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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), faces iron scarcity within the host due to immune defenses. This review explores the importance of iron for Mtb and its strategies to overcome iron restriction. We discuss how the host limits iron as an innate immune response and how Mtb utilizes various iron acquisition systems, particularly the siderophore-mediated pathway. The review illustrates the structure and biosynthesis of mycobactin, a key siderophore in Mtb , and the regulation of its production. We explore the potential of targeting siderophore biosynthesis and uptake as a novel therapeutic approach for TB. Finally, we summarize current knowledge on Mtb 's iron acquisition and highlight promising directions for future research to exploit this pathway for developing new TB interventions. • Iron vital for Mtb (TB bacteria) but limited by immune system. • This review explores how Mtb combats iron deficiency. • We discuss iron restriction by our body and Mtb 's acquisition strategies, including siderophores. • Siderophores (e.g., mycobactin) are high-affinity iron-grabbing molecules. We explore their structure, production, and regulation in Mtb. • Targeting this pathway holds promise for new TB treatments. [ABSTRACT FROM AUTHOR]

The Gram-negative marine bacterium Vibrio anguillarum is able to cause vibriosis with hemorrhagic septicemia in many fish species, and iron acquisition is a critical step for virulence. Despite the fact that genes specific to certain processes of iron transport have been studied, the iron-regulated circuits of the V. anguillarum strains remain poorly understood. In this study, we showed that in V. anguillarum strain 775, iron could affect the expression of a number of critical metabolic pathways and virulence factors. The global iron uptake regulator VaFur is the major actor to control these processes for the bacterium to respond to different iron conditions. A VaFur binding motif was identified to distinguish directly and indirectly regulated targets. The absence of VaFur resulted in the aberrant expression of most iron acquisition determinants under rich-iron conditions. A similar regulation pattern was also observed in the transcription of genes coding for the type VI secretion system. The expression of peroxidase genes is positively controlled by VaFur to prevent iron toxicity, and the deletion of Vafur caused impaired growth in the presence of iron and H2O2. VaFur also upregulates some virulence factors under limited-iron conditions, including metalloprotease EmpA and motility, which are likely critical for the high virulence of V. anguillarum 775. The deletion of VaFur led to reduced swimming motility and decreased extracellular protease activity under limited-iron conditions, thereby leading to attenuated pathogenicity. Our study provides more evidence to better understand the VaFur regulon and its role in the pathogenesis of V. anguillarum. IMPORTANCEVibriosis, the most common disease caused by marine bacteria belonging to the genus Vibrio, leads to massive mortality of economical aquatic organisms in Asia. Iron is one of the most important trace elements, and its acquisition is a critical battle occurring between the host and the pathogen. However, excess iron is harmful to cells, so iron utilization needs to be strictly controlled to adapt to different conditions. This process is mediated by the global iron uptake regulator Fur, which acts as a repressor when iron is replete. On the other hand, free iron in the host is limited, so the reduced virulence of the Δfur mutant should not be directly caused by abnormally regulated iron uptake. The significance of this work lies in uncovering the mechanism by which the deletion of Fur causes reduced virulence in Vibrio anguillarum and identifying the critical virulence factors that function under limited-iron conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT To discover new factors that are involved in iron acquisition by Legionella pneumophila, we used RNA-Seq to identify the genes that are most highly induced when virulent strain 130b is cultured in a low-iron chemically defined medium. Among other things, this revealed 14915, a heretofore uncharacterized gene that is predicted to be transcriptionally regulated by Fur and to encode a novel, ~15 kDa protein. 14915 was present in all L. pneumophila strains examined and had homologs in a subset of the other Legionella species. Compatible with it containing a classic signal sequence, the 14915 protein was detected in bacterial culture supernatants in a manner dependent upon the L. pneumophila type II secretion system. Thus, we designated 14915 as IrsA for iron-regulated, secreted protein A. Based on mutant analysis, the irsA gene was not required for optimal growth of strain 130b in low-iron media. However, after discovering that the commonly used laboratory-derived strain Lp02 has a much greater requirement for iron, we uncovered a growth-enhancing role for IrsA after examining an Lp02 mutant that lacked both IrsA and the Fe2+-transporter FeoB. The irsA mutant of 130b, but not its complemented derivative, did, however, display increased biofilm formation on both plastic and agar surfaces, and compatible with this, the mutant hyper-aggregated. Thus, IrsA is a novel, iron-regulated exoprotein that modulates biofilm formation and, under some circumstances, promotes growth in low-iron conditions. For this study, we determined and deposited in the database a complete and fully assembled genome sequence for strain 130b.IMPORTANCEThe bacterium Legionella pneumophila is the principal cause of Legionnaires’ disease, a potentially fatal form of pneumonia that is increasing in incidence. L. pneumophila exists in many natural and human-made water systems and can be transmitted to humans through inhalation of contaminated water droplets. L. pneumophila flourishes within its habitats by spreading planktonically, assembling into biofilms, and growing in larger host cells. Iron acquisition is a key determinant for L. pneumophila persistence in water and during infection. We previously demonstrated that L. pneumophila assimilates iron both by secreting a non-protein iron chelator (siderophore) and by importing iron through membrane transporters. In this study, we uncovered a novel, secreted protein that is highly iron-regulated, promotes L. pneumophila’s growth in low-iron media, and impacts biofilm formation. We also identified uncharacterized, IrsA-related proteins in other important human and animal pathogens. Thus, our results have important implications for understanding iron assimilation, biofilm formation, and pathogenesis.

