Identification of siderophores of Pseudomonas stutzeri

We have identified two types of siderophores produced by Pseudomonas, one of which has never before been found in the genus. Twelve strains of Pseudomonas stutzeri belonging to genomovars 1, 2, 3, 4, 5, and 9 produced proferrioxamines, the hydroxamate-type siderophores. Pseudomonas stutzeri JM 300 (genomovar 7) and DSM 50238 (genomovar 8) and Pseudomonas balearica DSM 6082 produced amonabactins, catecholate-type siderophores. The major proferrioxamines detected were the cyclic proferrioxamines E and D2. Pseudomonas stutzeri KC also produced cyclic (X1 and X2) and linear (G1 and G2a-c) proferrioxamines. Our data indicate that the catecholate-type siderophores belong to amonabactins P 750, P 693, T 789, and T 732. A mutant of P. stutzeri KC (strain CTN1) that no longer produced the secondary siderophore pyridine-2,6-dithiocarboxylic acid continued to produce all other siderophores in its normal spectrum. Siderophore profiles suggest that strain KC (genomovar 9) belongs to the proferrioxamine-producing P. stuzeri. Moreover, a putative ferrioxamine outer membrane receptor gene foxA was identified in strain KC, and colony hybridization showed the presence of homologous receptor genes in all P. stutzeri and P. balearica strains tested.     

The metagenomics of disease-suppressive soils - experiences from the METACONTROL project

Soil teems with microbial genetic information that can be exploited for biotechnological innovation. Because only a fraction of the soil microbiota is cultivable, our ability to unlock this genetic complement has been hampered. Recently developed molecular tools, which make it possible to utilize genomic DNA from soil, can bypass cultivation and provide information on the collective soil metagenome with the aim to explore genes that encode functions of key interest to biotechnology. The metagenome of disease-suppressive soils is of particular interest given the expected prevalence of antibiotic biosynthetic clusters. However, owing to the complexity of soil microbial communities, deciphering this key genetic information is challenging. Here, we examine crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.     

Bacterial siderophores: structures of pyoverdins Pt, siderophores of Pseudomonas tolaasii NCPPB 2192, and pyoverdins Pf, siderophores of Pseudomonas fluorescens CCM 2798. Identification of an unusual natural amino acid

Pyoverdins were isolated and characterized respectively from the cultures of Pseudomonas tolaasii NCPPB 2192 (pyoverdins Pt, Pt A, and Pt B) and Pseudomonas fluorescens CCM 2798 (Pyoverdins Pf/1, Pf/2, Pf, Pf/3/1, and Pf/3/2) each grown in iron-deficient conditions. Their structures were established by using FAB-MS, NMR, and CD techniques. These siderophores are chromopeptides, and all but one (pyoverdin Pf/3/3) possess at the N-terminal end of their peptide chain the same chromophore that has been reported in pyoverdin Pa from Pseudomonas aeruginosa ATCC 15692 [Wendenbaum, S., Demange, P., Dell, A., Meyer, J. M., & Abdallah, M. A. (1983) Tetrahedron Lett. 24, 4877-4880] and pseudobactin B 10 from Pseudomonas B10 [Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., & Van der Helm, D. (1981) Biochemistry 20, 6446-6457] which is derived from 2,3-diamino-6,7-dihydroxyquinoline. In pyoverdins Pt this chromophore is bound to a linear peptide chain D-Ser-L-Lys-L-Ser-D-Ser-L-Thr-D-Ser-L-OHOrn-L-Thr-D-Ser-D-OHOrn (cyclic) which has its C-terminal end blocked by cyclic D-N delta-hydroxyornithine. In pyoverdins Pf, the peptide chain is also linear, SerCTHPMD-Gly-L-Ser-D-threo-OHAsp-L-Ala-Gly-D-Ala-Gly-L-O HOrn(cyclic), and contains an unusual natural amino acid which is the result of the condensation of 1 mol of serine and 1 mol of 2,4-diaminobutyric acid, forming a cyclic amidine. The pyoverdins Pt differ only in substituent bound to the nitrogen on C-3 of the chromophore, which is succinic acid in pyoverdin Pt A, succinamide in pyoverdin Pt, and alpha-ketoglutaric acid bound to the chromophore by its C-5 carbon atom in pyoverdin Pt B. Similarly, pyoverdin Pf/1, pyoverdin Pf/2, pyoverdin Pf (the major compound), and pyoverdin Pf/3/2 are substituted respectively by L-malic acid, succinic acid, L-malic amide, and succinamide. Pyoverdin Pf/3/3 has the same chromophore as azotobactin, the peptidic siderophore of Azotobacter vinelandii. These pyoverdins are very similar to pseudobactin B 10, the siderophore of Pseudomonas B10: they are linear peptides containing three bidentate groups strongly chelating Fe(III) and blocked at their N-terminal end by the catecholic chromophore and at their C-terminal end by cyclic N delta-hydroxyornithine. They differ therefore from other pyoverdins such as those from P. aeruginosa ATCC 15692 which contain a partly cyclic peptide [Briskot, G., Taraz, K., & Budzikiewicz, H. (1989) Liebigs Ann. Chem., 375-384].     

