Molecular recognition of synthetic siderophore analogues: A study with receptor-deficient and fhu(A-B) deletion mutants of Escherichia coli

The biological activity of six synthetic siderophore analogues (two dihydroxamates, two trihydroxamates, one tetrahydroxamate and one 3-hydroxy-4(1H)pyridinone) has been studied in Escherichia coli, Morganella morganii 13 and Proteus mirabilis 8993 strains by using growth promotion tests. Various transport-deficient mutants of E. coli were used to study the route of entry into gram-negative bacteria. The results indicated that the synthetic hydroxamate compounds are transported via Fhu-mediated transport systems, although receptor specificity was low. This could be proven by using a delta (fhuA-B) E. coli mutant as a control in which growth promotion by natural hydroxamates was completely abolished, suggesting that a periplasmic binding-protein-dependent transport system (FhuB, C, D) is required for the transport of all synthetic ferric hydroxamate complexes. Although utilization of the synthetic hydroxamates was generally lower than that of the natural siderophores, differences in growth promotion could be detected. Highest activity was observed with the dihydroxamate DOCYDHAMA ligand which supported growth at concentrations <1 mM. In comparison with other polyamino-polyhydroxamate ligands studied, this dihydroxamate ligand has an extra diamide backbone that could be important for the interaction with the receptors or with FhuD. The synthetic trihydroxamate and tetrahydroxamate ligands showed a relatively low siderophore activity. Studies with Proteus and Morganella in the presence of increasing bipyridyl concentrations showed a decreased growth promotion with the synthetic ferric hydroxamates, suggesting the involvement of a reduction step during iron mobilization or an increased toxicity of bipyridyl. This was not observed in the case of the 3-hydroxy-4(1H)pyridinone where bipyridyl had no effect.     

The use of bis(mercaptoacetato-S,O)hydroxoiron(III) complex for the determination of hydroxamates

A rapid, indirect, spectrophometric procedure for the determination of hydroxamates, based on the competition for ferric ions of the bis(mercaptoacetato-S,O)hydroxoiron(III) complex, has been developed. The assay is remarkably free of interferences by common ions, thus rendering it useful in the quantitative determination of hydroxamates in culture fluids and crude preparations.     

Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases

Iron is required for microbial growth and proliferation. To survive in low-iron environments, some microorganisms secrete ferric iron chelators called siderophores. Siderophore biosynthesis occurs via two pathways: the non-ribosomal peptide synthetase (NRPS) pathway and the NRPS-independent siderophore (NIS) synthetase pathway. NIS enzymes function by adenylating a carboxylic acid substrate, typically citrate, or a derivative, followed by nucleophilic capture of an amine or alcohol and displacement of a citryl intermediate. In this review, we summarize recent advances in NIS biochemistry with a particular focus on structural biology and confirm the classification of NIS enzymes into Types A, A’, B, and C based on substrate specificity. Based on a phylogenetic analysis, we also propose a new subclass of NIS enzymes, Type C’, responsible for dimerization and macrocyclization of complex and substituted amine or amide intermediates. Finally, we describe the role of NIS enzymes in virulence of pathogenic microbes and discuss NIS inhibitors as potential anti-microbial agents.     

Synthesis and characterization of metal binding pseudotripeptides.

Metal complexes with peptide or pseudopeptide type ligands can serve as good model compounds for a deeper understanding of enzymatic catalysis, but ligands with a high selectivity for different transition metal cations are hard to find due to the rather flexible nature of peptides. Since such ligands would be the sine qua non condition for the synthesis of heterodinuclear peptide metal complexes with catalytic activity, the search for small, affine and selective metal chelating sequences is of interest. Using four different amino acids (His, Lys, Asp, Glu) a set of 16 pseudotripeptides of the common structure Bz-AS1-Sar-AS2-NH2 has been synthesized, purified and characterized by mass spectrometry and 1H-NMR. Their ability to form metal complexes has been investigated leading to short motifs capable of selectively binding only one or two transition metal cations with high affinity. As expected, the complexation of transition metal cations by pseudotripeptides is strongly dependent not only on the amino acid composition, but also on the sequence with regard to the stability of the resulting complexes, as well as the selectivity of the ligands towards Cu2+, Co2+, Ni2+, Zn2+ and Mn2+.     

Synthesis and iron-binding properties of quinolobactin,a siderophore from a pyoverdine-deficient <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Quinolobactin is a new siderophore produced by a pyoverdine deficient mutant of Pseudomonas fluorescens. A simple and efficient synthesis of quinolobactin is described, starting from xanthurenic acid. The protonation constants of quinolobactin were determined by potentiometric titrations as pK_a2=5.50 ± 0.07, pK_a1=10.30 ± 0.05. The equilibria of the metal complexes were studied by means of spectrophotometric and potentiometric titrations. The overall stability constants of the quinolobactin-Fe^III complexes was found to be log ₁₁₁=18.60 ± 0.10, log ₁₂₁=32.60 ± 0.20, log ₁₂₀=28.20 ± 0.25 resulting in a pFe^III value of 18.2 at pH 7.4. The UV-visible spectral parameters of the [FeL₂] are in agreement with a complex containing two ligands coordinated to one Fe³⁺ cation through the oxygen and nitrogen quinoline atoms.     

