RESULTS:
1 - 20 of 27 for ""siderophore""
Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation
A siderophore-dependent iron transport system of the pathogenic yersiniae plays a role in the pathogenesis of these organisms. The structure of the yersiniabactin (Ybt) siderophore produced by Yersinia enterocolitica has been elucidated. This paper reports the purification of Ybt from Yersinia pestis and demonstrates that it has the same structure as Ybt from Y. enterocolitica. Purified Ybt had a formation constant for Fe3+ of ~ 4×10-36. Addition of purified Ybt from Y. pestis enhanced iron uptake by a siderophore-negative (irp2)strain of Y. pestis. Maximal expression of the Ybt outer-membrane receptor Psn in this strain was dependent upon exogenously supplied Ybt. Regulation of Psn expression by Ybt occurred at the transcriptional level. Y. pestis DNA was used to construct irp2 and psn mutations in Yersinia pseudotuberculosis. The irp2 mutant strain no longer synthesized Ybt and the psn mutant strain could not use exogenously supplied Ybt. As in Y. pestis Ybt was required for maximal expression of Psn. Regulation by Ybt occurred at the transcriptional level. In contrast to Y. pestis in which a psn mutation does not repress synthesis of Ybt siderophore or expression of the iron-regulated HMWP1 and HMWP2 proteins the same mutation in Y. pseudotuberculosis partially repressed these products.
Stenotrophomonas maltophilia produces an EntC-dependent catecholate siderophore that is distinct from enterobactin
Stenotrophomonas maltophilia a Gram-negative multi-drug-resistant bacterium is increasingly recognized as a key opportunistic pathogen. Thus we embarked upon an investigation of S. maltophilia iron acquisition. To begin we determined that the genome of strain K279a is predicted to encode a complete siderophore system including a biosynthesis pathway an outer-membrane receptor for ferrisiderophore and other import and export machinery. Compatible with these data K279a and other clinical isolates of S. maltophilia secreted a siderophore-like activity when grown at 25–37 °C in low-iron media as demonstrated by a chrome azurol S assay which detects iron chelation and Arnow and Rioux assays which detect catecholate structures. Importantly these supernatants rescued the growth of iron-starved S. maltophilia documenting the presence of a biologically active siderophore. A mutation in one of the predicted biosynthesis genes (entC) abolished production of the siderophore and impaired bacterial growth in low-iron conditions. Inactivation of the putative receptor gene (fepA) prevented the utilization of siderophore-containing supernatants for growth in low-iron conditions. Although the biosynthesis and import loci showed some similarity to those of enterobactin a well-known catecholate made by enteric bacteria the siderophore of K279a was unable to rescue the growth of an enterobactin-utilizing indicator strain and conversely iron-starved S. maltophilia could not use purified enterobactin. Furthermore the S. maltophilia siderophore displayed patterns of solubility in organic compounds and mobility upon thin-layer chromatography that were distinct from those of enterobactin and its derivative salmochelin. Together these data demonstrate that S. maltophilia secretes a novel catecholate siderophore.
Siderophore uptake and use by the yeast Saccharomyces cerevisiae
The non-reductive uptake of several siderophores (ferrioxamine B ferrichrome triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied. Several aspects of siderophore transport were examined including specificity of transport regulation of transport and intracellular localization of the ferri-siderophores. Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product but the specificity was not absolute. Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product. The apparent specificity of transport was strongly dependent on the genetic background of the cells. Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate. Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6. Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome ferricrocin or ferrioxamine B but not for the uptake of triacetylfusarinine C. Cells bearing the AFT1 up mutation accumulated large amounts of ferric siderophores. Intracellular decomplexation of the siderophores occurred when transcription of the AFT1 up gene was repressed. Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome. Endocytosis was however not involved in the non-reductive uptake of siderophores.
Characterization of a tonB mutation in Erwinia chrysanthemi 3937: TonB Ech is a member of the enterobacterial TonB family The GenBank accession number for the sequence reported in this paper is Y15888.
The GenBank accession number for the sequence reported in this paper is Y15888.
