Feature sequence-based genome mining uncovers the hidden diversity of bacterial siderophore pathways.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

Identification and characterization of the siderochelin biosynthetic gene cluster via coculture.

Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs.

Predicting metallophore structure and function through genome mining.

Metallophores are small molecule chelators that many microbes use to obtain trace metals from their environment. Through genome mining, where genomes are scanned for metallophore biosynthesis genes, one can not only identify which organisms are likely to produce a metallophore, but also predict the metallophore structure, thus preventing undesired reisolation of known compounds and accelerating characterization. Furthermore, the presence of accessory genes for the transport, utilization, and regulation can suggest the biological function and fate of a metallophore. Modern, user-friendly tools have made powerful genomic analyses accessible to scientists with no bioinformatics experience, but these tools are often not utilized to their full potential. This chapter provides an introduction to metallophore genomics and demonstrates how to use the free, publicly available antiSMASH platform to infer metallophore function and structure.

A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

Functional and Evolutionary Integration of a Fungal Gene With a Bacterial Operon.

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determine the extant distribution of yeast enterobactin producers and cheaters.

Characterization and Statistical Optimization of Enterobatin Synthesized by Escherichia coli OQ866153.

Microorganisms produce siderophores, which are secondary metabolites with a high affinity for iron. Siderophores have received significant attention due to their diverse applications in ecological and clinical research. In this study, siderophores production by Escherichia coli OQ866153 was optimized using two-stage statistical approach involving Plackett-Burman design (PBD) and response surface methodology (RSM) using central composite design (CCD). Out of 23 variables, succinate, tryptophan, Na2HPO4, CaCl2, agitation, and KH2PO4 were found to have the most significant effect on siderophores production in the first optimization stage with the highest SU% of 43.67%. In the second stage, RSM using CCD was utilized, and the optimal conditions were determined to be 0.3 g/l succinate, 0 g/l tryptophan, 6 g/l Na2HPO4, 0.1 g/l CaCl2, 150 RPM agitation, and 0.6 g/l KH2PO4, resulting in a maximum siderophore units (SU%) of 89.13%. The model was significant, as indicated by the model f-value of 314.14 (p-value = 0.0004) and coefficient of determination R2 of 0.9950. During validation experiments, the obtained maximum SU% was increased up to 87.1472%, which was two times as the value obtained under ordinary conditions (46.62%). The produced siderophores were purified and characterized using 1H, 13C NMR, IR spectroscopy. The obtained results indicated that the compound was enterobactin and entABCDEF genes were further detected in Escherichia coli OQ866153 extracted DNA. To our knowledge, this is the first report of statistical optimization for enterobactin synthesis by an E. coli strain isolated from a clinical source in Egypt.

The Bradyrhizobium japonicum exporter ExsFGH is involved in efflux of ferric xenosiderophores from the periplasm.

The gram-negative bacterium Bradyrhizobium japonicum can take up structurally dissimilar ferric siderophores from the environment (xenosiderophores) to meet its nutritional iron requirements. Siderophore-bound iron transported into the periplasm is reduced to the ferrous form by FsrB, dissociated from the siderophore and the free ion is then transported into the cytoplasm by the ferrous iron transporter FeoAB. Here, we identified the RND family exporter genes exsFG and exsH in a selection for secondary site suppressor mutants that restore growth of an fsrB mutant on the siderophores ferrichrome or ferrioxamine. The low level of radiolabel accumulation from 55Fe-labeled ferrichrome or ferrioxamine observed in the fsrB mutant was restored to wild type levels in the fsrB exsG mutant. Moreover, the exsG mutant accumulated more radiolabel from the 55Fe-labeled siderophores than the wild type, but radiolabel accumulation from inorganic 55Fe was similar in the two strains. Thus, ExsFGH exports siderophore-bound iron, but not inorganic iron. The rescued fsrB exsG mutant required feoB for growth, indicating that ExsFGH acts on those siderophores in the periplasm. The exsG mutant was more sensitive to the siderophore antibiotic albomycin than the wild type, whereas the fsrB mutant was more resistant. This suggests ExsFGH normally exports ferrated albomycin. B. japonicum is naturally resistant to many antibiotics. The exsG strain was very sensitive to tetracycline, but not to six other antibiotics tested. We conclude that ExsFGH is a broad substrate exporter that is needed to maintain siderophore homeostasis in the periplasm.

An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 with 7-hydroxytropolone is pivotal for its antibacterial activity.