ABSTRACT Mycobacterium tuberculosis, the causative agent of the airborne infection tuberculosis (TB), contains 13 mycobacterial membrane protein large (MmpL) transporters that can be divided into two distinct subclasses. These MmpL proteins play important functional roles within the mycobacterium and subsequently are considered attractive drug targets to combat TB infection. Previously, we reported both X-ray and cryo-electron microscopy (cryo-EM) structures of the MmpL3 transporter, providing high-resolution structural information for this subclass of the MmpL proteins. Thus far, there is no structural information available for the other subclass, which includes MmpL5, an inner membrane transporter that plays a critical role in iron hemostasis. Here, we report the first cryo-EM structure of the Mycobacterium smegmatis MmpL5 transporter bound with the meromycolate extension acyl carrier protein M (AcpM) to a resolution of 2.81 Å. Our structural data reveals that MmpL5 and AcpM interact in the cytoplasm to form a complex, and this allows us to propose that MmpL5 may also associate with the mycobactin L (MbtL) protein in a similar fashion to form a heterocomplex important for iron acquisition, which enables the survival and replication of the mycobacterium.IMPORTANCEThe emergence and spread of multidrug-resistant tuberculosis (TB) present enormous challenges to the global public health. The causative agent, Mycobacterium tuberculosis, has now infected more than one-third of the world's population. Here, we report the first structure of the mycobacterial membrane protein large 5 (MmpL5), an essential transporter for iron acquisition, bound with the meromycolate extension acyl carrier protein M (AcpM), indicating a plausible pathway for mycobactin translocation. Our studies will ultimately inform an era in structure-guided drug design to combat TB infection.

ABSTRACT Streptococcus pneumoniae is a common member of the nasopharynx commensal microflora and the leading etiological agent of bacterial pneumonia in young children and aging adults. SPD_0739, a highly expressed lipoprotein, is the predicted substrate-binding component of an ABC transporter linked to the uptake of nucleosides and heme by independent studies (named PnrA or Spbhp-37, respectively). Here, we demonstrate that SPD_0739 binds heme in vitro and contributes to the bacterial binding to hemoglobin. A ∆spd_0739 strain exhibited growth attenuation that was relieved by the inactivation of the piuBCDA transporter. Knocking out spd_0739 in the wild type, or the ΔpiuBCDA strain resulted in heme accumulation, higher sensitivity to heme toxicity, and a small growth reduction compared to medium supplemented with a nucleoside mixture. In addition, spd_0739 loss results in higher iron- and heme-related gene expression and lower H2O2 production. Altogether, the data are consistent with a role in nucleoside import and show that SPD_0739 does not import heme. Instead, it indirectly influences iron and heme metabolism, linking nucleosides and iron status in S. pneumoniae.IMPORTANCES. pneumoniae obtains growth essential iron from hemoglobin and other host hemoproteins. Still, the bacterial mechanisms involved are only partially understood, and there are inconsistent reports regarding the function of several transporters implicated in iron uptake. In this study, we clarified the role of PnrA/Spbhp-37, a ligand-binding protein previously linked to nucleoside or heme by different studies. We present data supporting a role in nucleoside scavenging rather than heme import and reveal that PnrA/Spbhp-37 modulates iron and heme uptake, likely by influencing the nucleoside cellular pool. Hence, this work provides a new understanding of a process critical to the pathophysiology of a significant human pathogen. Moreover, PnrA/Spbhp-37 is an abundant and immunogenic surface protein that is highly conserved. Hence, this study also clarifies the function of a promising vaccine target.

During infection, the host employs nutritional immunity to restrict access to iron. Staphylococcus lugdunensis has been recognized for its ability to utilize host‐derived heme to overcome iron restriction. However, the mechanism behind this process involves the release of hemoglobin from erythrocytes, and the hemolytic factors of S. lugdunensis remain poorly understood. S. lugdunensis encodes four phenol‐soluble modulins (PSMs), short peptides with hemolytic activity. The peptides SLUSH A, SLUSH B, and SLUSH C are β‐type PSMs, and OrfX is an α‐type PSM. Our study shows the SLUSH locus to be essential for the hemolytic phenotype of S. lugdunensis. All four peptides individually exhibited hemolytic activity against human and sheep erythrocytes, but synergism with sphingomyelinase was observed exclusively against sheep erythrocytes. Furthermore, our findings demonstrate that SLUSH is crucial for allowing the utilization of erythrocytes as the sole source of nutritional iron and confirm the transcriptional regulation of SLUSH by Agr. Additionally, our study reveals that SLUSH peptides stimulate the human immune system. Our analysis identifies SLUSH as a pivotal hemolytic factor of S. lugdunensis and demonstrates its concerted action with heme acquisition systems to overcome iron limitation in the presence of host erythrocytes. [ABSTRACT FROM AUTHOR]

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS-as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions. [ABSTRACT FROM AUTHOR]

