Synthesis of Biosynthetic Intermediates of Vibrioferrin and Enzyme Reactions Using Them as Substrates.

Biosynthetic intermediates of siderophore vibrioferrin (VF), O-citryl-L-serine, 2-aminoethyl citrate, and alanine-2-amidoethyl citrate were respectively synthesized as a mixture of stereoisomers. These compounds were used as substrates for enzyme reactions using recombinant PvsA, PvsB, and PvsE proteins as corresponding enzyme equivalents. The results of our study show that each enzyme reacts with a respective substrate and produces VF along the proposed biosynthetic pathway. Furthermore, the results of this study will contribute to the understanding of VF biosynthetic enzymes and may help in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis.

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the 'phosphorylated' state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.

Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis.

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.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Deciphering Trifolium pratense L. holobiont reveals a microbiome resilient to future climate changes.

The plant microbiome supports plant growth, fitness, and resistance against climate change. Trifolium pratense (red clover), an important forage legume crop, positively contributes to ecosystem sustainability. However, T. pratense is known to have limited adaptive ability toward climate change. Here, the T. pratense microbiomes (including both bacteria and fungi) of the rhizosphere and the root, shoot, and flower endospheres were comparatively examined using metabarcoding in a field located in Central Germany that mimics the climate conditions projected for the next 50-70 years in comparison with the current climate conditions. Additionally, the ecological functions and metabolic genes of the microbial communities colonizing each plant compartment were predicted using FUNGuild, FAPROTAX, and Tax4Fun annotation tools. Our results showed that the individual plant compartments were colonized by specific microbes. The bacterial and fungal community compositions of the belowground plant compartments did not vary under future climate conditions. However, future climate conditions slightly altered the relative abundances of specific fungal classes of the aboveground compartments. We predicted several microbial functional genes of the T. pratense microbiome involved in plant growth processes, such as biofertilization (nitrogen fixation, phosphorus solubilization, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production). Our findings indicated that T. pratense microbiomes show a degree of resilience to future climate changes. Additionally, microbes inhabiting T. pratense may not only contribute to plant growth promotion but also to ecosystem sustainability.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

Easy detection of siderophore production in diluted growth media using an improved CAS reagent.

Siderophores are low molecular weight organic compounds produced by various microorganisms, especially pathogenic bacteria including rhizobacteria, and have a high affinity for iron. Although most microorganisms are thought to secrete siderophores under iron-depleted conditions, it is unclear how many microorganisms produce siderophores in the natural environment. Also, the chrome azurol sulfonate (CAS) assay, which is widely used for the detection of siderophores, needs to be improved for wider applicability. We developed a simple, high-throughput CAS assay in a 96-well microplate with a concentrated CAS reagent and commonly used diluted growth media in the absence of artificial iron depletion. The improved microplate CAS shuttle assay revealed that it could easily detect siderophores released from Pseudomonas (P.) fluorescence, P. putida, Burlkholderia stabilis, and Ottowia oryzae, as models of siderophore-producing bacteria. This CAS shuttle assay employed along with diluted growth media is a promising tool to screen new siderophore-producing bacteria.

Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX.

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe3+), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe3+ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (-Fe3+) and 8-fold (+Fe3+) higher than that of the wild-type A. bisporus grown without Fe3+ ions, while no siderophore was detected in the wild-type supernatant grown with Fe3+. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

Cyanochelins, an Overlooked Class of Widely Distributed Cyanobacterial Siderophores, Discovered by Silent Gene Cluster Awakening.

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring ß-hydroxyaspartate (ß-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of ß-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe3+ to Fe2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread ß-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, ß-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

Scorpion Venom Antimicrobial Peptides Induce Siderophore Biosynthesis and Oxidative Stress Responses in Escherichia coli.

The increasing development of microbial resistance to classical antimicrobial agents has led to the search for novel antimicrobials. Antimicrobial peptides (AMPs) derived from scorpion and snake venoms offer an attractive source for the development of novel therapeutics. Smp24 (24 amino acids [aa]) and Smp43 (43 aa) are broad-spectrum AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio mauruspalmatus and subsequently characterized. Using a DNA microarray approach, we examined the transcriptomic responses of Escherichia coli to subinhibitory concentrations of Smp24 and Smp43 peptides following 5 h of incubation. Seventy-two genes were downregulated by Smp24, and 79 genes were downregulated by Smp43. Of these genes, 14 genes were downregulated in common and were associated with bacterial respiration. Fifty-two genes were specifically upregulated by Smp24. These genes were predominantly related to cation transport, particularly iron transport. Three diverse genes were independently upregulated by Smp43. Strains with knockouts of differentially regulated genes were screened to assess the effect on susceptibility to Smp peptides. Ten mutants in the knockout library had increased levels of resistance to Smp24. These genes were predominantly associated with cation transport and binding. Two mutants increased resistance to Smp43. There was no cross-resistance in mutants resistant to Smp24 or Smp43. Five mutants showed increased susceptibility to Smp24, and seven mutants showed increased susceptibility to Smp43. Of these mutants, formate dehydrogenase knockout (fdnG) resulted in increased susceptibility to both peptides. While the electrostatic association between pore-forming AMPs and bacterial membranes followed by integration of the peptide into the membrane is the initial starting point, it is clear that there are numerous subsequent additional intracellular mechanisms that contribute to their overall antimicrobial effect.IMPORTANCE The development of life-threatening resistance of pathogenic bacteria to the antibiotics typically in use in hospitals and the community today has led to an urgent need to discover novel antimicrobial agents with different mechanisms of action. As an ancient host defense mechanism of the innate immune system, antimicrobial peptides (AMPs) are attractive candidates to fill that role. Scorpion venoms have proven to be a rich source of AMPs. Smp24 and Smp43 are new AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, and these peptides can kill a wide range of bacterial pathogens. By better understanding how these AMPs affect bacterial cells, we can modify their structure to make better drugs in the future.

