Determination of bacterial viability by selective capture using surface-bound siderophores

A significant challenge in bacterial detection is the identification of viable bacteria over debris, specifically post decontamination. Of increasing concern are antibiotic resistant strains that require accurate and rapid post decontamination analysis. Current strategies are fraught with disadvantages and most of them are not selective for viable bacteria. However, bacteria are critically dependent upon iron sequestration, synthesizing and releasing siderophores (SDPs) to tightly bind iron, with the subsequent uptake of iron bound SDPs. This is a highly conserved process that occurs only in intact bacteria. Herein we report a facile method to use bacterial SDPs to selectively and rapidly identify only viable bacteria in complex matrices, and discriminate them from their dead counterparts. Desferrioxamine B (Desf B) tethered to a glass slide is used to specifically capture viable bacteria from a mixture of viable and dead Escherichia coli, as demonstrated by fluorescence microscopy. We re- port both direct conjugation of Desf B on thin-film-coated glass slides as well as biotin-streptavidin conjugation strategies, both of which are successful in the said goal. We have analyzed the density of images obtained upon fluorescence staining using edge detection with a Canny edge detector. This novel application of a software analysis tool originally developed for satellite imaging to biological staining allows for accurate quantitation of observed data.

Nobachelins,new siderophores from Nocardiopsis baichengensis protecting Caenorhabditis elegans from Pseudomonas aeruginosa infection

The biosynthetic potential of actinobacteria to produce novel natural products is still regarded as immense.In this paper,we correlated a cryptic biosynthetic gene cluster to chemical molecules by genome mining and chemical analyses,leading to the discovery of a new group of catecholate-hydroxamate siderophores,nobachelins,from Nocardiopsis baichengensis DSM 44845.Nobachelin biosynthesis genes are conserved in several bacteria from the family Nocardiopsidaceae.Structurally,nobachelins feature fatty-acylated hydroxy-ornithine and a rare chlorinated catecholate group.Intriguingly,nobachelins rescued Caenorhabditis elegans from Pseudomonas aeruginosa-mediated killing.

Recombineering enables genome mining of novel siderophores in a non-model Burkholderiales strain

Iron is essential for bacterial survival,and most bacteria capture iron by producing siderophores.Burkholde-riales bacteria produce various types of bioactive secondary metabolites,such as ornibactin and malleobactin siderophores.In this study,the genome analysis of Burkholderiales genomes showed a putative novel siderophore gene cluster crb,which is highly similar to the ornibactin and malleobactin gene clusters but does not have pvdF,a gene encoding a formyltransferase for N-δ-hydroxy-ornithine formylation.Establishing the bacteriophage recom-binase Redγ-Redδβ7029 mediated genome editing system in a non-model Burkholderiales strain Paraburkholderia caribensis CICC 10960 allowed the rapid identification of the products of crb gene cluster,caribactins A-F(1-6).Caribactins contain a special amino acid residue N-δ-hydroxy-N-δ-acetylornithine(haOrn),which differs from the counterpart N-δ-hydroxy-N-δ-formylornithine(hOrn)in ornibactin and malleobactin,owing to the absence of pvdF.Gene inactivation showed that the acetylation of hOrn is catalyzed by CrbK,whose homologs proba-bly not be involved in the biosynthesis of ornibactin and malleobactin,showing possible evolutionary clues of these siderophore biosynthetic pathways from different genera.Caribactins promote biofilm production and en-hance swarming and swimming abilities,suggesting that they may play crucial roles in biofilm formation.This study also revealed that recombineering has the capability to mine novel secondary metabolites from non-model Burkholderiales species.

Biochemical Insights into Siderophore Esterases

Iron is an essential but excessively toxic nutrient. Although iron is rich in nature, the acquisition of iron is a challenge to life. Its solubility is very low because it is mostly in the form of oxidation or hydroxide. In order to overcome this, microorganisms have evolved a variety of iron absorption pathways, the most important of which is the siderophore-dependent iron absorption pathway. Both bacteria and fungi require specific siderophore esterases to encourage the release of iron within the cell. A deeper understanding of siderophore esterases is crucial for the development of new antibacterial and antifungal diagnostic and therapeutic approaches. There have been many recent studies on anti-infectives via siderophore antibiotic couplers in which siderophore esterases have also played an important role, and in this review, we provide an overview of several of the more common iron carriers as well as siderophore esterases in terms of structure as well as function.

Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective:To characterize biofilm production by clinical(n=21)and environmental(n=11)isolates of Burkholderia pseudomallei and evaluate the production of proteases,hemolysins and siderophores.Methods:Initially,the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1%glucose(MH-1%glucose)and BHI broth-1%glucose,using the crystal violet staining technique.Subsequently,growing(48 h)and mature(72 h)biofilms were evaluated by confocal microscopy.Finally,the production of proteases,hemolysins and siderophores by planktonic aggregates,growing biofilms and mature biofilms was evaluated.Results:All isolates produced biofilms,but clinical isolates had significantly higher biomass in both MH-1%glucose(P<0.001)and BHI-glucose 1%(P=0.005).The structural analyses by confocal microscopy showed thick biofilms,composed of multiple layers of cells,homogeneously arranged,with mature biofilms of clinical isolates presenting higher biomass(P=0.019)and thickness of the entire area(P=0.029),and lower roughness coefficient(P=0.007)than those of environmental isolates.Protease production by growing biofilms was significantly greater than that of planktonic(P<0.001)and mature biofilms(P<0.001).Hemolysin release by planktonic aggregates was higher than that of biofilms(P<0.001).Regarding siderophores,mature biofilms presented higher production than growing biofilms(P<0.001)and planktonic aggregates(P<0.001).Conclusions:Clinical isolates have higher production of biofilms than their environmental counterparts;protease and siderophores seem important for growth and maintenance of Burkholderia pseu­domallei biofilms.

β-cyclopiazonic acid binds iron demonstrating siderophorelike activity and promotes growth in Pseudomonas aeruginosa

This chemical study reports a novel siderophore-like compound,β-cyclopiazonic acid(1,β-CPA)extracted from marine fungus Aspergillus flavus.The chemical structure ofβ-CPA was elucidated by a combination of extensive spectroscopic analyses and TDDFT-ECD calculations.The iron-binding ability and CAS assays demonstrate thatβ-CPA is a novel siderophore that features a different chemical structure from those of traditional siderophores.Theβ-CPA has no obvious influence on the growth of bacterium Pseudomonas aeruginosa PAO1.However,its iron chelator could promote the growth of P.aeruginosa PAO1,suggesting that P.aeruginosa employed siderophores to sequester iron,which is vital for their survival.The study provides the physiochemical evaluation ofβ-CPA,an unusual skeletonstructure siderophore,which for the first time,was proven to have the ability to bind iron and affect P.aeruginosa growth.This new discovery of siderophore provides an opportunity for developing novel anti-P.aeruginosa drugs.

Hypermucoviscosity in Clinical Isolates of Klebsiella pneumoniae Correlates with High Multiple Antibiotic Resistance (MAR) Index

Klebsiella pneumoniae is an opportunistic pathogen of medical importance and the capsule and mucoid phenotype in this organism are considered as requisite virulence determinants. A total of 62 clinical samples from ATBUTH were collected and screened for K. pneumoniae. The isolates were identified using standard tests for this organism. The string test was used to detect the mucoid (hypermucoviscous) phenotype and the antimicrobial susceptibility test to 10 antibiotics was carried out with the disk diffusion technique after standardizing inoculum. A K. pneumoniae prevalence of 24% (15/62) was obtained of which 47% (7/15) were mucoid (hypermucoviscous) and 53% (8/15) were non-mucoid. Colonial sizes of the two strains do not reveal any significant differences in growth fitness of the strains. On blood agar, the mucoid and non-mucoid strains had a mean colonial size ± standard deviations of 4.41 ± 0.58 mm and 4.27 ± 0.42 mm respectively. The antibiotic susceptibility rate showed that the mucoid strains compared to the non-mucoid were more resistant to nine out of 10 antibiotics. The mucoid strains were outrightly resistant to augmentin, amoxicillin, septrin, sparfloxacin and perfloxacin. The non-mucoid strains showed no complete resistant to any antibiotic tested but had a higher resistant rate to chloramphenicol only. The Multiple Antibiotic Resistance (MAR) index shows the themucoid strains with a high MAR index range of 0.7 - 1.0 with a median MAR index of 0.8, while the non-mucoid strains had a MAR index of 0.2 - 0.8 with a median MAR index of 0.35. The data suggest that the mucoid phenotype could be associated with extrachromsomal element(s) carrying resistance genes to antibiotics and that these extrachromosomal elements may not harbour resistance determinants to chloramphenicol. Furthermore, the extrachromosomal elements bearing the mucoid phenotype and the resistance elements in the mucoid strains do not significantly impact on the fitness of the cognate strain. Whether these phenotype and resistances that had no fitness cost to the bacterium could significantly affect the virulence of the bacteria in vivo remains to be investigated.

