmVIT1 is expressed in soybean leaves, stems, and roots. The expression of GmVIT1 was significantly induced by excessive Fe/Mn in leaves and stems. GUS assay showed that excess Fe/Mn enhanced GmVIT1 promoter activity. Furthermore, overexpression of GmVIT1 in Arabidopsis seedlings showed reduced phytotoxic effects induced by excess Fe/Mn stress, including yellowing in leaves, decreased chlorophyll content, and accumulated MDA. GmVIT1 overexpression in Arabidopsis showed relatively higher soluble sugar content and SOD, POD, and CAT activity. In addition, the ferric reductase activity in GmVIT1 overexpression in Arabidopsis decreased under excess Fe, while it increased under excess Mn. By integrating all these results, we found that GmVIT1 plays a vital role in plant response to excess Fe/Mn. The results showed that GmVIT1 was worthy of metal homeostasis mechanism research in plants and could be applied in the metal toxic-tolerance improvement in crops.

The role of site-2-proteases in bacteria: a review on physiology, virulence, and therapeutic potential.

Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored. Site-2-proteases are highly conserved among bacteria and play a key role in multiple processes, including iron uptake, stress response, and pheromone production. Additionally, an increasing number of site-2-proteases have been found to play a pivotal role in the virulence properties of multiple human pathogens, such as alginate production in Pseudomonas aeruginosa, toxin production in Vibrio cholerae, resistance to lysozyme in enterococci and antimicrobials in several Bacillus spp, and cell-envelope lipid composition in Mycobacterium tuberculosis. The prominent role of site-2-proteases in bacterial pathogenicity highlights the potential of site-2-proteases as novel targets for therapeutic intervention. In this review, we summarize the role of site-2-proteases in bacterial physiology and virulence, as well as evaluate the therapeutic potential of site-2-proteases.

Coupling VIGS with Short- and Long-Term Stress Exposure to Understand the Fiskeby III Iron Deficiency Stress Response

Yield loss due to abiotic stress is an increasing problem in agriculture. Soybean is a major crop for the upper Midwestern United States and calcareous soils exacerbate iron deficiency for growers, resulting in substantial yield losses. Fiskeby III is a soybean variety uniquely resistant to a variety of abiotic stresses, including iron deficiency. Previous studies identified a MATE transporter (Glyma.05G001700) associated with iron stress tolerance in Fiskeby III. To understand the function of this gene in the Fiskeby III response to iron deficiency, we coupled its silencing using virus-induced gene silencing with RNAseq analyses at two timepoints. Analyses of these data confirm a role for the MATE transporter in Fiskeby III iron stress responses. Further, they reveal that Fiskeby III induces transcriptional reprogramming within 24 h of iron deficiency stress, confirming that like other soybean varieties, Fiskeby III is able to quickly respond to stress. However, Fiskeby III utilizes novel genes and pathways in its iron deficiency response. Identifying and characterizing these genes and pathways in Fiskeby III provides novel targets for improving abiotic stress tolerance in elite soybean lines.

Iron stress response and bioaccumulation potential of three fungal strains isolated from sewage-irrigated soil.

Contamination with heavy metal (HM) is a severe environmental issue. Therefore, there is a pressing need to create environmentally safe and cost-effective HM bioremediation approaches. Three iron-tolerant fungal strains were isolated from sewage-irrigated soils, molecularly identified and deposited in the GenBank as Aspergillus flavus MT639638, A. terreus MT605370 and Fusarium oxysporum MT605399. The fungal growth, minimum inhibitory concentration (MIC), tolerance index (TI), removal efficiency, bioaccumulation, and enzymatic and non-enzymatic antioxidants were determined. Based on MIC values, A. flavus MT639638 was the most resistant strain. F. oxysporum displayed the highest percent removal efficiency (93.65% at 4000mg L-1 ) followed by A. flavus (92.92%, at 11,000mg L-1 ), and A. terreus (91.18% at 3000mg L-1 ). F. oxysporum was selected based on its highly sensitivity for further characterization of its response to Fe(II) stress using TEM, SEM and EDX, in addition to HPLC analysis of organic acids. These analyses demonstrated the localization of bioaccumulated Fe(II) and ultrastructural changes induced by iron and indicated induction release of organic acids. Our fungal strains showed an effective capacity for removal of Fe(II) via bioaccumulation and biosorption mechanisms which were supported by instrumental analyses. The iron tolerance potentiality was mediated by induction of selected antioxidative enzymes and biomolecules. This study depicts a potential utilization of the three fungal strains for the bioremediation of iron-contaminated soils.

Genome-Wide Identification, Characterization and Expression Analysis of Soybean CHYR Gene Family.

The CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) proteins have been functionally characterized in iron regulation and stress response in Arabidopsis, rice and Populus. However, their roles in soybean have not yet been systematically investigated. Here, in this study, 16 GmCHYR genes with conserved Zinc_ribbon, CHY zinc finger and Ring finger domains were obtained and divided into three groups. Moreover, additional 2–3 hemerythrin domains could be found in the N terminus of Group III. Phylogenetic and homology analysis of CHYRs in green plants indicated that three groups might originate from different ancestors. Expectedly, GmCHYR genes shared similar conserved domains/motifs distribution within the same group. Gene expression analysis uncovered their special expression patterns in different soybean tissues/organs and under various abiotic stresses. Group I and II members were mainly involved in salt and alkaline stresses. The expression of Group III members was induced/repressed by dehydration, salt and alkaline stresses, indicating their diverse roles in response to abiotic stress. In conclusion, our work will benefit for further revealing the biological roles of GmCHYRs.

New insights into the formation and recovery of sublethally injured Escherichia coli O157:H7 induced by lactic acid

Escherichia coli O157:H7 can be injured by the action of lactic acid (LA) and injured cells can be recovered under suitable condition. In this study, RNA sequencing analysis revealed the overall genes change of sublethally injured (4 mM LA, 60 min; SI) and initial recovered (minA, 20 min; R) cells. Compared with untreated samples, 53 up-regulated and 98 down-regulated differentially expressed genes (DEGs; Padj < 0.05, change fold ≥2) were found in SI. Meanwhile, Genes related to carbohydrate transport and metabolic were up-regulated and the addition of carbohydrate increased cells resistance to LA. Genes involved in osmotic stress response and cell membrane integrity were down-regulated and E. coli O157:H7 cells were sensitive to osmotic stress during sublethal injury. Genes related to iron stress response and cation transport were changed and cation may affect sublethal injury formation by influencing production of ROS and cellular processes. In R, 1370 up-regulated and 1110 down-regulated DEGs were subdivided into various GO terms and membrane, biological adhesion, cell projection, oxidation-reduction process and catalytic activity, etc., showed significant enrichment (corrected P < 0.05). Particularly, genes related to fimbrial, flagellum and type III secretion system were up-regulated, which may improve infection ability and virulence property during recovery of injured cells. These findings provide novel insights into formation and recovery of sublethally injured E. coli O157:H7 induced by LA.

Characterizing short and long term iron stress responses in iron deficiency tolerant and susceptible soybean (Glycine max L. Merr.)

Nutrient deficiencies limit growth and yield in many different crop species. The calcareous soils in the upper Midwestern United States favor the development of iron deficiency chlorosis (IDC) in soybean (Glycine max L. Merr.). Even minor symptoms result in end of season yield loss. To identify molecular pathways and networks underlying tolerance to iron deficiency stress in soybean, we leveraged two near isogenic lines (Clark and Isoclark) that differ in their tolerance to iron stress. Clark and Isoclark were grown in hydroponics in one of three treatments: iron sufficient media for ten days, iron deficient media for ten days, or iron sufficient media for eight days followed by transfer to iron deficient media for two days. Following phenotyping, plant tissues were harvested for RNA-seq analysis. To facilitate analyses, we clustered differentially expressed genes across genotypes and timepoints in both roots and leaves. With this experimental design, we could identify iron stress response differences between Clark and Isoclark, evaluate the impact of iron stress duration, and link expression clusters with discrete biological functions and the transcription factor families that regulate them. We identified thousands of differentially expressed genes associated with soybean stress tolerant responses including the cell cycle, gene silencing, iron acquisition, and defense. In Clark, the number of differentially expressed genes and magnitude of expression increased with increasing iron stress duration. In contrast, Isoclark decreased the number and magnitude of differentially expressed genes across time. Differences in gene expression corresponded to phenotypic differences. The shifting expression patterns between timepoints and tissues suggests novel mechanisms for iron stress signaling between source and sink tissues.

Effects of environmental factors and coexisting substrates on PAH degradation and transcriptomic responses of the defined bacterial consortium OPK.

The present study showed that syntrophic associations in a defined bacterial consortium, named OPK, containing Mycolicibacterium strains PO1 and PO2, Novosphingobium pentaromativorans PY1 and Bacillus subtilis FW1, led to effective pyrene degradation over a wide range of pH values, temperatures and salinities, as well as in the presence of a second polycyclic aromatic hydrocarbon (PAH). Anthracene, phenanthrene or fluorene facilitated complete pyrene degradation within 9 days, while fluoranthene delayed pyrene degradation. Interestingly, fluoranthene degradation was enhanced in the presence of pyrene. Transcriptome analysis confirmed that Mycolicibacterium strains were the key PAH-degraders during the cometabolism of pyrene and fluoranthene. Notably, the transcription of genes encoding pyrene-degrading enzymes were shown to be important for enhanced fluoranthene degradation. NidAB was the major initial oxygenase involved in the degradation of pyrene and fluoranthene mixture. Other functional genes encoding ribosomal proteins, an iron transporter, ABC transporters and stress response proteins were induced in strains PO1 and PO2. Furthermore, an intermediate pyrene-degrading Novosphingobium strain contributed to protocatechuate degradation. The results demonstrated that synergistic interactions among the bacterial members (PO1, PO2 and PY1) of the consortium OPK promoted the simultaneous degradation of two high molecular weight (HMW) PAHs.