Alcohol-induced changes in hepatic estrogen-binding proteins: A mechanism explaining feminization in alcoholics

Chronic alcoholic men frequently display an apparent hyperestrogenization manifested by enhanced hepatic synthesis of estrogen-responsive proteins as well as many other estrogen-linked tissue alterations. Because of these clinical observations, we assessed the effect of chronic alcohol ingestion, using a rat model, on the levels of two estrogen-binding proteins of male rat liver cytosol. These two estrogen-binding proteins, the estrogen receptor, and an unusual male-specific estrogen binder, differ in specificity for the non-steroidal estrogen diethylstilbestrol (DES), permitting development of an assay for each using unfractionated cytosol. The estrogen receptor was labeled with [³H]DES, and the male-specific estrogen binder with [³H]estradiol in the presence of unlabeled DES, since the latter protein does not recognize DES. The specificity of labeling under these conditions was verified by gel filtration chromatography. The livers of rats fed either an alcohol-containing (AF) or isocalorically matched control diet were assayed for the levels of both proteins. The livers of the AF animals had twice the content of estrogen receptor, as compared to the isocalorically matched control group (105 vs 52 fmol/mg cytosol protein). Conversely, the livers of the AF animals had only one-third as much male-specific estrogen binder as did those of the isocalorically matched control group (22 vs 62 pmol/mg cytosol protein). Alcohol feeding also resulted in those animals having smaller testes, seminal vesicles, and prostates, as well as decreased serum testosterone levels. No change in serum estradiol levels occurred after 34 days of alcohol feeding; however, 61 days of alcohol feeding resulted in an increase in serum estradiol levels in the AF animals. These results are incorporated into a proposed model of feminization of the chronic alcoholic male.     

Ferribactins as the biosynthetic precursors of the Pseudomonas siderophores pyoverdins

By a feeding experiment with a 15N-labelled precursor it is shown that ferribactins are transformed into pyoverdins and thus are their biogenetic precursors.     

高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究

采用改进的CAS检测平板从东湖中筛选得到了一株高产铁载体细菌sp-f,并用CAS检测液定量检测其分泌铁载体量,发现其As/Ar仅0.09(OD680),Su(Siderophore Unit)为90%,达到产铁载体菌最高级。用BIOLOG检测板,结合细菌生理生化反应、形态观察和16S rDNA序列比对分析等分类鉴定方法,确定sp-f为一株荧光假单胞菌。P.fluorescenssp-f生长过程中胞外铁载体的量在对数生长前期累积达到最高后有所减少,至稳定期时菌液中铁载体量达到稳定。在已知铁载体特异吸收峰波长下,用反向高效液相色谱检测无铁环境和高铁环境下培养液上清,比较发现sp-f上清含有3种含儿茶酚胺类基团铁载体,其中包括荧光和非荧光性的脓菌素,200μmol/L Fe2 可完全抑制荧光性质脓菌素的分泌,但非荧光脓菌素的分泌不受抑制,并且对非脓菌素的儿茶酚胺类铁载体的合成分泌反而具有一定的诱导作用。     

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

High-performance liquid chromatography analyses of pyoverdin siderophores differentiate among phytopathogenic fluorescent Pseudomonas Species

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two beta-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.     

Bacterial siderophores promote plant growth: Screening of catechol and hydroxamate siderophores

The aim of the study was to determine the quality and quantity of siderophores produced by bacteria isolated from plants' roots. The second aim was to determine the effect of siderophores on plants growth (Festuca rubra L. and Brassica napus L.). The study was carried out using bacteria isolated from roots of: Arabidopsis thaliana L., F. rubra, and Agrostis capillaris L., growing on the heavy metals contaminated area. The chrome azurol sulfonate (CAS) test, Arnow's test for catechol siderophores, and Csaksy's test for hydroxamate siderophores were performed. Among the bacteria, 42 isolates (39%) had a positive result in the CAS. Endophytic bacteria were mostly producing the catechol siderophores. It was found that F. rubra is the plant which is linked with the highest number of siderophores producing bacteria. The highest concentration of siderophores was noted for ectorhizospheric bacteria associated with A. thaliana, hyperaccumulating plant. It was found that hydroxamate siderophores are mainly produced by ectorhizosphere and rhizoplane bacteria. The siderophores producing bacteria reduced the toxicity of metals and improved the phytoremediation. Siderophores treatment increased the growth of plants in the biological assay, growing on two different soils: one highly contaminated with heavy metals and the second strongly alkaline soil.     