Photoactive siderophores: Structure, function and biology.

It is well known that bacteria and fungi have evolved sophisticated systems for acquiring the abundant but biologically inaccessible trace element iron. These systems are based on high affinity Fe(III)-specific binding compounds called siderophores which function to acquire, transport, and process this essential metal ion. Many hundreds of siderophores are now known and their numbers continue to grow. Extensive studies of their isolation, structure, transport, and molecular genetics have been undertaken in the last three decades and have been comprehensively reviewed many times. In this review we focus on a unique subset of siderophores that has only been recognized in the last 20 years, namely those whose iron complexes display photoactivity. This photoactivity, which typically results in the photooxidation of the siderophore ligand with concomitant reduction of Fe(III) to Fe(II), seemingly upsets the siderophore paradigm of forming and transporting only extremely stable Fe(III) complexes into microbial cells. Here we review their structure, synthesis, photochemistry, photoproduct coordination chemistry and explore the potential biological and ecological consequences of this photoactivity.

An Assessment of Siderophore Production, Mucoviscosity, and Mouse Infection Models for Defining the Virulence Spectrum of Hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) bacteria are more virulent than classical K. pneumoniae (cKp) with resultant differences in clinical manifestations and management. It is unclear whether all hvKp isolates share a similar pathogenic potential. This report assessed the utility of siderophore production, mucoviscosity, and murine infection for defining the virulence spectrum of hvKp. Three strain cohorts were identified and defined based on the CD1 mouse subcutaneous (SQ) challenge model: (i) fully virulent hvKp strains (fvhvKp), lethal at a challenge inoculum (CI) of ≤103 CFU; (ii) partially virulent hvKp strains (pvhvKp), lethal at a CI of >103 to 107 CFU; (iii) classical K. pneumoniae, not lethal at a CI of 107 CFU. Quantitative siderophore and mucoviscosity assays differentiated fvhvKp and pvhvKp strains from cKp strains but were unable to differentiate between the fvhvKP and pvhvKP strain cohorts. However, SQ challenge of CD1 mice and intraperitoneal (IP) challenge of CD1 and BALB/c mice, but not C57BL/6 mice, were able to discriminate between an fvhvKp and a pvhvKp strain; SQ challenge of CD1 mice may have the greatest sensitivity. cKp was differentiated from hvKp both by SQ challenge of CD1 mice and IP challenge of all three mouse strains. These data identify a means to define the relative virulence of hvKP strains. It remains unclear whether the observed differences of hvKp virulence in mice translates to human infection. However, these data can be used to sort random collections of K. pneumoniae strains into fvhvKp and pvhvKp strain cohorts and assess for differences in clinical manifestations and outcomes.IMPORTANCE The pathogenic potential of hvKp strains is primarily mediated by a large virulence plasmid. The minimal set of genes required for the full expression of the hypervirulent phenotype is undefined. A number of reports describe hvKp strains possessing only a portion of the virulence plasmid; the clinical consequences of this are unclear. Therefore, the goal of this report was to determine whether virulence among hvKp strains varied and, if so, how to best identify the relative virulence of hvKp isolates. Data demonstrate hvKp pathogenic potential varies in CD1 and BALB/c murine infection models. In contrast, measurements of siderophore production and mucoviscosity were unable to discriminate the differences in hvKp isolate virulence observed in mice. This information can be used in future studies to determine the mechanisms responsible for differences between fully virulent hvKp and partially virulent hvKp and whether the differences observed in mice translate to disease in humans.

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.

Biosurfactant based formulation of Pseudomonas guariconensis LE3 with multifarious plant growth promoting traits controls charcoal rot disease in Helianthus annus.