四株儿茶酚类铁载体高产菌株产消化酶活性及其益生特性

【背景】儿茶酚类铁载体对胃肠道菌群的生长代谢具有重要作用,研究儿茶酚类铁载体高产菌株的消化酶活性,挖掘其潜在益生特性具有重要意义。【目的】分析4株分离自健康成人粪样的儿茶酚类铁载体高产菌的产酶特性,通过分析菌株耐酸耐胆盐能力、粘附定殖能力、抗生素耐受性和急性毒性研究其益生特性。【方法】测定4株菌的蛋白酶、淀粉酶、脂肪酶、纤维素酶、植酸酶、乳糖酶和β-葡萄糖苷酶的酶活性。4株菌经人工模拟胃、肠液连续培养后分别计算其活菌数;分析4株菌的自凝集率、黏蛋白粘附率和表面疏水率;对小鼠连续7 d灌胃不同剂量的4株高产菌,观察并记录小鼠的一般体征,计算小鼠脏器指数,进行阳性细菌移位试验。【结果】在试验所测的7种消化酶中,E. coli Gut 07、E. coli Gut 12无蛋白酶和脂肪酶活性,B. cereus Gut 16无乳糖酶活性,E. coli Gut 20无蛋白酶活性,其余均具有。4株菌经人工模拟胃液培养6h后存活率均大于60%,转移到人工模拟肠液培养24 h后活菌数均大于初始菌落数;该4株菌具备在胃肠道中粘附定殖的能力,对大多数抗生素敏感,在浓度低于4.5×10~(11)CFU/m L、灌胃剂量为20m L/kg-bw时对小鼠无急性毒性,无阳性菌株移位现象。【结论】4株儿茶酚类铁载体高产菌株可作为潜在益生菌进行进一步的安全性和功能性研究。     

四株碱性环境真菌铁载体活性筛选

【目的】铁载体(Siderophore)是由微生物产生的一类低分子量金属离子螯合物。在生物医药、环境修复、健康食品等领域均具有广泛应用前景。文献调查显示,针对碱性环境真核微生物铁载体研究尚无相关报道。从中筛选高活性铁载体真菌具有重要意义。【方法】采用铬天青S显色法对分离于云南省碱性湖泊-程海和个旧大屯碱性尾矿土壤的99株真菌进行筛选;利用"分光光度法"对菌株产铁载体能力和铁载体类型进行考察;采用固相萃取(SPE)手段对菌株铁载体富集效果进行考察;根据菌体形态电镜观察和ITS基因系统发育树构建,对菌株进行生物学鉴定。【结果】通过初筛和复筛,确定菌株FEDT-866、FEDT-145、FECH-998和FECH-595均为铁载体高产菌株;所产铁载体类型主要为异羟肟酸型和羧酸型。除菌株FEDT-866外,铁载体活性产物适合采用固相萃取(SPE)方法进行富集。经生物学鉴定,菌株FEDT-866和FECH-998属于曲霉属,分别与Aspergillus tubigensis和A.nomius相似性较高;菌株FECH-595和FEDT-145属于青霉属,分别与Penicillium svalbardense和P.chrysogenum相似性较高。【结论】P.chrysogenum是1种常见的产铁载体真菌。而A.tubigensis、A.nomius及P.svalbardense菌株尚无产铁载体相关报道,可作为铁载体研究良好材料。     

Involvement of the siderophore of cowpea Rhizobium in the iron nutrition of the peanut

An iron-inefficient variety of peanut plant, when grown hydroponically with the catechol siderophore of Rhizobium (peanut isolate), showed increased growth and chlorophyll content compared with plants grown with Fe alone. The siderophore, when used in concentrations less than the concentrations of Fe, was still effective in grwoth promotion, indicating that it might function as a shuttle agent, solubilizing and supplying Fe to the plant. Similar results were also obtained with desferrioxamine B.The authors are with the Department of Microbiology and Biotechnology Centre, Faculty of Science, M.S. University of Baroda, Baroda-390 002, Gujarat, India.     