The hmuR operon encodes proteins for the uptake and utilization of heme as a nutritional iron source in Bradyrhizobium japonicum. The hmuR operon is transcriptionally activated by the Irr protein and is also positively controlled by HmuP by an unknown mechanism. An hmuP mutant does not express the hmuR operon genes nor does it grow on heme. Here, we show that hmuR expression from a heterologous promoter still requires hmuP, suggesting that HmuP does not regulate at the transcriptional level. Replacement of the 5′ untranslated region (5′UTR) of an HmuP‐independent gene with the hmuR 5′UTR conferred HmuP‐dependent expression on that gene. Recombinant HmuP bound an HmuP‐responsive RNA element (HPRE) within the hmuR 5′UTR. A 2 nt substitution predicted to destabilize the secondary structure of the HPRE abolished both HmuP binding activity in vitro and hmuR expression in cells. However, deletion of the HPRE partially restored hmuR expression in an hmuP mutant, and it rescued growth of the hmuP mutant on heme. These findings suggest that the HPRE is a negative regulatory RNA element that is suppressed when bound by HmuP to express the hmuR operon. [ABSTRACT FROM AUTHOR]

ABSTRACT Salmonella enterica is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As S. enterica serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of Salmonella infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated Salmonella. Despite decades of research, many aspects of Salmonella infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated S. Typhimurium strains. We reveal differential essential pathways between strains in vitro and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCESalmonella enterica is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, S. enterica is used widely as a model to understand host responses to infection. Understanding how Salmonella survives in vivo is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, aroA, is thought to attenuate Salmonella by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of Salmonella to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of aroA in vaccine development.

Iron is an essential micronutrient for most living organisms, including cyanobacteria. These microorganisms have been found in Earths driest polar and non-polar deserts, including the Atacama Desert, Chile. Iron-containing minerals were identified in colonized rock substrates from the Atacama Desert, however, the interactions between microorganisms and iron minerals remain unclear. In the current study, we determined that colonized gypsum rocks collected from the Atacama Desert contained both magnetite and hematite phases. A cyanobacteria isolate was cultured on substrates consisting of gypsum with embedded magnetite nanoparticles. Transmission electron microscopy imaging revealed a significant reduction in the size of magnetite nanoparticles due to their dissolution, which occurred around the microbial biofilms. Concurrently, hematite was detected, likely from the oxidation of the magnetite nanoparticles. Higher cell counts and production of siderophores were observed in cultures with magnetite nanoparticles suggesting that cyanobacteria were actively acquiring iron from the magnetite nanoparticles. Magnetite dissolution and iron acquisition by the cyanobacteria was further confirmed using large bulk magnetite crystals, uncovering a survival strategy of cyanobacteria in these extreme environments.

Background: Staphylococcus aureus remains a prominent pathogen within health-care environments due to its extensive repertoire of virulence factors. The escalating prevalence of antibiotic-resistant strains has underscored the urgency of identifying alternative treatment strategies. The study focuses on the HtsA siderophore receptor, an integral cell wall component of all S. aureus strains, responsible for iron acquisition from the host environment. Here, we investigate the potential of Bacillus sp. derived macrolides as inhibitors of this critical iron uptake mechanism. Methods: Sixteen distinct macrolide ligands were evaluated for their ability to disrupt iron acquisition by S. aureus through interaction with the HtsA receptor. Employing molecular docking and subsequent molecular dynamics (MDs) simulations, the binding interactions and dynamic behaviors of these ligands with the receptor were assessed. Results: Notably, the complex formed between 7-O-succinyl macrolactin F and HtsA demonstrated a good binding affinity of − 8.5 kcal/mol, outperforming the binding affinity of staphyloferrin A, which exhibited a binding affinity of − 6.9 kcal/mol. MD simulations further corroborated the stability of the 7-O-succinyl macrolactin F-HtsA complex over time, suggesting the potential for this interaction to be harnessed as a novel therapeutic strategy not only for existing antibiotic-resistant strains but also for those that may arise in the future. Conclusion: The findings propose Bacillus species-derived macrolides as potential adjuvant therapies for S. aureus infections, pending comprehensive safety assessments. This research lays the foundation for the development of innovative antimicrobial agents targeting essential virulence-associated mechanisms in S. aureus.

This article, titled "Editorial: Sensing of and Acclimations to Environmental pH in Plants," discusses the importance of pH in plant growth and adaptation. It highlights the discovery of pH sensors in plants and their role in responding to changes in external pH. The article also explores the impact of soil pH on plant performance and the availability of nutrients. It concludes that external pH is a crucial factor in plant function and adaptation, affecting growth, defense, and development. However, the mechanisms and consequences of pH sensing in plants are not fully understood. [Extracted from the article]

Background: Staphylococcus aureus remains a prominent pathogen within health-care environments due to its extensive repertoire of virulence factors. The escalating prevalence of antibiotic-resistant strains has underscored the urgency of identifying alternative treatment strategies. The study focuses on the HtsA siderophore receptor, an integral cell wall component of all S. aureus strains, responsible for iron acquisition from the host environment. Here, we investigate the potential of Bacillus sp. derived macrolides as inhibitors of this critical iron uptake mechanism. Methods: Sixteen distinct macrolide ligands were evaluated for their ability to disrupt iron acquisition by S. aureus through interaction with the HtsA receptor. Employing molecular docking and subsequent molecular dynamics (MDs) simulations, the binding interactions and dynamic behaviors of these ligands with the receptor were assessed. Results: Notably, the complex formed between 7-O-succinyl macrolactin F and HtsA demonstrated a good binding affinity of - 8.5 kcal/mol, outperforming the binding affinity of staphyloferrin A, which exhibited a binding affinity of - 6.9 kcal/mol. MD simulations further corroborated the stability of the 7-O-succinyl macrolactin F-HtsA complex over time, suggesting the potential for this interaction to be harnessed as a novel therapeutic strategy not only for existing antibiotic-resistant strains but also for those that may arise in the future. Conclusion: The findings propose Bacillus species-derived macrolides as potential adjuvant therapies for S. aureus infections, pending comprehensive safety assessments. This research lays the foundation for the development of innovative antimicrobial agents targeting essential virulence-associated mechanisms in S. aureus. [ABSTRACT FROM AUTHOR]