Synthesis of a Novel Chiral Trihydroxamic Acid Containing L-Pro-L-Ala-L-Ala-β-(HO)Ala-OMe Unit

An N-hydroxyl peptide unit of H-L-Pro-L-Ala-L-Ala-β-（BnO）Ala-OMe was synthesized by stepwise chain building method in solution. Then, based on nitrilotriacetic acid backbone and this N-hydroxyl peptide unit, a novel chiral tripodal ligand P as an analogue of desferrichrome, has been synthesized. In neutral pH, ligand P formed stable tri（hydroxamato）- iron（Ⅲ） complexes which showed a maximum absorptiota （∑max=2750 M^-lcm^-1） at 422 nm in UV-Vis spectra. The characteristic absorption spectra are similar to those of natural trihydroxamate-type siderophores.

鸭疫里默氏杆菌siderophore基因的克隆与表达

根据鸭疫里默氏杆菌全基因组序列,设计一对特异性引物,扩增了siderophore基因序列,克隆至pGEM-Teasy载体后进行序列测定及分析。结果表明,阅读框架全长为729bp,编码243个氨基酸。将该基因克隆到原核表达载体pET-28a后,转化大肠杆菌BL21(DE3),IPTG诱导表达,经SDS-PAGE分析结果显示,已成功表达28ku的融合蛋白。

Isolation, Screening and Molecular Characterization of Multifunctional Plant Growth Promoting Rhizobacteria for a Sustainable Agriculture

The use of PGPR as a multifunctional biofertilizer or biostimulant is an alternative way to prevent soil pollution and preserve agricultural for sustainable economy. In this study, 102 bacterial strains were isolated from rhizospheric soil of different crop fields. Among them, 15 bacterial isolates rich of NPK were selected to screen for PGP activity. It was found that 4 out 15 isolates were able to fix atmospheric nitrogen, 14 could solubilize phosphate and 5 could solubilize potassium. They were further examined for the production of hydrolytic enzymes (amylase, cellulose, chitinase, etc.), plant hormone (IAA) and plant defense substances (HCN, siderophore, etc.). All PGPR isolates were able to produce IAA, siderophore and ammonia while 2 isolates could produce HCN. Among them, 73.33% of selected isolates produced amylase, 80% produced cellulase, 66.67% produced pectinase, 93.33% produced chitinase and β-glucanase. For salt stress tolerance, all the isolates grew well in 5% NaCl while only 4 tolerated 9% NaCl. Among all isolates, 2 have antifungal activity and 5 have antibacterial activity. The best 6 isolates and consortium were tested to promote plant growth in green-gram and maize germination. Seed germination of green-gram and maize was observed the best in Acromobacter insolitus S3 compared with other treatments. Pseudomonas plecoglossicida B3 was found the best in fresh weight for both crops. The highest root formation was observed in Acromobacter insolitus S3 treatment in maize and Enterobacter hormaechei W1 treatment in green-gram.