The pectinolytic enterobacterium Erwinia chrysanthemi 3937 causes a systemic disease in its natural host the African violet (Saintpaulia ionantha). It produces two structurally unrelated siderophores chrysobactin and achromobactin. Chrysobactin makes a large contribution to invasive growth of the bacterium in its host. Insertion mutants of a chrysobactin-defective strain were constructed and screened on the universal CAS-agar medium used for siderophore detection. A set of mutants affected in the production of achromobactin were identified. This paper describes a mutant affected in the transport of all the ferrisiderophores used by the bacterium as iron sources. Molecular analysis revealed that the insertion mutation disrupts the tonB gene. The predicted Er. chrysanthemi TonB protein has a molecular mass of 27600 Da and shares 20–58% identity with the TonB proteins from 20 other bacterial species. The pathogenicity of the tonB mutant was assessed by inoculation of African violets. The impairment in the spread of symptoms was similar in the tonB mutant to that in chrysobactin-defective mutants. However the pectinolytic activity the major pathogenicity determinant in Er. chrysanthemi appeared to be stimulated twofold in the tonB mutant.
Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor
Ferric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low. Thus it is a limiting resource for which microbes must compete in natural habitats. Since competition for iron occurs at the level of individual cells knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats. Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy. Cells of this iron biosensor exhibited iron-dependent GFP fluorescence that was inversely proportional to the amount of iron added to the media and which differed by over 20-fold in iron-replete compared to iron-deplete culture media. Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities. In contrast the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly. The distribution of fluorescence intensities was strongly right-hand skewed with about 10% of the cells exhibiting substantially higher GFP fluorescence than that of the median cell. Cells of a positive control strain harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene exhibited uniform GFP fluorescence both in culture media and on plants. These results indicate that there is substantial heterogeneity of iron biovailability to cells of P. syringae on plants with only a small subset of cells experiencing low iron availability. Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.
Functional analysis of yersiniabactin transport genes of Yersinia enterocolitica
Yersinia enterocolitica O:8 biogroup (BG) IB strain WA-C carries a high-pathogenicity island (HPI) including iron-repressible genes (irp1–9 fyuA) for biosynthesis and uptake of the siderophore yersiniabactin (Ybt). The authors report the functional analysis of irp678 which show 98–99% similarity to the corresponding genes ybtPQX on the HPI of Yersinia pestis. It was demonstrated that irp67 are involved in ferric (Fe)-Ybt utilization and mouse virulence of Y. enterocolitica thus confirming corresponding results for Y. pestis. Additionally it was shown that inactivation of the ampG-like gene irp8 did not affect either Fe-Ybt utilization or mouse virulence. To determine whether irp6 irp7 and fyuA (encoding the outer-membrane Fe-Ybt/pesticin receptor FyuA) are sufficient to mediate Fe-Ybt transport/utilization these genes were transferred into Escherichia coli entDF and into non-pathogenic Y. enterocolitica BG IA strain NF-O. Surprisingly E. coli entDF but not Y. enterocolitica NF-O gained the capability to utilize exogenous Fe-Ybt as a result of this gene transfer although both strains expressed functional FyuA (pesticin sensitivity). These results suggest that besides irp6 irp7 and fyuA additional genes are required for sufficient Fe-Ybt transport/utilization. Finally it was shown that irp6 irp7 and fyuA but not irp8 are involved in controlling Ybt biosynthesis and fyuA gene expression: irp6 and/or irp7 mutation leads to upregulation whereas fyuA mutation leads to downregulation. However fyuA-dependent control of Ybt biosynthesis could be bypassed in a fyuA mutant by ingredients of chrome azurol S (CAS) siderophore indicator agar.