ABSTRACT Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

ABSTRACT In the presence of molecular oxygen, the human pathogen Streptococcus pneumoniae produces and secretes large amounts of hydrogen peroxide (H2O2), which can readily interact with free and heme-bound iron. Here, we investigated the role of the endogenously produced H2O2 in iron acquisition. The data revealed that S. pneumoniae uses H2O2 to liberate iron from met-hemoglobin (Hb-Fe3+) extracellularly, allowing the bacterium to import and grow on free iron even when cultivated on met-hemoglobin as the only iron source. The loss of H2O2 production leads to a dramatic pneumococcal intake of heme and is associated with a robust upregulation of most iron uptake machinery (indicating an iron starvation signal). These and other data reveal a close and previously unexplored relation between H2O2 production and iron metabolism in S. pneumoniae. The data also show that, in addition to extracellular degradation, pneumococci are armed with H2O2-independent mechanisms for intracellular heme catabolism. IMPORTANCE Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

ESX‐3 is a secretion pathway which is essential for mycobactin‐mediated iron acquisition under iron‐limited conditions. Although present in all Mycobacterium sp., ESX‐3 remains to be elucidated in Mycobacterium abscessus. In the study reported here, impaired ESX‐3 seriously restricts the growth of M. abscesses under iron‐limited conditions; growth is salvaged by functional ESX‐3 or iron supplementation. Notably, impaired ESX‐3 does not kill M. abscesses when environmental iron is insufficient but induces persistence to bedaquiline, a diarylquinoline class antibiotic used to treat multidrug‐resistant mycobacteria. One potential mechanism contributing to persistence is the iron deficiency due to impaired ESX‐3 suppressing succinate dehydrogenase activity, which dysregulates the tricarboxylic acid cycle and inactivates bedaquiline. Experiments conducted here also demonstrate that the regulator, MtrA, can bind ESX‐3 and promote the survival of M. abscessus. As such, this study suggests that a novel pathway involving MtrA, ESX‐3, iron metabolism, and the TCA cycle contributes to bedaquiline persistence in M. abscesses growing under iron‐limited conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT Successful pathogens must be able to adapt to a multitude of stressors imposed by their host. Acinetobacter baumannii has emerged as a major global health threat due to its exceptional ability to adapt to hostile environments and skyrocketing rates of multidrug resistance. Recent studies have begun to explore the importance of tRNA methylation in the regulation of bacterial stress responses, including adaptation to antibiotic and oxidative stresses. However, tRNA methyltransferases (trms) have not been investigated in A. baumannii. Bioinformatic analyses revealed nine putative, SAM-dependent trms conserved across clinical A. baumannii isolates and laboratory strains. We generated eight trm mutants in a modern, colistin-resistant clinical isolate, ARC6851, and analyzed the mutants’ stress responses. One mutant, ΔtrmB, was vastly more sensitive to oxidative stress and displayed a growth defect at low pH. Accordingly, ΔtrmB was unable to replicate in J774A.1 macrophages and had decreased virulence in an acute pneumonia murine model. Subsequently, we showed that A. baumannii TrmB makes the m7G tRNA modification. A proteomic analysis revealed that ARC6851 significantly upregulates a siderophore biosynthesis and uptake cluster, acinetobactin, under oxidative stress. In contrast, the upregulation of the acinetobactin proteins in ΔtrmB was only modest, which impacted its ability to withstand iron deprivation under oxidative stress. qRT-PCR data showed that TrmB-dependent regulation of acinetobactin is post-transcriptional. Our results indicate that TrmB-mediated stress responses are important for A. baumannii pathogenesis, highlighting the therapeutic potential of targeting trms to combat the rise of multidrug-resistant A. baumannii. IMPORTANCE As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications’ impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH. [ABSTRACT FROM AUTHOR]

ABSTRACT Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

ABSTRACT Bacterial cells form and release membrane vesicles (MVs) originating from cellular membranes. In recent years, many biological functions of bacterial MVs have been identified. Here, we show that MVs derived from Corynebacterium glutamicum, a model organism for mycolic acid-containing bacteria, can mediate iron acquisition and other phylogenetically related bacteria. Lipid/protein analysis and iron quantification assay indicate that C. glutamicum MVs formed by outer mycomembrane blebbing can load ferric iron (Fe3+) as its cargo. Iron-loaded C. glutamicum MVs promoted the growth of producer bacteria in iron-limited liquid media. MVs were received by C. glutamicum cells, suggesting a direct transfer of iron to the recipient cells. Cross-feeding of C. glutamicum MVs with phylogenetically close (Mycobacterium smegmatis and Rhodococcus erythropolis) or distant (Bacillus subtilis) bacteria indicated that C. glutamicum MVs could be received by the different species tested, while iron uptake is limited to M. smegmatis and R. erythropolis. In addition, our results indicate that iron loading on MVs in C. glutamicum does not depend on membrane-associated proteins or siderophores, which is different from what has been shown in other mycobacterial species. Our findings illustrate the biological importance of MV-associated extracellular iron for C. glutamicum growth and suggest its ecological impact on selected members of microbial communities. IMPORTANCE Iron is an essential element of life. Many bacteria have developed iron acquisition systems, such as siderophores, for external iron uptake. Corynebacterium glutamicum, a soil bacterium known for its potential for industrial applications, was shown to lack the ability to produce extracellular, low-molecular-weight iron carriers, and it remains elusive how this bacterium acquires iron. Here, we demonstrated that MVs released from C. glutamicum cells could act as extracellular iron carriers that mediate iron uptake. Although MV-associated proteins or siderophores have been shown to play critical roles in MV-mediated iron uptake by other mycobacterial species, the iron delivery through C. glutamicum MVs is not dependent on these factors. Moreover, our results suggest that there is an unidentified mechanism that determines the species specificity of MV-mediated iron acquisition. Our results further demonstrated the important role of MV-associated iron.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH.