Biosurfactants are environment compatible surface-active biomolecules with multifunctional properties which can be utilized in various industries. In this study a biosurfactant producing novel plant growth promoting isolate Pseudomonas guariconensis LE3 from the rhizosphere of Lycopersicon esculentum is presented as biostimulant and biocontrol agent. Biosurfactant extracted from culture was characterized to be mixture of various mono- and di-rhamnolipids with antagonistic activity against Macrophomina phaseolina, causal agent of charcoal rot in diverse crops. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) analysis confirmed the rhamnolipid nature of biosurfactant. PCR analysis established the presence of genes involved in synthesis of antibiotics diacetylphloroglucinol, phenazine 1-carboxylic acid and pyocyanin, and lytic enzymes chitinase and endoglucanase suggesting biocontrol potential of the isolate. Plant growth promoting activities shown by LE3 were phosphate solubilization and production of siderophores, indole acetic acid (IAA), ammonia and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). To assemble all the characteristics of LE3 various bioformuations were developed. Amendment of biosurfactant in bioformulation of LE3 cells improved the shelf life. Biosurfactant amended formulation of LE3 cells was most effective in biocontrol of charcoal rot disease of sunflower and growth promotion in field conditions. The root adhered soil mass of plantlets inoculated with LE3 plus biosurfactant was significantly higher over control. Biosurfactant amended formulation of LE3 cells caused maximum yield enhancement (80.80%) and biocontrol activity (75.45%), indicating that addition of biosurfactant improves the plant-bacterial interaction and soil properties leading to better control of disease and overall improvement of plant health and yield.

Healthy Cotwins Share Gut Microbiome Signatures With Their Inflammatory Bowel Disease Twins and Unrelated Patients.

BACKGROUND & AIMS: It is currently unclear whether reported changes in the gut microbiome are cause or consequence of inflammatory bowel disease (IBD). Therefore, we studied the gut microbiome of IBD-discordant and -concordant twin pairs, which offers the unique opportunity to assess individuals at increased risk of developing IBD, namely healthy cotwins from IBD-discordant twin pairs. METHODS: Fecal samples were obtained from 99 twins (belonging to 51 twin pairs), 495 healthy age-, sex-, and body mass index-matched controls, and 99 unrelated patients with IBD. Whole-genome metagenomic shotgun sequencing was performed. Taxonomic and functional (pathways) composition was compared among healthy cotwins, IBD-twins, unrelated patients with IBD, and healthy controls with multivariable (ie, adjusted for potential confounding) generalized linear models. RESULTS: No significant differences were observed in the relative abundance of species and pathways between healthy cotwins and their IBD-twins (false discovery rate <0.10). Compared with healthy controls, 13, 19, and 18 species, and 78, 105, and 153 pathways were found to be differentially abundant in healthy cotwins, IBD-twins, and unrelated patients with IBD, respectively (false discovery rate <0.10). Of these, 8 (42.1%) of 19 and 1 (5.6%) of 18 species, and 37 (35.2%) of 105 and 30 (19.6%) of 153 pathways overlapped between healthy cotwins and IBD-twins, and healthy cotwins and unrelated patients with IBD, respectively. Many of the shared species and pathways have previously been associated with IBD. The shared pathways include potentially inflammation-related pathways, for example, an increase in propionate degradation and L-arginine degradation pathways. CONCLUSIONS: The gut microbiome of healthy cotwins from IBD-discordant twin pairs displays IBD-like signatures. These IBD-like microbiome signatures might precede the onset of IBD. However, longitudinal follow-up studies are needed to infer a causal relationship.

The nucleoid-associated protein IHF acts as a 'transcriptional domainin' protein coordinating the bacterial virulence traits with global transcription.

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

Structural and Biosynthetic Analysis of the Fabrubactins, Unusual Siderophores from Agrobacterium fabrum Strain C58.

Siderophores are iron-chelating molecules produced by microorganisms and plants to acquire exogenous iron. Siderophore biosynthetic enzymology often produces elaborate and unique molecules through unusual reactions to enable specific recognition by the producing organisms. Herein, we report the structure of two siderophore analogs from Agrobacterium fabrum strain C58, which we named fabrubactin (FBN) A and FBN B. Additionally, we characterized the substrate specificities of the NRPS and PKS components. The structures suggest unique Favorskii-like rearrangements of the molecular backbone that we propose are catalyzed by the flavin-dependent monooxygenase, FbnE. FBN A and B contain a 1,1-dimethyl-3-amino-1,2,3,4-tetrahydro-7,8-dihydroxy-quinolin (Dmaq) moiety previously seen only in the anachelin cyanobacterial siderophores. We provide evidence that Dmaq is derived from l-DOPA and propose a mechanism for the formation of the mature Dmaq moiety. Our bioinformatic analyses suggest that FBN A and B and the anachelins belong to a large and diverse siderophore family widespread throughout the Rhizobium/Agrobacterium group, α-proteobacteria, and cyanobacteria.