The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.     

Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352

The production of peptide siderophores and the variation in siderophore production among strains of Pseudomonas syringae and Pseudomonas viridiflava were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two P. syringae strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent Pseudomonas strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of P. syringae pv. syringae, P. syringae pv. aptata, P. syringae pv. morsprunorum, P. syringae pv. tomato, and P. viridiflava had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of P. syringae pv. aptata and P. syringae pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of P. syringae and P. viridiflava; siderotyping between pathovars of P. syringae was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.     

Structural information for explaining the molecular mechanism of protein biosynthesis.

Protein biosynthesis is controlled by a number of proteins external to the ribosome. Of these, extensive structural investigations have been performed on elongation factor-Tu and elongation factor-G. This now gives a rather complete structural picture of the functional cycle of elongation factor-Tu and especially of the elongation phase of protein biosynthesis. The discovery that three domains of elongation factor-G are structurally mimicking the amino-acylated tRNA in the ternary complex of elongation factor-Tu has been the basis of much discussion of the functional similarities and functional differences of elongation factor-Tu and elongation factor-G in their interactions with the ribosome. Elongation factor-G:GDP is now thought to leave the ribosome in a state ready for checking the codon-anticodon interaction of the aminoacyl-tRNA contained in the ternary complex of elongation factor-Tu. Elongation factor-G does this by mimicking the shape of the ternary complex. Other translation factors such as the initiation factor-2 and the release factor 1 or 2 are also thought to mimic tRNA. These observations raise questions concerning the possible evolution of G-proteins involved in protein biosynthesis.     

Corticosterone inhibits feather growth: potential mechanism explaining seasonal down regulation of corticosterone during molt

Corticosterone (CORT) is seasonally modulated in many passerines, with plasma CORT concentrations lowest during the prebasic molt when all feathers are replaced. To explain why, we proposed that the birds downregulate natural CORT release during molt in order to avoid CORT's degradative effects on proteins and its inhibition of protein synthesis. If CORT exerted these effects during molt, it could slow protein deposition during feather production and potentially result in a longer period of degraded flight performance. To test this hypothesis, either empty or CORT-filled silastic implants were inserted into captive European starlings (Sturnus vulgaris) and white-crowned sparrows (Zonotrichia leucophrys) undergoing induced (feather replacement after plucking) and natural molts. We then measured the rate of feather re-growth by regularly measuring the length of primary, secondary, and tail feathers. CORT implanted birds showed a significantly decreased rate of feather growth compared to control animals. Basal CORT concentrations of induced molt and non-molting birds were also compared but no difference was noted. The results suggest a tradeoff; a complete set of new feathers may be more important to the survival of a bird than the ability of CORT to respond maximally to a stressor.     

A unifying hypothesis for explaining the mechanism of amyloid formation under conditions of increased oxidative stress

Amyloid related organ dysfunction is a common feature of conditions associated with chronic oxidative injury such as diabetes, inflammation, neurodegenerative disorders, renal failure, and natural aging. Matrix metalloproteinases (MMPs) are a family of calcium and zinc-dependent endopeptidases comprised of 23 enzymes in the human. Among these, MMPs 2 and 9 are known as secretable forms, present in all body fluids and susceptible to activation by oxidants. Although MMPs are generally accepted and named for their effect on extracellular matrix turnover, their non-extracellular-matrix targets have emerged recently. Cystatin C (CysC) is a very potent inhibitor of cysteine proteinases, present in all body fluids. Its solubility is determined by its N-terminal sequence. CysC is known to polimerize and form fibrils and has been isolated from amyloids. The CysC isolated from amyloids is in the N-terminal truncated form. My hypothesis regarding amyloid formation is that CysC could be a substrate for MMPs 2 and 9, which upon cleaving the N-terminal off the CysC protein will render it insoluble and promote amyloid formation. Several in vitro studies have demonstrated degradation of CysC by MMPs. The implications of such a degradation in kidney glomerules (where the clearance of CysC occurs) could be of importance for understanding the mechanism of kidney failure e.g. in diabetes. This proposed mechanism for amyloid formation through degradation of CysC by MMPs, can be proposed for all cases of CysC related amyloid formation, such as those seen in cerebrovascular, cardiac and rheumatoid disorders.     

A new mechanism for membrane iron transport in Pseudomonas aeruginosa

Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli. This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e. the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore. One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.