One of the most significant constraints on agricultural productivity is the low availability of iron (Fe) in soil, which is directly related to biological, physical, and chemical activities in the rhizosphere. The rhizosphere has a high iron requirement due to plant absorption and microorganism density. Plant roots and microbes in the rhizosphere play a significant role in promoting plant iron (Fe) uptake, which impacts plant development and physiology by influencing nutritional, biochemical, and soil components. The concentration of iron accessible to these live organisms in most cultivated soil is quite low due to its solubility being limited by stable oxyhydroxide, hydroxide, and oxides. The dissolution and solubility rates of iron are also significantly affected by soil pH, microbial population, organic matter content, redox processes, and particle size of the soil. In Fe-limiting situations, plants and soil microbes have used active strategies such as acidification, chelation, and reduction, which have an important role to play in enhancing soil iron availability to plants. In response to iron deficiency, plant and soil organisms produce organic (carbohydrates, amino acids, organic acids, phytosiderophores, microbial siderophores, and phenolics) and inorganic (protons) chemicals in the rhizosphere to improve the solubility of poorly accessible Fe pools. The investigation of iron-mediated associations among plants and microorganisms influences plant development and health, providing a distinctive prospect to further our understanding of rhizosphere ecology and iron dynamics. This review clarifies current knowledge of the intricate dynamics of iron with the end goal of presenting an overview of the rhizosphere mechanisms that are involved in the uptake of iron by plants and microorganisms. [ABSTRACT FROM AUTHOR]

Iron is one of the essential metals required by almost all living organisms. However, nature has certain constraints in distributing this element among tissues. Since polymeric oxide-bridged Fe (III) is the prominent source of Fe (III) ions, the insolubility of Fe (III) ions in aqueous systems reduces the direct uptake by cells. Secondly, the free-Fe entities which generate .OH radicals pave the way to the destruction of the cells. Hence, a protective coordination environment via sophisticated chemical systems is required for the acquisition of Fe, its successive transport, storage, and effective utilization in various tissues. Siderophores are polydentate ligands used by bacterial cells for Fe acquisition, with a relatively high affinity for Fe (III) ions. Secreted from the bacterial cells into the external aqueous medium, they sequester Fe to give a soluble complex which re-enters the organism at a specific receptor. Once it gets inside the cell, the Fe is released from the complex and utilized for essential biochemical reactions. The medicinal applications of these natural ligands, developing progressively in various research groups, necessitate the theoretical aspects of their coordination chemistry. This research paper deals with the coordination chemistry of one of the siderophores, cepabactin (Cep). The chemical computations confirm that the FeIII(Cep)3 complex is octahedral and high spin. The oxygen atoms of Cep, which are hard and negatively charged, thus act as electron donors in the FeIII(Cep)3 complex formation. This in turn makes the siderophores relatively less attractive towards Fe (II) ions. [ABSTRACT FROM AUTHOR]

The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits the nocardamine xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 Å resolution the crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore–iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which have implications for the development of siderophore–antibiotic conjugates to treat difficult infections. [ABSTRACT FROM AUTHOR]

• Sequence type (ST) 2 is dominant in hypervirulent extensively-drug-resistant Acinetobacter baumannii (XDRAB), followed by ST129. • Higher iron acquisition and capsule production may contribute to the virulence of XDRAB. • Higher prevalence of tonB, hemO, abaI and ptk in XDRAB than in non-multi-drug-resistant A. baumanii. • Isolates that cause high mortality rates in larvae also exhibit higher virulence in mice. Individuals infected with extensively-drug-resistant (XDR) Acinetobacter baumannii are difficult to cure and have a high mortality rate. This study compared the genomic and phenotypic differences between XDR and non-multi-drug-resistant (MDR) A. baumannii , and further characterized hypervirulent XDR A. baumannii. In total, 1403 acinetobacter isolates were collected from patients with bacteraemia between 1997 and 2015. Antimicrobial susceptibility tests were performed to categorize isolates into non-MDR, MDR and XDR groups. The presence of selected virulence-associated genes was determined by polymerase chain reaction. Bacterial phenotypes, including iron acquisition, biofilm formation, capsule production, and virulence to larvae and mice, were determined. Multi-locus sequence typing revealed a high prevalence of sequence type (ST) 2 (81.6%) and ST129 (18.4%) among 49 XDR isolates, and the STs of 18 non-MDR isolates were more diverse. Virulence-associated phenotypic assays showed that XDR isolates had higher iron acquisition ability, greater capsule production, and virulence to Galleria mellonella larvae. However, their ability to form biofilm was lower compared with that of non-MDR isolates. XDR isolates were more likely to have virulence genes (tonB, hemO, abaI and ptk), while non-MDR isolates were more likely to have pld and ompA genes. Twenty-one XDR isolates that had a <20% larvae survival rate after 7 days post-infection were defined as hypervirulent XDR isolates. Among them, isolates 1677 (ST129) and 929-1 (ST2) caused the death of all infected mice within 2 days. Some subpopulations of highly-drug-resistant ST2 isolates exhibit high virulence. As such, it is of utmost importance to continue monitoring the spread of hypervirulent XDR A. baumannii isolates. [ABSTRACT FROM AUTHOR]