Streptomyces pini sp. nov., an actinomycete isolated from phylloplane of pine (Pinus sylvestris L.) needle-like leaves
A novel siderophore-producing actinomycete designated PL19T was isolated from the Scots-pine needle-like leaves collected from TNAU campus Coimbatore India. The isolate was chemoorganotrophic in nutrition and able to grow at 30 °C and the optimum pH and NaCl facilitated the growth pH 6–11 and 0–8 % (w/v) respectively. The cells are filamentous and the mycelia formed are basically of wide and intricately branched substrate mycelium from which aerial mycelia arises later gets differentiated into spores that are warty and arranged spirally. The 16S rRNA gene of strain PL19T was sequenced and was highly similar to the type strains of species of the genus Streptomyces including Streptomyces barkulensis RC1831T (98.8 % pairwise similarity) Streptomyces fenghuangensis GIMN4.003T (98.2 %) Streptomyces nanhaiensis SCSIO 01248T (98.0 %) Streptomyces radiopugnans R97T (97.9 %) Streptomyces atacamensis C60T (97.8 %) and Streptomyces macrosporus NBRC 14749T (97.2 %) all of which were subjected to taxonomical characterization using a polyphasic approach. The strains showed unique carbon utilization patterns and it possesses iso-C16 : 0 anteiso-C15 : 0 and anteiso-C17 : 0 as a major cellular fatty acids. The cell-wall was dominated with ll-type diaminopimelic acid and the menaquinone type was MK-9(H6 H8). These chemotaxonomic evidences placed strain PL19T within the genus Streptomyces . The determination of G+C ratio (69.5 mol%) and DNA–DNA hybridization values (13.4–31.8 % with the phylogenetically related species) helped in further hierarchical classification of strain PL19T. Based on morphological physiological and chemotaxonomic data as well as DNA–DNA hybridization values strain PL19T could be distinguished from the evolutionarily closest species currently available. All these collective data show that strain PL19T represents a novel species of the genus Streptomyces for which the name Streptomyces pini sp. nov. is proposed. The type strain is PL19T (=NRRL B-24728T=ICMP 17783T).
Insights into plant-beneficial traits of probiotic Pseudomonas chlororaphis isolates
Pseudomonas chlororaphis isolates have been studied intensively for their beneficial traits. P. chlororaphis species function as probiotics in plants and fish offering plants protection against microbes nematodes and insects. In this review we discuss the classification of P. chlororaphis isolates within four subspecies; the shared traits include the production of coloured antimicrobial phenazines high sequence identity between housekeeping genes and similar cellular fatty acid composition. The direct antimicrobial insecticidal and nematocidal effects of P. chlororaphis isolates are correlated with known metabolites. Other metabolites prime the plants for stress tolerance and participate in microbial cell signalling events and biofilm formation among other things. Formulations of P. chlororaphis isolates and their metabolites are currently being commercialized for agricultural use.
Chryseobacterium lecithinasegens sp. nov., a siderophore-producing bacterium isolated from soil at the bottom of a pond
Bacterial strain PAGU 2197T which was isolated from soil collected from the bottom of a pond in Japan is characterized in this study. Cells of strain PAGU 2197T were aerobic Gram-negative short rod-shaped non-motile flexirubin-producing oxidase-positive catalase-positive and lecithinase-negative. A phylogenetic study based on 16S rRNA gene sequences and multilocus sequence analysis (gyrB rpoB and rpoD) indicated that strain PAGU 2197T belongs to the genus Chryseobacterium and is a member of an independent lineage including Chryseobacterium tructae CCUG 60111T (sequence similarity 95.9 %) Chryseobacterium lactis CCUG 60566T (93.4 %) and Chryseobacterium viscerum CCUG 60103T (91.6 %). The average nucleotide identity values were 80.83–85.04 %. Because average nucleotide identity values of 95–96 % exceed the 70 % DNA–DNA hybridization cutoff value for species discrimination strain PAGU 2197T represents a novel species in the genus Chryseobacterium . The genome of strain PAGU 2197T was 4 967 738 bp with a G+C content of 35.5 mol%. The sole respiratory quinone of strain PAGU 2197T was MK-6; the major cellular fatty acids were iso-C15 : 0 iso-C17 : 0 3OH summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl); and the major polar lipids were phosphoglycolipids and phosphatidylethanolamine. These results indicate that strain PAGU 2197T should be classified as representing a novel species in the genus Chryseobacterium for which the name Chryseobacterium lecithinasegens sp. nov. is proposed with strain PAGU 2197T (=NBRC 114264T=CCUG 75150T) as the type strain.