Pseudomonas donghuensis P482 exhibits broad antimicrobial activity against phytopathogens, including the soft rot bacteria of the Dickeya genus. Here, we report that under limited nutrient availability, the antibacterial activity of P. donghuensis P482 against Dickeya solani requires the reciprocal action of two iron scavengers: 7-hydroxytropolone (7-HT) and a newly characterized pyoverdine (PVDP482 ) and is quenched in the iron-augmented environment. Further, we show that the biosynthesis of pyoverdine and 7-HT is metabolically coordinated, and the functional BV82_4709 gene involved in 7-HT synthesis is pivotal for expressing the BV82_3755 gene, essential for pyoverdine biosynthesis and vice versa. The synthesis of both scavengers is under the control of Gac/Rsm, but only PVD is controlled by Fur. The isoelectric focusing profile of the P482 siderophore differs from that of the other Pseudomonas spp. tested. This finding led to the unveiling of the chemical structure of the new pyoverdine PVDP482 . To summarize, the antibacterial activity of P. donghuensis P482 is attributed to 7-HT and PVDP482 varies depending on the nutrient and iron availability, highlighting the importance of these factors in the competition between P482 and D. solani.

Effects of iron concentration and DFB (Desferrioxamine-B) on transcriptional profiles of an ecologically relevant marine bacterium.

Research into marine iron cycles and biogeochemistry has commonly relied on the use of chelators (including siderophores) to manipulate iron bioavailability. To test whether a commonly used chelator, desferrioxamine B (DFB) caused effects beyond changing the iron-status of cells, cultures of the environmentally relevant marine heterotrophic bacterium, Ruegeria pomeroyii, were grown in media with different concentrations of iron and/or DFB, resulting in a gradient of iron availability. To determine how cells responded, transcriptomes were generated for cells from the different treatments and analyzed to determine how cells reacted to these to perturbations. Analyses were also performed to look for cellular responses specific to the presence of DFB in the culture medium. As expected, cells experiencing different levels of iron availability had different transcriptomic profiles. While many genes related to iron acquisition were differentially expressed between treatments, there were many other genes that were also differentially expressed between different sample types, including those related to the uptake and metabolism of other metals as well as genes related to metabolism of other types of molecules like amino acids and carbohydrates. We conclude that while DFB certainly altered iron availability to cells, it also appears to have had a general effect on the homeostasis of other metals as well as influenced metabolic processes outside of metal acquisition.

Experimental and computational methods to highlight behavioural variations in TonB-dependent transporter expression in Pseudomonas aeruginosa versus siderophore concentration.

Iron is a key nutrient for bacterial growth. The source can be either heme or siderophore-Fe complexes. Siderophores are small molecules synthesized by bacteria to scavenge iron from the bacterial environment. The pathogen Pseudomonas aeruginosa can express at least 15 different iron uptake pathways and all but one involve a TonB-dependent transporter (TBDT) for the uptake of iron across the outer membrane. Little is known about how bacteria modulate and adapt the expression of their different iron import pathways according to their environment. Here, we have developed fluorescent reporters between the promoter region of genes encoding a TBDT and the fluorescent reporter mCherry. With these constructs, we can follow the expression of TBDTs under different growth conditions. Mathematical modelling of the data obtained showed the transcription and expression of the gene encoding the TBDT PfeA to have a sigmoidal shape, whereas it was logarithmic for the TBDT gene foxA. Maximum transcription for pfeA was reached in the presence of 3 µM enterobactin, the siderophore recognized by PfeA, whereas the maximum was not reached for foxA with 100 µM nocardamine, the siderophore of FoxA.

HapX-mediated H2B deub1 and SreA-mediated H2A.Z deposition coordinate in fungal iron resistance.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.

Regulation of High-Affinity Iron Acquisition, Including Acquisition Mediated by the Iron Permease FtrA, Is Coordinated by AtrR, SrbA, and SreA in Aspergillus fumigatus.

Iron acquisition is crucial for virulence of the human pathogen Aspergillus fumigatus. Previous studies indicated that this mold regulates iron uptake via both siderophores and reductive iron assimilation by the GATA factor SreA and the SREBP regulator SrbA. Here, characterization of loss of function as well as hyperactive alleles revealed that transcriptional activation of iron uptake depends additionally on the Zn2Cys6 regulator AtrR, most likely via cooperation with SrbA. Mutational analysis of the promoter of the iron permease-encoding ftrA gene identified a 210-bp sequence, which is both essential and sufficient to impart iron regulation. Further studies located functional sequences, densely packed within 75 bp, that largely resemble binding motifs for SrbA, SreA, and AtrR. The latter, confirmed by chromatin immunoprecipitation (ChIP) analysis, is the first one not fully matching the 5'-CGGN12CCG-3' consensus sequence. The results presented here emphasize for the first time the direct involvement of SrbA, AtrR, and SreA in iron regulation. The essential role of both AtrR and SrbA in activation of iron acquisition underlines the coordination of iron homeostasis with biosynthesis of ergosterol and heme as well as adaptation to hypoxia. The rationale is most likely the iron dependence of these pathways along with the enzymatic link of biosynthesis of ergosterol and siderophores. IMPORTANCE Aspergillus fumigatus is the most common filamentous fungal pathogen infecting humans. Iron acquisition via siderophores has previously been shown to be essential for virulence of this mold species. Here, we demonstrate that AtrR, a transcription factor previously shown to control ergosterol biosynthesis, azole resistance, and adaptation to hypoxia, is essential for activation of iron acquisition, including siderophore biosynthesis and uptake. Dissection of an iron-regulated promoter identified binding motifs for AtrR and the two previously identified regulators of iron acquisition, SrbA and SreA. Altogether, this study identified a new regulator required for maintenance of iron homeostasis, revealed insights into promoter architecture for iron regulation, and emphasized the coordinated regulation of iron homeostasis ergosterol biosynthesis and adaptation to hypoxia.