Background: Stenotrophomonas maltophilia, a species of highly genetic diversity, has emerged as an important nosocomial pathogen. S. maltophilia and Pseudomonas aeruginosa are often co-isolated from pneumonia patients. In our previous study, we have demonstrated that the pacIRA cluster present in some but not all clinical S. maltophilia isolates. Proteins encoded by pacIRA operon are an extracytoplasmic function (ECF) sigma factor, a transmembrane anti-sigma regulator, and a TonB-dependent receptor. This study aimed to elucidate PacIRA system function and its significance to S. maltophilia. Methods: The pacI, pacR, and pacA genes were individually or totally deleted from the chromosome of KJΔEnt, a pacIRA-positive and siderophore-null strain. Growth promotion assay was performed to examine the implication of pacIRA system in iron utilization. Gene expression was quantified by quantitative real time PCR (qRT-PCR). Growth competition assay was executed to investigate the significance of pacIRA operon to S. maltophilia. Results: PacIRA system contributed to utilize ferri-pyochelin of P. aeruginosa as iron sources for growth in an iron-depleted condition, but hardly utilized ferric citrate, hemin, ferri-stenobactin, and ferri-pyoverdine. PacIRA was founded to belong to Fur regulon and upregulated in response to iron-depleted stress. Growth competition assay demonstrated that pacIRA-positive S. maltophilia had a superiority over pacIRA-negative S. maltophilia in iron acquisition when they were co-cultured in P. aeruginosa ferri-pyochelin-supplemented medium. Conclusions: PacIRA system of S. maltophilia is a xenosiderophore uptake implement, involving in the acquisition of pyochelin of P. aeruginosa.

The ability to acquire iron from the environment is often an important virulence factor for pathogenic bacteria and Vibrios are no exception to this. Vibrios are reported mainly from marine habitats and most of the species are pathogenic. Among those, the pathogenic vibrios eg. V cholerae, V. parahaemolyticus, V. vulnificus causes foodborne illnesses. Vibrios are capable of producing all different classes of siderophores like hydroxamate (aerobactin), catecholate (vibriobactin, fluvibactin), carboxylate (vibrioferrin), and amphiphilic (amphibactin). Every different species of vibrios are capable of utilizing some endogenous or xenosiderophores. Being Gram-negative bacteria, Vibrios import iron siderophore via TonB dependent transport system and unlike other Gamma proteobacteria these usually possess two or even three partially redundant TonB systems for iron siderophore transport. Other than selected few iron siderophores, most pathogenic Vibrios are known to be able to utilize heme as the sole iron source, while some species are capable of importing free iron from the environment. As per the present knowledge, the spectrum of iron compound transport and utilization in Vibrios is better understood than the siderophore biosynthetic capability of individual species.

ABSTRACT Mycobacterium tuberculosis (Mtb) is transmitted through aerosols and primarily colonizes within the lung. The World Health Organization estimates that Mtb kills ~1.4 million people every year. A key aspect that makes Mtb such a successful pathogen is its ability to overcome iron limitation mounted by the host immune response. In our previous studies, we have shown that Mtb can utilize iron from heme, the largest source of iron in the human host, and that it uses two redundant heme utilization pathways. In this study, we show that the ESX-4 type VII secretion system (T7SS) is necessary for extracellular heme uptake into the Mtb cell through both heme utilization pathways. ESX-4 influences the secretion of the culture filtrate proteins Rv0125 and Rv1085c, which are also necessary for efficient heme utilization. We also discovered that deletion of the alternative sigma factor SigM significantly reduced Mtb heme utilization through both pathways and predict that SigM is a global positive regulator of core heme utilization genes of both pathways. Finally, we present the first direct evidence that some mycobacterial PPE (proline-proline-glutamate motif) proteins of the PPE protein family are pore-forming membrane proteins. Altogether, we identified core components of both Mtb Heme utilization pathways that were previously unknown and identified a novel channel-forming membrane protein of Mtb. IMPORTANCE M. tuberculosis (Mtb) is completely dependent on iron acquisition in the host to cause disease. The largest source of iron for Mtb in the human host is heme. Here, we show that the ancestral ESX-4 type VII secretion system is required for the efficient utilization of heme as a source of iron, which is an essential nutrient. This is another biological function identified for ESX-4 in Mtb, whose contribution to Mtb physiology is poorly understood. A most exciting finding is that some mycobacterial PPE (proline-proline-glutamate motif) proteins that have been implicated in the nutrient acquisition are membrane proteins that can form channels in a lipid bilayer. These observations have far-reaching implications because they support an emerging theme that PPE proteins can function as channel proteins in the outer mycomembrane for nutrient acquisition. Mtb has evolved a heme uptake system that is drastically different from all other known bacterial heme acquisition systems.