Adaptive strategies to minimise iron limiting stress under climate change scenario: A study pertinent to iron stress response of two soybean (Glycine max (L.) Merr.) Genotypes

The response of soybean genotypes to iron deficiency stress under elevated CO2 and temperature condition could be guided by three critical rhizospheric processes viz. proton (H+) extrusion, root exudation and ferric chelate reductase (FCR) activity. In the present study, the iron efficient and responsive (FeER) genotype recorded an impressive performance over iron inefficient and responsive (FeIR) genotype in counteracting iron deficiency stress but experienced modest stress caused by the combined interaction between the genotype (G), environment (E) and HCO3- ion (B). The antagonistic interaction between Fe2+ with HCO3- ion resulted in greater iron stress. Plants grown in the absence of bicarbonate have significantly higher total chlorophyll content (1.79±0.04 mg g-1, mean ± SE) than plants grown in presence of bicarbonate (1.48 ± 0.06 mg g-1). To warfare the constraints in Fe availability, especially under more stressed e-[CO2+T] environmental condition in the presence of bicarbonate ion, the root system of iron efficient genotype of soybean (FeER) exuded out significantly higher amount of low molecular weight organic acids. Furthermore, the presence of bicarbonate ion in the nutrient solution exacerbated the iron deficiency stress and consequently resulted in higher proton extrusion (~1.2 fold increase), lower ferric chelate reductase activity (~1.3 fold decrease) and higher organic acid exudation (up to ~1.9 fold increase in malic acid). The intra-specific variability between contrasting genotypes and their response to elevated CO2 and temperature could be exploited to remediate emerging Fe deficiency of soybean plants under climate change scenario.

Commonalities and Differences in the Transcriptional Response of the Model Fungus Saccharomyces cerevisiae to Different Commercial Graphene Oxide Materials.

Graphene oxide has become a very appealing nanomaterial during the last years for many different applications, but its possible impact in different biological systems remains unclear. Here, an assessment to understand the toxicity of different commercial graphene oxide nanomaterials on the unicellular fungal model organism Saccharomyces cerevisiae was performed. For this task, an RNA purification protocol was optimized to avoid the high nucleic acid absorption capacity of graphene oxide. The developed protocol is based on a sorbitol gradient separation process for the isolation of adequate ribonucleic acid levels (in concentration and purity) from yeast cultures exposed to the carbon derived nanomaterial. To pinpoint potential toxicity mechanisms and pathways, the transcriptome of S. cerevisiae exposed to 160 mg L–1 of monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) was studied and compared. Both graphene oxide products induced expression changes in a common group of genes (104), many of them related to iron homeostasis, starvation and stress response, amino acid metabolism and formate catabolism. Also, a high number of genes were only differentially expressed in either GO (236) or GOC (1077) exposures, indicating that different commercial products can induce specific changes in the physiological state of the fungus.

A Method for Deciphering Major Drivers of Bacterial Iron Stress Response in the Presence of Oxidative Stressors.

This paper describes a method for deciphering major drivers of bacterial stress response using an empirically informed computational approach. We develop a working model of iron flux regulation and concomitant oxidative stress response in Escherichia coli. The integrated model is used to investigate the temporal effects of iron and hydrogen peroxide stress on bacterial growth and metabolism. We employ a sensitivity analysis platform and, using various measures, probe for major mechanistic drivers of the bacterial response to iron stress.

Examining Short-Term Responses to a Long-Term Problem: RNA-Seq Analyses of Iron Deficiency Chlorosis Tolerant Soybean.

Iron deficiency chlorosis (IDC) is a global crop production problem, significantly impacting yield. However, most IDC studies have focused on model species, not agronomically important crops. Soybean is the second largest crop grown in the United States, yet the calcareous soils across most of the upper U.S. Midwest limit soybean growth and profitability. To understand early soybean iron stress responses, we conducted whole genome expression analyses (RNA-sequencing) of leaf and root tissue from the iron efficient soybean (Glycine max) cultivar Clark, at 30, 60 and 120 min after transfer to iron stress conditions. We identified over 10,000 differentially expressed genes (DEGs), with the number of DEGs increasing over time in leaves, but decreasing over time in roots. To investigate these responses, we clustered our expression data across time to identify suites of genes, their biological functions, and the transcription factors (TFs) that regulate their expression. These analyses reveal the hallmarks of the soybean iron stress response (iron uptake and homeostasis, defense, and DNA replication and methylation) can be detected within 30 min. Furthermore, they suggest root to shoot signaling initiates early iron stress responses representing a novel paradigm for crop stress adaptations.