Discovery of New Siderophores from a Marine Streptomycetes sp. via Combined Metabolomics and Analysis of Iron-Chelating Activity.

The marine-derived Streptomyces sp. FIMYZ-003 strain was found to produce novel siderophores with yields negatively correlated with the iron concentration in the medium. Mass spectrometry (MS)-based metabolomics coupled with metallophore assays identified two novel α-hydroxycarboxylate-type siderophores, fradiamines C and D (3 and 4), together with two related known siderophores, fradiamines A and B (1 and 2). Their chemical structures were elucidated by nuclear magnetic resonance (NMR) and MS experiments. The annotation of a putative fra biosynthetic gene cluster enabled us to propose the biosynthetic pathway of fradiamines A-D. Furthermore, the solution-phase iron-binding activity of fradiamines was evaluated using metabolomics, confirming them as general iron scavengers. Fradiamines A-D exhibited Fe(III) binding activity equivalent to that of deferoxamine B mesylate. Growth analysis of pathogenic microbes demonstrated that fradiamine C promoted the growth of Escherichia coli and Staphylococcus aureus, but fradiamines A, B, and D did not. The results indicate that fradiamine C may serve as a novel iron carrier applicable to antibiotic delivery strategies to treat and prevent foodborne pathogens.

De novogenomic analysis ofEnterobacter asburiaeEBRJ12, a plant growth-promoting rhizobacteria isolated from the rhizosphere of Phaseolus vulgarisL.

AIM: Environmental stresses such as water deficit induced stress are one of the major limiting factors in crop production. However, some plant growth-promoting rhizobacteria (PGPR) can promote plant growth in such adverse condition. Therefore, the objective was to isolate rhizospheric bacteria from Phaseolus vulgaris L. growing in a drought-affected soil and to analyze its plant growth promoting (PGP) efficacy to black gram (Vigna mungo L.) and Bhut jolokia (Capsicum chinense Jacq.). Whole-genome sequencing of the potential bacteria was targeted to analyze the genetic potential of the isolate as a plant growth-promoting agent. METHODS AND RESULTS: The isolate Enterobacter asburiae EBRJ12 was selected based on its PGP efficacy, which significantly improved plant growth and development. The genomic analysis revealed the presence of one circular chromosome of size 4.8 Mb containing 16 genes for osmotic stress regulation including osmotically inducible protein osmY, outer membrane protein A precursor ompA, aquaporin Z, and an operon for osmoprotectant ABC transporter yehZYXW. Moreover, the genome has a complete genetic cluster for biosynthesis of siderophore Enterobactin and siderophore Aerobactin.The PGP effects were verified with black gram and Bhut jolokia in pot experiments. The isolate significantly increased the shoot length by 35.0% and root length by 58.0% of black gram, while 41.0% and 57.0% of elevation in shoot and root length were observed in Bhut jolokia compared to non-inoculated plants. CONCLUSIONS: The EBRJ12 has PGP features that could improve the growth in host plants, and the genomic characterization revealed the presence of genetic potential for plant growth promotion.

Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections.

Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

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.

Mining elements of siderophore chirality encoded in microbial genomes.

The vast majority of bacteria require iron to grow. A significant iron acquisition strategy is the production of siderophores, which are secondary microbial metabolites synthesized to sequester iron(III). Siderophore structures encompass a variety of forms, of which highly modified peptidic siderophores are of interest herein. State-of-the-art genome mining tools, such as antiSMASH (antibiotics & Secondary Metabolite Analysis SHell), hold the potential to predict and discover new peptidic siderophores, including a combinatoric suite of triscatechol siderophores framed on a triserine-ester backbone of the general class, (DHB-l/d CAA-l Ser)3 (CAA, cationic amino acid). Siderophores with l/d Arg, l/d Lys and l Orn, but not d Orn, were predicted in bacterial genomes. Fortuitously the d Orn siderophore was identified, yet its lack of prediction highlights the limitation of current genome mining tools. The full combinatoric suite of these siderophores, which form chiral iron(III) complexes, reveals stereospecific coordination chemistry encoded in microbial genomes. The chirality embedded in this suite of Fe(III)-siderophores raises the question of whether the relevant siderophore-mediated iron acquisition pathways are stereospecific and selective for ferric siderophore complexes of a defined configuration.