ABSTRACT The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of infections and mortality in immunocompromised patients. This organism can overcome iron deprivation during infection via the synthesis of two iron-chelating siderophores, pyoverdine and pyochelin, which scavenge iron from host proteins. P. aeruginosa can also uptake xenosiderophores produced by other bacteria or fungi using dedicated transporter systems. The precise substrate specificity of these siderophore transporters remains to be determined. The thiopeptide antibiotic thiostrepton exploits the pyoverdine transporters FpvA and FpvB to cross the outer membrane and reach intracellular targets. Using a series of intricate biochemical experiments, a recent study by Chan and Burrows capitalized on the specificity of thiostrepton to uncover that FpvB transports the xenosiderophores ferrichrome and ferrioxamine B with higher affinity than pyoverdine. This surprising result highlights an alternative uptake pathway for these siderophores and has significant implications for our understanding of iron acquisition in this organism.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity. [ABSTRACT FROM AUTHOR]

Pastoral nomadic groups in the Eurasian steppe were in existence for more than a few millennia, paving the way for the emergence of steppe polities that maintained close and dynamic interactions with their sedentary farming neighbors. Despite the critical role of iron materials in the genesis of these mobile political entities, surprisingly little is known about the mode of iron acquisition adopted by mobile pastoralist groups. This is particularly true for mobile communities located far away from the population centers where technological capabilities and material resources were more likely to converge in innovative ways. In this article, we present a microscopic examination of small cast iron objects bearing evidence of a unique thermal treatment in the molten state to obtain rapid decarburization, specifically for small-scale steelmaking from a non-local material, cast iron. This highly sophisticated technique was carried out on-site at a Mongol period habitation located in north-central Mongolia. These findings are important as they demonstrate that mobile pastoralists of the Mongolian steppe developed various methods of small-scale iron production that allowed them to enjoy a degree of self-sufficiency in one of the most critical areas of material culture, iron, and steel production. [ABSTRACT FROM AUTHOR]

The ever-evolving research in the field of antitubercular agents has led to the identification of several new potential drug classes. Among them, 5-phenyl-furan-2-carboxylic acids have emerged as innovative potential therapeutics, targeting iron acquisition in mycobacterial species. In our efforts to characterize the molecular interactions between these compounds and their protein target (MbtI from M. tuberculosis) by means of co-crystallization experiments, we unexpectedly obtained the structure of 5-(4-nitrophenyl)furan-2-carboxylic acid (1). Herein, we describe the preparation of the compound and its analysis by 1H NMR, 13C NMR, HRMS, and SC-XRD. [ABSTRACT FROM AUTHOR]

5-Phenyl-furan-2-carboxylic acids have emerged as a new, promising class of antimycobacterial agents that have the ability to interfere with iron homeostasis. Considering the lack of structural data on these compounds, we analyzed the crystal of a fluorinated ester derivative of 5-(4-nitrophenyl)furan-2-carboxylic acid, one of the most potent candidates in the series. Here, we describe the preparation of methyl 5-(2-fluoro-4-nitrophenyl)furan-2-carboxylate (1) and its analysis by 1H-NMR, 13C-NMR, HRMS, and SC-XRD. [ABSTRACT FROM AUTHOR]

You Lan, Mao Zhou, Xin Li, Xuan Liu, Jun Li, Wenen Liu Department of Clinical Laboratory, Xiangya Hospital Central South University, Changsha, Hunan, 410008, People’s Republic of ChinaCorrespondence: Wenen LiuDepartment of Clinical Laboratory, Xiangya Hospital Central South University, No. 87 Xiangya Road, Changsha, Hunan, 410008, People’s Republic of China, Tel +86 731 8432 7437, Fax +86 731 8432 7332, Email wenenliu@163.comObjective: To investigate the role of outer membrane vesicles (OMVs) and related proteins in iron acquisition of hypervirulent Klebsiella pneumoniae (HVKP) and classic Klebsiella pneumoniae (cKP).Methods: The OMVs of HVKP and cKP under iron-deficient and iron-sufficient media were extracted and purified by ultracentrifugation. Transmission electron microscopy (TEM) was used to identify OMVs. The quantitative proteomics were performed based on mass spectrometry.Results: Four OMVs samples secreted by HVKP and cKP under iron-deficient and iron-sufficient environment were isolated and collected (HVKP OMVs under iron-deficient environment (A1), HVKP OMVs under iron-sufficient environment (A2), cKP OMVs under iron-deficient environment (B1), cKP OMVs under iron-sufficient environment (B2)). The amount of OMVs released by HVKP in iron-deficient medium was significantly larger than that in iron-sufficient medium (P < 0.05). HVKP secreted more OMVs than cKP in iron-deficient medium (P < 0.05). A total of 1074 kinds of proteins were identified in four samples. A comparison between the iron-deficient vs iron-sufficient environment showed that 61 proteins in HVKP OMVs were identified with a significant change in abundance under iron-deficient environment. Among them, 17 proteins were related to iron acquisition and transportation systems. While in cKP OMVs, 62 proteins significantly changed under iron-deficient environment in which 5 proteins were related to iron acquisition and transportation systems. Upon comparison of the HVKP vs cKP OMVs under iron deficiency, 81 proteins were detected with a significant change in which 8 proteins were related to iron acquisition and transportation systems.Conclusion: Above all, the results of this study suggest a potential role for OMVs in iron acquisition of HVKP and provide evidence of potential connections between OMVs and strong iron-acquisition ability of HVKP during iron limitation.Keywords: hypervirulent Klebsiella pneumoniae, iron acquisition, proteomics