Production of the triacetecholate siderophore protochelin by Azotobacter vinelandii

Azotobacter vinelandii grown in iron-limited medium containing 1 m molybdate released the catecholate siderophores azotochelin and aminochelin [bis(2,3-dihydroxybenzoyl-lysine) and 2,3-dihydroxybenzoyl-putrescine, respectively] into the culture fluid. However these catecholates were not observed when the medium contained 1 mm molybdate, but were replaced by another catecholate compound. The appearance of this new compound was not an artifact of extraction of the catecholates from the culture fluid in the presence of high molybdate. Full and partial acid hydrolysis and fast atom bombardment mass spectroscopy showed that the new compound was the tricatecholate protochelin, a product of the condensation of azotochelin and aminochelin. The production of protochelin was iron-repressible and protochelin very rapidly decolorized the Chrome Azurol-S assay. Protochelin promoted the growth of the siderophore-deficient A. vinelandii strain P100 under iron-restricted conditions and promoted ⁵⁵Fe uptake into iron-limited cells, confirming that protochelin can be used as a siderophore by A. vinelandii.     

Alkali and alkaline-earth metal complexes of different nuclearities with azamacrocyclic pendant-arms ligands

A new series of alkali and alkaline-earth complexes of Na(I), Ca(II), Sr(II) and Ba(II) with two pyridyl pendant-arms azamacrocyclic ligands, L¹ and L², were synthesized by reaction of the ligand and the appropriate perchlorate salt in refluxing acetonitrile. The reactions gave analytically pure products that were characterized by elemental analysis, FAB mass spectrometry, IR, conductivity measurements and ¹H NMR spectroscopy. The crystal structure of [NaL¹](ClO₄), [BaL¹](ClO₄)₂ · 2CH₃CN and [Ca₂L²(μ-ClO₄)(H₂O)₂](ClO₄)₂ · Cl · (H₂O)_0.5 could be also determined. The crystal structure of the Na(I) and Ba(II) complexes with L¹ show endomacrocyclic mononuclear species, where the metal ions are in N8 and N10 coordination environment, respectively. The geometry around the metal ions can be described as cube and bicapped cube geometry for Na(I) and Ba(II), respectively. Instead, the crystal structure of the Ca(II) compound with L² shows an exomacrocyclic dinuclear complex where both metal ions are in a similar N5O2 environment joined through a bidentate perchlorate anion. The coordination geometry for both Ca(II) ions can be described as distorted pentagonal bipyramid.     

棉花黄萎病拮抗细菌产铁载体测定及其对抑菌活性的影响

【背景】棉花是新疆农业的支柱产业,棉花黄萎病严重影响棉花产量和纤维品质,有效的防治途径是目前解决的首要任务。【目的】探究BacillusvelezensisBHZ-29、Bacillusatrophaeus SHZ-24、Bacillus subtilis SHT-15、Bacillus vanillea SMT-24菌株产铁载体能力及其对抑菌活性的影响。【方法】MKB-CAS双层平板检测铁载体,Arnow法与高氯酸铁法鉴定铁载体类型,CAS染液定量铁载体,琼脂扩散法测定低铁条件下铁载体对抑菌物质的影响。【结果】MKB-CAS固体平板菌体周围出现橙紫色显色反应,Arnow实验4株拮抗菌反应液颜色为红色,高氯酸铁实验反应液颜色为黄色;BHZ-29、SHZ-24、SHT-15、SMT-24菌株铁载体定量活性单位最大值分别为8.27%±0.61%、31.80%±2.06%、16.06%±3.61%、15.53%±0.51%,吸光度值As/Ar比值最小分别为0.917±0.092、0.682±0.021、0.845±0.005、0.846±0.008。BHZ-29、SHZ-24、SMT-24菌株含铁离子浓度0、10、20μmol/L 3组处理发酵无菌滤液均未出现抑菌圈,SHT-15菌株3组处理出现抑菌圈,抑菌圈直径大小无差异性。【结论】4株拮抗菌株均产铁载体,铁载体类型为儿茶酚型,4株拮抗菌株整体产铁载体能力较低,最高达31.80%±2.06%,产铁能力As/Ar比值高于0.682时,BHZ-29、SHZ-24、SMT-24菌株含铁离子浓度0、10、20μmol/L 3组处理发酵无菌滤液对抑菌活性无影响,SHT-15菌株3组不同铁离子浓度处理对抑菌物质影响差异不显著。     

微生物铁载体转运调控机制及其在环境污染修复中的应用

铁载体是微生物在胞内低铁浓度下分泌的螯合铁的物质,可分为儿茶酚盐类、氧肟酸盐类、羧酸盐类三大类。铁载体的转运分别受Fur、σ因子、群体感应信号这3种机制调控。近年来铁载体在石油污染修复、重金属污染修复和纸浆生物漂白等领域得到了应用,受到广泛关注。文中综述了铁载体的分类及其转运调控机制,以及铁载体在环境污染治理与修复中的应用,并展望了铁载体今后的应用前景。     

Synthesis and mycobactericidal properties of metal complexes of isonicotinoyldithiocarbazic acid

An isonicotinoyldithiocarbazic acid (IN-DtczH) ligand, synthesized from isoniazid, was complexed with transition metals and evaluated for anti-mycobacterial activity as well as toxicity towards human-transformed rhabdomyosarcoma (RD) cells in vitro. Complexes with Ni, Co and Zn showed MIC of 2, 2 and 50 μg/ml against Mycobacterium tuberculosis H₃₇Rv, and 10, 100 and 50 μg/ml against a multidrug-resistant strain of M. tuberculosis. They had little cytotoxic effect on the RD cells. In contrast, the Cu complex was highly cytotoxic with a low anti-mycobacterial activity.     