Propolis potentiates the effect of cranberry (Vaccinium macrocarpon) against the virulence of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the most prevalent bacteria isolated in urinary tract infections (UTI), is now frequently resistant to antibiotics used to treat this pathology. The antibacterial properties of cranberry and propolis could reduce the frequency of UTIs and thus the use of antibiotics, helping in the fight against the emergence of antibiotic resistance. Transcriptomic profiles of a clinical UPEC strain exposed to cranberry proanthocyanidins alone (190 µg/mL), propolis alone (102.4 µg/mL) and a combination of both were determined. Cranberry alone, but more so cranberry + propolis combined, modified the expression of genes involved in different essential pathways: down-expression of genes involved in adhesion, motility, and biofilm formation, and up-regulation of genes involved in iron metabolism and stress response. Phenotypic assays confirmed the decrease of motility (swarming and swimming) and biofilm formation (early formation and formed biofilm). This study showed for the first time that propolis potentiated the effect of cranberry proanthocyanidins on adhesion, motility, biofilm formation, iron metabolism and stress response of UPEC. Cranberry + propolis treatment could represent an interesting new strategy to prevent recurrent UTI.

Transcriptional regulation of chickpea ferritin CaFer1 influences its role in iron homeostasis and stress response

Ferritin, ubiquitous among all living organisms except yeast, exhibits iron-regulated expression. In plants, this regulation is applied through transcriptional control. Previous studies established the presence of two types of cis-acting elements in the promoter region: the iron regulatory element (FRE) in soybean and the iron-dependent regulatory sequence (IDRS) in maize and Arabidopsis. Adverse environmental conditions (e.g. water-deficit and oxidative stress) are known to modulate the expression of phytoferritin genes. In this study, we cloned and investigated the promoter sequence of a chickpea ferritin, designated CaFer1. Phylogenetic analysis of the CaFer1 promoter revealed its evolutionary relationship with other phytoferritins. The CaFer1 promoter exhibited several putative regulatory elements including two known transcription factor (TF) binding sites, Athb-1 and Myb.Ph. Electrophoretic mobility shift assay confirmed the sequence-specific binding of Athb-1 and Myb.Ph on the CaFer1 promoter. The TF-binding dynamics of CaFer1 showed high induction under conditions of iron-deficiency and water-deficit. We also demonstrated the possible interaction of CaFer1 with IRT1, a key component of the iron uptake system in plants, indicating its involvement in maintaining cellular iron levels. These results provide new insights into the underlying mechanisms of function of these interacting factors in CaFer1-mediated iron homeostasis and the stress response in plants.

A novel antibacterial peptide derived fromCrocodylus siamensishaemoglobin hydrolysate induces membrane permeabilization causing iron dysregulation, oxidative stress and bacterial death

A novel antibacterial peptide from Crocodylus siamensis haemoglobin hydrolysate (CHH) was characterized for antimicrobial activity. CHHs were hydrolysed for 2h (2h-CHH), 4h (4h-CHH), 6h (6h-CHH) and 8h (8h-CHH). The 8h-CHH showed antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa at concentrations of 20, 20, 20 and 10mgml-1 (w/v) respectively. Fluorescent microscopy revealed that the 8h-CHH had bactericidal activity against E.coli and P. aeruginosa. β-galactosidase assay supported by RT-qPCR demonstrated that the 8h-CHH resulted in differential expression of genes involved in iron homeostasis (ftnA and bfd) and oxidative stress (sodA, soxR and oxyR). Siderophore assay indicated that the 8h-CHH also impaired siderophore production with diminished expression of pvdF. This pattern of gene expression suggests that the 8h-CHH triggers the release of free ferric ions in the cytoplasm. However, decreased expression of genes associated with the SOS response (recA and lexA) in combination with neutral comet revealed that no DNA damage was caused by 8h-CHH. Membrane permeabilization assay indicated that 8h-CHH caused membrane leakage thought to mediate the antibacterial and iron-stress responses observed, due to loss of regulated iron transport. The novel active peptide from 8h-CHH was determined as QAIIHNEKVQAHGKKVL (QL17), with 41% hydrophobicity and +2 net charge. The QAIIHNEKVQAHGKKVL fragment of C. siamensis haemoglobin is antibacterial via a mechanism that likely relies on iron dysregulation and oxidative stress which results in bacterial death. We have described for the first time, a novel peptide derived from C. siamensis haemoglobin hydrolysate that has the potential to be developed as a novel antimicrobial peptide.