ABSTRACT Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets—including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach. IMPORTANCE Gram-negative bacteria express a variety of outer membrane transporters to import critical nutrients such as iron. Due to its insolubility, iron is taken up while bound to small-molecule chelators called siderophores. Pseudomonas aeruginosa takes up its own siderophores pyoverdine and pyochelin but can also steal siderophores produced by other bacteria and fungi, giving it a competitive advantage in iron-limited environments. Here, we used whole-cell reporter assays to show that FpvB, originally identified as a secondary transporter for pyoverdine, transports the chemically distinct fungal siderophore ferrichrome and the bacterial siderophore ferrioxamine B with high affinity. FpvB is also used by thiopeptide antibiotic thiostrepton for uptake. We predicted that all of these ligands bind to a common hydrophobic pocket in FpvB and used site-directed mutagenesis coupled with phenotypic assays to identify residues required for uptake. These analyses showed that siderophore and antibiotic uptake could be uncoupled. Our data show that FpvB is a promiscuous transporter of multiple chemically distinct ligands and fills in missing details of ferrichrome transport by P. aeruginosa. A clearer picture of the spectrum of outer membrane transporter substrate specificity is useful for the design of novel siderophore-antibiotic conjugates that can exploit nutrient uptake pathways to kill challenging Gram-negative pathogens.

ABSTRACT Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the mbt gene cluster, but the role of this gene cluster in the adaption of M. abscessus to iron limitation is not characterized. We identified an M. abscessus Tn_mutant with interruption of the mbtE gene (MAB_2248c), a central component of mycobactin biosynthesis. We tested this isolate growth characteristic, dependency on supplements, and transcriptomic response, comparing it to the response of wild-type (WT) bacteria in iron-limiting conditions. We also compare the structure of the mbt gene cluster across several mycobacteria. The Tn_mbtE mutant had a substantial, but not absolute, growth defect, which was more substantial in iron-limited media. Supplementation with mycobactin-J, hemin, blood, and surprisingly, albumin, salvaged the poor growth. Similarly, secreted mature (carboxy)-mycobactins from WT bacteria rescued the Tn_mbtE mutant during iron deprivation. The transcriptomic response of the Tn_mbtE mutant involved the upregulation of genes known to be implicated in iron homeostasis and was comparable to that of WT bacteria grown in iron-limiting conditions. Interestingly, the response was not identical to the response of M. tuberculosis to iron limitation. The mbt gene cluster and mycobactins play important roles in the physiology of M. abscessus. (Carboxy)-mycobactin is secreted from WT bacteria and can serve as “public good.” The role of several iron-homeostasis related genes (like ideR) may differ between M. abscessus and Mtb. IMPORTANCE Mycobacterium abscessus is an emerging human pathogen belonging to the nontuberculous mycobacteria (NTM) family, causing severe pulmonary disease in compromised individuals. How this bacterium acquires iron is poorly understood. Here, we provide the first characterization of the role(s) the mbtE gene required for the biosynthesis of siderophore mycobactin in M. abscessus. We show that the gene mbtE is required for growth during iron deprivation and can be compensated by several supplements, including, surprisingly, albumin. We also show the transcriptomic response of the mbtE-mutant is comparable to the response of the parental strain to iron starvation and seems different from the response of M. tuberculosis. These results indicate the importance of studying mycobactin in M. abscessus and NTM strains. Understanding this pathway is central to understanding the acquisition of iron within hosts and its role in pathogenesis, which in turn may facilitate the development of antimycobacterial therapeutics.

Iron is an essential micronutrient for most living organisms, including cyanobacteria. These microorganisms have been found in Earth's driest polar and non-polar deserts, including the Atacama Desert, Chile. Iron-containing minerals were identified in colonized rock substrates from the Atacama Desert, however, the interactions between microorganisms and iron minerals remain unclear. In the current study, we determined that colonized gypsum rocks collected from the Atacama Desert contained both magnetite and hematite phases. A cyanobacteria isolate was cultured on substrates consisting of gypsum with embedded magnetite nanoparticles. Transmission electron microscopy imaging revealed a significant reduction in the size of magnetite nanoparticles due to their dissolution, which occurred around the microbial biofilms. Concurrently, hematite was detected, likely from the oxidation of the magnetite nanoparticles. Higher cell counts and production of siderophores were observed in cultures with magnetite nanoparticles suggesting that cyanobacteria were actively acquiring iron from the magnetite nanoparticles. Magnetite dissolution and iron acquisition by the cyanobacteria was further confirmed using large bulk magnetite crystals, uncovering a survival strategy of cyanobacteria in these extreme environments.

Acinetobacter baumannii is an opportunistic pathogen that has become difficult to eradicate mainly because of its high level of antibiotic resistance. Other features that contribute to this organism's success are the ability to compete for nutrients and iron. Recently, several novel Tn7-family transposons that encode synthesis and transport of siderophore and iron uptake systems were characterised. Here, another Tn7-type transposon (named Tn6553) is described. Tn6553 contains a set of iron utilisation genes with a transposition module related to Tn7. Tn7-family transposons that carry iron uptake systems facilitate the spread of these functions in Acinetobacter strains. Given that Tn7 is known to transpose efficiently into its preferred target site, finding siderophore functions on Tn7 family transposons is important in the context of dissemination of virulence genes amongst Acinetobacter strains. [ABSTRACT FROM AUTHOR]

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted hemebinding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare. [ABSTRACT FROM AUTHOR]