<i>Pseudomonas aeruginosa</i>BUP2—A Novel Strain Isolated from Malabari Goat Produces Type 2 Pyoverdine

This study focuses on the isolation and characterization of a novel strain of siderophore producing bacterium, i.e., Pseudomonas aeruginosa BUP2 (Pa BUP2) from the rumen of Malabari goat, coupled with qualitative and quantitative analyses of the siderophore produced by it. Pa BUP2—a facultative anaerobe was tuned to be an aerobe by repeatedly growing in Benjamin flask. The new isolate was grown in a specially designed semi-synthetic medium, designated as BUP medium, and the yellowish-green pigment produced was identified as a typical siderophore by spectrophotometry, Chromazurol-S assay, thin layer chromatography and isolectric focusing (IEF). The characteristic orange fluorescence upon UV irradiation on chromatogram and absorption maximum at λ404 confirmed that the characteristic siderophore produced by Pa BUP2 was a typical pyoverdine (PVD). This PVD was further categorized under type 2 by comparing its profile on the IEF gel with that of the representative strains of each PVD types, viz., Pa O1, Pa ATCC 27853 and Pa6. Moreover, the type 2 PVD was purified by XAD-4 Amberlite column chromatography and quantified;maximum yield (11.17 mg/ml) was observed on day 4 of incubation (37°C). Thus, it was confirmed that the bacterium isolated from the rumen content of Malabari goat is a novel strain of Pa capable of producing large quantity of PVD type 2 in specially designed BUP medium under aerobic condition, and that its clinical and industrial implications remain elusive.

Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB

The adenosine 5'-triphosphate(ATP)-binding cassette(ABC)transporter,IrtAB,plays a vital role in the replication and viability of Mycobacterium tuberculosis(Mtb),where its function is to import iron-loaded siderophores.Unusually,it adopts the canonical type IV exporter fold.Herein,we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP,ADP,or ATP analogue(AMP-PNP)at resolutions ranging from 2.8 to 3.5Å.The structure of IrtAB bound ATP-Mg2+shows a“head-to-tail”dimer of nucleotide-binding domains(NBDs),a closed amphipathic cavity within the transmembrane domains(TMDs),and a metal ion liganded to three histidine residues of IrtA in the cavity.Cryo-electron microscopy(Cryo-EM)structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB.Moreover,the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle.This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Halophile plant growth-promoting rhizobacteria induce salt tolerance traits in wheat seedlings(Triticum aestivum L.)

Salinity is one of the most important growth-limiting factors for most crops in arid and semi-arid regions;however,the use of plant growth-promoting rhizobacteria isolated from saline soils could reduce the effects of saline stress in crops.This study aimed to evaluate the efficiency of plant growth-promoting rhizobacteria(PGPRs),isolated from the rhizosphere of halophile plants,for the growth,Na^+/K^+balance,ethylene emission,and gene expression of wheat seedlings{Triticum aestivum L.)grown under saline conditions(100 mmol L^-1 NaCl)for 14 d.A total of 118 isolates obtained from saline soils of the deserts of Iran were tested for their capacity as PGPRs.Out of the 118 isolates,17 could solubilize phosphate(Ca3(P〇4)2),5 could produce siderophores,and 16 could synthesize indole-3-acetic acid.Additionally,PGPRs were also evaluated for aminocyclopropane-l-carboxylate deaminase activity.A pot experiment was conducted to evaluate the ability of 28 PGPR isolates to promote growth,regulate Na^+/K^+balance,and decrease ethylene emissions in plants.The most efficient PGPRs were Arthrobacter aurescens.Bacillus atrophaeus,Enterobacter ashuriae,and Pseudomonas fluorescens.Gene expression analysis revealed the up-regulation of H^+-PPase,HKT1,NHX7,CAT,and APX expression in roots of Enterobacter-inocuVdied salt-stressed plants.Salt-tolerant rhizobacteria exhibiting plant growth-promoting traits can facilitate the growth of wheat plants under saline conditions.Our results indicate that the isolation of these bacteria may be useful for formulating new inoculants to improve wheat cropping systems in saline soils.

Advances in Mycobacterium siderophore-based drug discovery

Tuberculosis remains a public health threat of global proportions.Iron is a scarce resource indispensable to both host and pathogen during infection with Mycobacterium tuberculosis,the causative agent of tuberculosis.Siderophores are critical molecules for iron acquisition under iron-limiting conditions and,as a very efficient pathogen,M.tuberculosis has evolved elaborate siderophores knowledge of which is being intensively translated into clinical practice.This paper summarizes the structures,types and physiological functions of Mycobacterium siderophores with emphasis on siderophore-inspired design of drugs and drug delivery vehicles.