Biochemical genetics of aerobactin biosynthesis in Escherichia coli

Abstract Single-gene mutants of Escherichia coli defective in aerobactin biosynthesis were incubated under non-growing conditions for 2 h with radiolabelled lysine. Analysis of the intermediates produced suggested that acetylation of lysine may be the first step in aerobactin production.     

Yersiniabactin metal binding characterization and removal of nickel from industrial wastewater

Yersiniabactin (Ybt) is a metal‐binding natural product that has been re‐purposed for water treatment. The early focus of this study was the characterization of metal binding breadth attributed to Ybt. Using LC‐MS analysis of water samples exposed to aqueous and surface‐localized Ybt, quantitative assessment of binding was completed with metals that included Pd²⁺, Mg²⁺, and Zn²⁺. In total, Ybt showed affinity for 10 metals. Next, Ybt‐modified XAD‐16N resin (Ybt‐XAD) was utilized to quantify the affinity for metal removal, showing a rank order of Fe³⁺ > Ga³⁺ > Ni²⁺ > Cu²⁺ > Cr²⁺≈Zn²⁺ > Co²⁺ > Pd²⁺ > Mg²⁺ > Al³⁺, and in the applied treatment of wastewater from a local precious metal plating company, showing selective removal of nickel from the aqueous effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1548–1554, 2017     

Purification and chemical characterization of staphyloferrin B,a hydrophilic siderophore from staphylococci

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicus DSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 mHCl revealed the presence of l-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.     

Peptide siderophores

Siderophores are low molecular weight iron chelators, produced by virtually all bacteria, fungi and some plants. They serve to deliver the essential element iron, barely soluble under aerobic conditions, into microbial cells. Siderophores are therefore important secondary metabolites which are very often based on amino acids and their derivatives. Biosynthesis, transport, regulation and chemical synthesis of natural siderophores and their analogues is of considerable interest for the protein and peptide chemist. This review gives an overview of the structural classes of peptidic siderophores, along with data on their biosynthesis. On a number of representative examples, strategies and schemes of their chemical synthesis are described. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

The membrane potential is the driving force for siderophore iron transport in fungi

Abstract Respiratory inhibitors and uncouplers severely impair [⁵⁵Fe]ferricrocin uptake by Neurospora crassa . parallel measurements of ATP decay and ferricrocin uptake, however, disprove the idea that direct input of metabolic energy in the form of ATP is required for transmembrane movement of siderophores. The role of the membrane potential for siderophore uptake was demonstrated using iron-deficient cells, which were derepressed in the glucose-II uptake system. Addition of high amounts of glucose (1 mM) to glu-II-derepressed cells leads to a membrane depolarization of about 120 mV, followed by a significant inhibition of ferricrocin uptake, which recovered after some minutes. Full transport inhibition occurred after membrane depolarization in the presence of plasma membrane ATP-ase inhibitors (DCCD or DES), indicating that the membrane potential is essential for siderophore transport in fungi.     

Identification of erythrobactin, a hydroxamate-type siderophore produced by Saccharopolyspora erythraea

AIMS: To investigate the production of siderophores by Saccharopolyspora erythraea SGT2 and how this production is affected by the inoculum. METHODS AND RESULTS: When grown in a low-iron, chemically defined medium (CDM), the soil dwelling actinomycete S. erythraea secretes a substance that is reactive in the nonspecific chrome azurol S (CAS) assay. Importantly, the production of CAS-reactive substance is highly reduced upon the addition of 0.925 micromol l(-1) iron to the cultures and has a peak of production in the late-log to early stationary growth phase. In addition, the culture supernatants tested were negative in the Arnow and Rioux assays but positive in the Csáky procedure. Interestingly, we also found evidence that the production of this CAS-reactive substance in CDM was highly reduced, when inoculated with cells that had been previously grown to late-stationary phase. Conversely, inocula derived from late-log to early stationary cultures presented high levels of CAS activity. CONCLUSIONS: These results indicate that S. erythraea produces a hydroxamate-type siderophore that we have generically designated as erythrobactin. Additionally, the inocula growth stage plays a key role in siderophore production in S. erythraea. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first evidence for siderophore synthesis in S. erythraea and one of the first examples of non-polyketide secondary metabolite production by this organism.