In silico analysis of comparative affinity of phytosiderophore and bacillibactin for iron uptake by YSL15 and YSL18 receptors of Oryza sativa.

Iron is an important micronutrient for plant growth and development. In the case of Oryza sativa, iron is made available primarily with the help of iron chelators called phytosiderophores i.e. variants of deoxymugineic acid (DMA). They bind with ferric ions and get internalized through Yellow Stripe Like transporters viz. YSL15 and YSL18. However, due to low amount of secretion of phytosiderophores, rice suffers from iron deficiency. Alternatively, siderophores of plant growth promoting rhizobacteria may support iron uptake and make it available to plants via transporting ferric ions possibly through the same transporters. Present study aims to assess comparative binding of DMA and a xenosiderophore (siderophores used by organisms other than the ones producing them) of rhizobacteria i.e. bacillibactin with Fe3+ ion and subsequent transporters of rice. Protein-protein interaction and gene expression analysis predicts uptake of Fe3+ by YSL15 from the rhizosphere region and further distribution through YSL18 with the help of various predicted functional partners. Docking studies confirm the thermodynamically more favourable structure of bacillibactin-Fe3+ complex than DMA-Fe3+ complex. Molecular modelling of YSL15 and YSL18 was done through ab initio method and their evaluation by Ramachandran plot, ProSA, ERRAT value and verify 3 D score revealed a good quality models. Comparative binding assessment through docking and molecular dynamics simulation suggests better binding energies of YSL transporters with bacillibactin-Fe3+ complex as compared to DMA-Fe3+ complex. The current study suggests possible application of xenosiderophores of PGPR origin in supporting plant growth via iron uptake and distribution in rice.Communicated by Ramaswamy H. Sarma.

Genome mining, antimicrobial and plant growth-promoting potentials of halotolerant Bacillus paralicheniformis ES-1 isolated from salt mine.

Salinity severely affects crop yield by hindering nitrogen uptake and reducing plant growth. Plant growth-promoting bacteria (PGPB) are capable of providing cross-protection against biotic/abiotic stresses and facilitating plant growth. Genome-level knowledge of PGPB is necessary to translate the knowledge into a product as efficient biofertilizers and biocontrol agents. The current study aimed to isolate and characterize indigenous plant growth-promoting strains with the potential to promote plant growth under various stress conditions. In this regard, 72 bacterial strains were isolated from various saline-sodic soil/lakes; 19 exhibited multiple in vitro plant growth-promoting traits, including indole 3 acetic acid production, phosphate solubilization, siderophore synthesis, lytic enzymes production, biofilm formation, and antibacterial activities. To get an in-depth insight into genome composition and diversity, whole-genome sequence and genome mining of one promising Bacillus paralicheniformis strain ES-1 were performed. The strain ES-1 genome carries 12 biosynthetic gene clusters, at least six genomic islands, and four prophage regions. Genome mining identified plant growth-promoting conferring genes such as phosphate solubilization, nitrogen fixation, tryptophan production, siderophore, acetoin, butanediol, chitinase, hydrogen sulfate synthesis, chemotaxis, and motility. Comparative genome analysis indicates the region of genome plasticity which shapes the structure and function of B. paralicheniformis and plays a crucial role in habitat adaptation. The strain ES-1 has a relatively large accessory genome of 649 genes (~ 19%) and 180 unique genes. Overall, these results provide valuable insight into the bioactivity and genomic insight into B. paralicheniformis strain ES-1 with its potential use in sustainable agriculture.

Genomic insight into iron acquisition by sulfate-reducing bacteria in microaerophilic environments.

Historically, sulfate-reducing bacteria (SRB) have been considered to be strict anaerobes, but reports in the past couple of decades indicate that SRB tolerate exposure to O2 and can even grow in aerophilic environments. With the transition from anaerobic to microaerophilic conditions, the uptake of Fe(III) from the environment by SRB would become important. In evaluating the metabolic capability for the uptake of iron, the genomes of 26 SRB, representing eight families, were examined. All SRB reviewed carry genes (feoA and feoB) for the ferrous uptake system to transport Fe(II) across the plasma membrane into the cytoplasm. In addition, all of the SRB genomes examined have putative genes for a canonical ABC transporter that may transport ferric siderophore or ferric chelated species from the environment. Gram-negative SRB have additional machinery to import ferric siderophores and ferric chelated species since they have the TonB system that can work alongside any of the outer membrane porins annotated in the genome. Included in this review is the discussion that SRB may use the putative siderophore uptake system to import metals other than iron.