A new iron(Ⅲ) chelator of coprogen-type siderophore from the deep-sea-derived fungus Mycosphaerella sp. SCSIO z059

Mycosphazine A(1), a new iron(Ⅲ) chelator of coprogen-type siderophore, and mycosphamide A(2), a new cyclic amide benzoate, together with six known aryl amides(3-8), were isolated from the fermentation broth of the deep-sea-derived fungus Mycosphaerella sp. SCSIO z059. Alkaline hydrolysis of 1 afforded a new epimer of dimerum acid, mycosphazine B(1 a), and a new bi-fusarinine-type siderophore, mycosphazine C(1 b). The planar structures of the new compounds were elucidated by extensive spectroscopic data analysis. The absolute configurations of amino acid residues in 1 a and 1 b were determined by acid hydrolysis. And the absolute configuration of 2 was established by quantum chemical calculations of the electronic circular dichroism(ECD) spectra. Compound 1 is the first siderophore-Fe(Ⅲ) chelator incorporating both L-ornithine and D-ornithine unites. Compounds 3-8 were reported as natural products for the first time, and the 1 H and 13 C NMR data of 6 and 8 were assigned for the first time. Compounds 1 and 1 a could greatly promote the biofilm formation of bacterium Bacillus amyloliquefaciens with the rate of about 249% and 524% at concentration of 100 μg·mL-1, respectively.

A zero-sum game or an interactive frame?Iron competition between bacteriaand humans ininfectionwar

Iron is an essential trace element for both humans and bacteria.It plays a vital role in life,such as in redox reactions and electron transport.Strict regulatory mechanisms are necessary to maintain iron homeostasis because both excess and insufficient iron are harmful to life.Competition for iron is a war between humans and bacteria.To grow,reproduce,colonize,and successfully cause infection,pathogens have evolved various mechanisms for iron uptake from humans,principally Fe^(3+)-siderophore and Fe^(2+)-heme transport systems.Humans have many innate immune mechanisms that regulate the distribution of iron and inhibit bacterial iron uptake to help resist bacterial invasion and colonization.Meanwhile,researchers have invented detection test strips and coupled antibiotics with siderophores to create tools that take advantage of this battle for iron,to help eliminate pathogens.In this review,we summarize bacterial and human iron metabolism,competition for iron between humans and bacteria,siderophore sensors,antibiotics coupled with siderophores,and related phenomena.We also discuss how competition for iron can be used for diagnosis and treatment of infection inthefuture.

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis,bacterial pneumonia, endocarditis, erysipelas, and necro-tizing fasciitis. To survive in the host environment withlimited free iron available, Streptococcus species havedeveloped various mechanisms to uptake iron as anessential nutrient. They can directly extract the metalions from host iron-containing proteins such as ferritin,transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in thestrains, include the employment of specialized secretedhemophores to acquire heme and the usage of smallmolecules called siderophores as high-affinity ferricchelators. This review intends to discuss the most recentdiscoveries of these iron acquisition systems and theirrelevant regulators in Streptococcus species.

Isolation,characterization and identification of plant growth-promoting rhizobacteria from the rhizosphere of Acacia mangium at sandy BRIS soil

This study has isolated,characterized,and identified potential plant growthpromoting rhizobacteria(PGPR)with multiple PGP characteristics(N_(2)-fixation,P-and K-solubilization,IAA,and siderophores production)from the rhizosphere BRIS soil of Acacia mangium.A total of 24 pure colonies were isolated and only 8 colonies were selected for further evaluation of the growth rate in 5%organic molasses medium supplemented with 2%KNO_(3).Based on the biochemical,potential PGP characteristics and growth performance,3 superior PGPR strains were selected and identified as Paraburkholderia unamae(UA1),Bacillus amyloliquefaciens(UA6),and Enterobacter asburiae(UAA2)by partial sequencing of the 16S rRNA gene.The selected bacterial strains either in single or mixed(UA1+UA6+􀀁UAA2)cultures have shown a significant biochemical estimation of the PGP characteristics.Each strain has its own PGPR traits superiority with UA1 showing the best PGP characteristic followed by UA6 and UAA2.The use of mixed bacterial strains was beneficial as it showed the best performance in N_(2)-fixation,siderophores production,and significant effect on corn phenology,growth and yield compared to using a single strain.These types of microbes showed potential to be used as biofertilizer and should be exploited more.