Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci

Abstract A highly hydrophilic compound with siderophore activity has been isolated from the supernatant of Staphylococcus hyicus DSM 20459 grown under iron-restricted conditions. The metabolite, named staphyloferrin B, is strictly iron-regulated and produced by a large variety of staphylococci strains. In vivo iron transport measurements and the growth-promoting activity in a bioassay establish staphyloferrin B as the second siderophore for staphylococci besides the previously described staphyloferrin A. The structure elucidation revealed 2,3-diaminopropionic acid, citrate, ethylenediamine and 2-ketoglutaric acid as structural components of the compound. Thus, staphyloferrin B is a structurally new siderophore of the complexone type.     

Isolation and partial characterization of a compound with siderophore activity from Vibrio parahaemolyticus

A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.     

Evidence for importance of the Staphylococcus hyicus lipase pro-peptide in lipase secretion, stability and activity

Abstract To investigate the function of the pro-peptide (PP) region of the Staphylococcus hyicus exolipase, restriction sites were created in the lipase gene to facilitate the construction of deletions in this region. Lipase gene expression was carried out in Staphylococcus carnosus . In the presence of the entire PP region, the 86-kDa pro-lipase was efficiently exported, had high lipolytic activity, and hardly any degradation products were seen in Western blot analysis. In addition to the 86-kDa pro-lipase, the membrane fraction contained a 106-kDa immunoreactive form. If the PP was completely or partially deleted, signal peptide processing, lipase secretion, lipase activity and/or lipase stability were impaired. The results obtained with lipase PP deletion mutants indicate that the PP region may have two functional domains. The N-terminal region of the lipase PP appears to be more important for lipase activity and the C-terminal portion for lipase secretion and proteolytic stability. In the presence of only the C-terminal part of the PP lipase, secretion was hardly affected. However, the activity of the extracellular lipase was markedly reduced. If only a small portion of the C-terminal part of the PP was present, lipase secretion was again markedly reduced and no lipase activity was detectable. In the presence of the N-terminal half of the PP region, lipase secretion was affected to a lesser extent. However, the resulting 60-kDa form, which showed comparably good specific lipase activity, suffered severe proteolytic degradation.     

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.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.     

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.     

Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis

Exfoliative toxins of approximately 30 kDa produced by Staphylococcus hyicus strains NCTC 10350, 1289D-88 and 842A-88 were purified and specific polyclonal antisera were raised against each of the toxins. It was shown by immunoblot analysis and ELISA that three exfoliative toxins from S. hyicus were antigenically distinct. The three toxins were designated ExhA, ExhB and ExhC. From 60 diseased pigs, each representing an outbreak of exudative epidermitis, a total of 584 isolates of S. hyicus were phage typed and tested for production of exfoliative toxin. ExhA-, ExhB- and ExhC-producing S. hyicus isolates were found in 12 (20%), 20 (33%) and 11 (18%), respectively, of the 60 pig herds investigated. Production of the different types of exfoliative toxin was predominantly associated with certain phage groups. However, toxin production was found in all of the six phage groups defined by the phage typing system. Some changes in the distribution of isolates between phage groups were observed when the results of this study were compared to previous investigations. In this study two new antigenically distinct exfoliative toxins were isolated and tools for in vitro detection of toxin producing S. hyicus isolates and for further studies on the exfoliative toxins from S. hyicus have been provided.     

Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus

Abstract Growth rates, siderophore secretion, and bacterial proteins of two clinical isolates of Staphylococcus aureus were studied over 72 h of growth in iron-supplemented and iron-restricted chemically defined media. Under iron restriction the growth rates were decreased to different extents depending on the strain. Production of siderophore was detected in the mid-exponential and stationary phases of growth. The expression of iron-regulated proteins of 81, 23, and 17 kDa was time-dependent, associated with the same stage of growth, and might be involved in siderophore efficiency.     

Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi

A catechcol-type siderophore, assigned the trivial name chrysobactin, was isolated from the phytopathogenic bacterium Erwinia chrysanthemi and characterized by degradation and spectroscopic techniques as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine. Chrysobactin, which was also obtained by chemical synthesis, was shown to be active in supplying iron to a group of mutants of E. chrysanthemi defective in biosynthesis of the siderophore.     

Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: indications of ExhA and ExhB being metalloproteins

The exfoliative toxins ExhA and ExhB produced by Staphylococcus hyicus strains NCTC10350 and 1289D-88, respectively, were investigated with regard to the effect of divalent metal ions on toxin production as measured in indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Data were obtained as endpoint titer values and used as semiquantitative measures for the amount of exfoliative toxin detected in culture supernatants. It was shown that the endpoint titers of ExhA in supernatants from cultures of strain NCTC10350 grown in the presence of 0.5 mM CaCl2, Cu(NO3)2 or ZnSO4 were higher compared to titers obtained by growth in medium supplemented with a number of other divalent metal salts. The titer of ExhB as determined in the indirect ELISA was increased by addition of 0.5 mM CoCl2, Cu(NO3)2 or CuSO4 to the growth medium. When ExhA or ExhB, prepared without addition of metal salt to the liquid growth medium, was subsequently incubated with 25 mM of Co2+, Cu2+ or Zn2+, the endpoint titers of the toxins were increased. Dialysis of ExhA and ExhB prepared with Zn2+ and Co2+, respectively, against certain metal chelators, resulted in a reduction of the titer determined in ELISA. Other metal chelators had varied effect in the detection of the toxins in ELISA. It was, however, not possible to restore the recognition of toxins by the monoclonal antibodies by incubation of EDDHA-dialyzed toxin preparations with Co2+, Cu2+ or Zn2+. The results of this study suggest that ExhA and ExhB are metalloproteins.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

Specificity of Staphyloferrin B Recognition by the SirA Receptor from Staphylococcus aureus

Many organisms use sophisticated systems to acquire growth-limiting iron. Iron limitation is especially apparent in bacterial pathogens of mammalian hosts where free iron concentrations are physiologically negligible. A common strategy is to secrete low molecular weight iron chelators, termed siderophores, and express high affinity receptors for the siderophore-iron complex. Staphylococcus aureus, a widespread pathogen, produces two siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). We have determined the crystal structure of the staphyloferrin B receptor, SirA, at high resolution in both the apo and Fe(III)-SB (FeSB)-bound forms. SirA, a member of the class III binding protein family of metal receptors, has N- and C-terminal domains, each composed of mainly a β-stranded core and α-helical periphery. The domains are bridged by a single α-helix and together form the FeSB binding site. SB coordinates Fe(III) through five oxygen atoms and one nitrogen atom in distorted octahedral geometry. SirA undergoes conformational change upon siderophore binding, largely securing two loops from the C-terminal domain to enclose FeSB with a low nanomolar dissociation constant. The staphyloferrin A receptor, HtsA, homologous to SirA, also encloses its cognate siderophore (FeSA); however, the largest conformational rearrangements involve a different region of the C-terminal domain. FeSB is uniquely situated in the binding pocket of SirA with few of the contacting residues being conserved with those of HtsA interacting with FeSA. Although both SirA and HtsA bind siderophores from the same α-hydroxycarboxylate class, the unique structural features of each receptor provides an explanation for their distinct specificity.     

Characterisation of a siderophore from Acinetobacter calcoaceticus

Abstract The Gram-negatice bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu ¹H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.     

Isolation,structural characterization,and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.     

Structural basis of phospholipase activity of Staphylococcus hyicus lipase

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.     

Isolation and characterization of siderophore,with antimicrobial activity,fromAzospirillum lipoferum M

Azospirillum lipoferum M was found to produce catechol-type of siderophores under iron-starved conditions. Chemical characterization of siderophores revealed the presence of salicylic acid, 2,3-dihydroxybenzoic acid (DHBA), and 3,5-DHBA conjugated with threonine and lysine. Siderophore production was found to be maximum after 28 h of growth. In addition to their established role in iron transport, the siderophores exhibited antimicrobial activity against various bacterial and fungal isolates.     

一株金黄色葡萄球菌噬菌体的分离鉴定与生物学特性

[背景]金黄色葡萄球菌作为一种条件致病菌,在临床感染中扮演着重要的角色,需要研究更多更有效的防治手段.[目的]分离金黄色葡萄球菌噬菌体,研究其生物学特性,从而为金黄色葡萄球菌的替代防治提供理论借鉴.[方法]以金黄色葡萄球菌D085为宿主从污水中分离得到一株噬菌体,命名为vB_SauS_SAP3,用PEG8000浓缩、氯...     

Identification and characterization of a novel-type ferric siderophore reductase from a gram-positive extremophile

Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with K(i) values in the micromolar range. The principal catalytic mechanism was found to couple increasing K(m) and K(D) values of substrate binding with increasing k(cat) values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.