Acquisition of iron by the non-siderophore-producing Pseudomonas fragi
The iron requirement siderophore production and iron uptake mechanisms of the type strain Pseudomonas fragi ATCC 4973 and five P. fragi isolates from meat were analysed. The strains exhibited a high sensitivity to iron starvation: their growth was strongly inhibited in medium supplemented with the iron chelator ethylenediamine di(hydroxyphenylacetic acid) or in medium treated with 8-hydroxyquinoline to remove contaminating iron. No siderophores were detectable in the growth supernatants of iron-starved cells. Cross-feeding experiments in iron-depleted medium showed however that the bacterial growth could be strongly stimulated by siderophores of foreign origin including desferriferrioxamine B enterobactin and some pyoverdines. Moreover all the strains were capable of efficiently using the iron sources present in their natural environment i.e. transferrin lactoferrin and haemoglobin. Iron starvation led to the specific production of supplementary outer-membrane proteins of apparent molecular mass ranging from 80 to 88 kDa. Furthermore growth in the presence of exogenous siderophores resulted in some strains in the induction of siderophore-mediated iron uptake systems. For one strain the concomitant synthesis of an iron-regulated siderophore-inducible outer-membrane protein was observed.
Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily
Summary: Uptake of iron from various siderophores by a Δfet3Δfet4 strain of Saccharomyces cerevisiae was investigated. The catecholate enterobactin and the hydroxamate coprogen were taken up by the cells by passive diffusion whereas the hydroxamates ferrioxamine B (FOB) and ferricrocin (FC) were taken up via a high-affinity energy-dependent mechanism. The kinetics of FOB and FC uptake showed reciprocal competitive inhibition. The transport was regulated by iron availability but was independent of the Aft1p and Mac1p transcriptional activators. Mutants affected in the transport of FOB were isolated. The transport of FC was not impaired in these mutants. Functional complementation of one mutant allowed the identification of the SIT1 gene (Siderophore iron Transport) encoding a putative permease belonging to the major facilitator superfamily. The Sit1 protein is probably a permease specific for the transport of ferrioxamine-type siderophores. The evidence suggests that the uptake of ferrichrome-type siderophores like FC involves other specific permease(s) although there seems to be a common handling of FOB and FC following their internalization by the cell.
Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF, are induced by extracellular pyochelin in Pseudornonas aeruginosa
SUMMARY: The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes pChEF8 encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha) the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE(156 kDa) and PchF (197 kDa) proteins had adenylation thiolation and condensationkyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchf-'/acZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin the end product of the pathway. Iron replete conditions led t o Fur (ferric uptake regulator)-dependent repression of the pchE -laciZ fusion. A translational pchD-lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.
High Affinity Iron Acquisition in Rhizobium Leguminosarum Requires the cycHJKL Operon and the feuPQ Gene Products, which belong to the Family of Two-Component Transcriptional Regulators
The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene lipA which is predicted to encode an outer-membrane lipoprotein. Further upstream of lipA there are two other genes whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes feuP and feuQ did not affect the transcription of lipA or of the cycHJKL operon. However a mutation in feuQ also led to the loss of the high affinity iron uptake system although siderophores were still produced.
Use of Siderophores to Type Pseudomonads: The Three Pseudomonas Aeruginosa Pyoverdine Systems
Eighty-eight Pseudomonas aeruginosa isolates most of them from the Collection of Bacterial Strains of the Institut Pasteur Paris were analysed for their pyoverdine-mediated iron incorporation system by different methods including pyoverdine isoelectrofocusing analysis pyoverdine-mediated growth stimulation immunoblot detection of (ferri)pyoverdine outer-membrane receptor and pyoverdine-facilitated iron uptake. The same grouping of the strains was reached by each of these methods resulting in the classification of the P. aeruginosa isolates even those which were devoid of pyoverdine production into three different siderophore types. Forty-two percent of the strains were identified with the type-strain P. aeruginosa ATCC 15692 (group I). 42% were identical with the second type-strain P. aeruginosa ATCC 27853 (group II) and 16% reacted identically with the clinical isolate P. aeruginosa Pa6 whose pyoverdine was recognized in this study to be identical in structure to the pyoverdine produced by a natural isolate P. aeruginosa strain R. No new pyoverdine species was detected among these strains.
Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO
Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid bank (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mutation in the 47 min region of the chromosome yielded one clone which restored yellow-green pigmentation and fluorescence when grown on iron-deficient medium. The relevant pMMB33-derivative cosmid pPYP17 contained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 kb Sacl-Clal fragment conferring the same phenotype. This derivative pPYP180 like pPYP17 also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of the P. aeruginosa PAO chromosome. Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PAO chromosome demonstrating that the restored apparent wild-type phenotype observed for the transconjugants was not the result of a true gene complementation. A gene interruption was obtained by replacing a 0.6 kb Bglll-Bglll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype by a Hgr interposon (ΩHg). After conjugational transfer and introduction of the mutagenized fragment into the PAO1 chromosome by gene replacement pyoverdine-deficient mutants were recovered indicating that the fragment indeed contained at least one gene involved in pyoverdine synthesis. The yellow-green fluorescent compound produced by such cells harbouring plasmids pPYP17 or pPYP180 differed from pyoverdine in several aspects and was consequently named pseudoverdine. Although pseudoverdine was able to complex iron it was unable to restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) or to mediate iron uptake into PAO1. Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine suggesting that it was structurally related to the chromophore of the pyoverdine molecule. The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthetic pathway of the pyoverdine chromophore.
Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality
The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD E F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However Western blot analyses showed that EntE F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and for EntB/G the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial loose or transient) with cytoplasmic membrane; therefore the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least the data indicate that EntE F and B/G are compartmentalized in E. coli and because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins suggest that this entire protein class may be sequestered in vivo.
Siderotyping of fluorescent pseudomonads:characterization of pyoverdines of Pseudornonas fluorescens and Pseudornonas putida strains from Antarctica
SUMMARY: Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping which involved pyoverdine-induced growth stimulation pyoverdine-mediated iron uptake pyoverdine analysis by electrophoresis and isoelectric focusing revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype including Pseudomonas fluorescens 1W and P. fluorescens lOcW was identical to that of P. fluorescens ATCC 13525. Two other strains P. fluorescens 9AW and Pseudomonas putida 9BW showed identical pyoverdine-related behaviour t o each other whereas the fifth strain P. fluorescens 5lW had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 1OcW had the same structures as the P. fluorescens ATCC 13525 pyoverdine whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared t o be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.
The Pseudomonas aeruginosa tonB gene encodes a novel TonB protein
The Pseudomonas aeruginosa tonB gene was cloned by complementation of the tonB mutation of Pseudomonas putida strain TE516 (W. Bitter J. Tommassen & P. J. Weisbeek 1993 Mol Microbiol 7 117-130). The gene was 1025 bp in length capable of encoding a protein of 36860 Da. As with previously described TonB proteins the P. aeruginosa TonB (TonBp.a.) was rich in Pro residues (18.1 %) and contained Glu-Pro/Lys-Pro repeats. Unlike previously described TonB proteins however TonBp.a. lacked an N-terminal membrane anchor (signal) sequence and contained instead a predicted internal signal/anchor sequence expected to yield an atypical N-terminal cytoplasmic domain in this protein. TonB proteins are essential components in iron-siderophore uptake in bacteria apparently functioning as energy transducers in coupling the energized state of the cytoplasmic membrane to outer-membrane receptor function. As expected tonB derivatives of P. aeruginosa were defective in siderophore-mediated iron acquisition. tonB gene expression was inducible by iron-limitation consistent with the identification of a Fur consensus binding sequence upstream of the gene. TonBp.a. showed substantially greater similarity to the Escherichia coli TonB protein than the Pseudomonas putida protein (31 % identity vs. 20 % identity) and tonBP.a. was able to complement deficiencies in the acquisition of ferric enterobactin and vitamin B12# and sensitivity to phage φ80 of an E. coli tonB strain. The larger size of TonBP.a. and its ability to function in both E. coli and P. putida make it a unique TonB protein whose characterization should enhance our understanding of TonB function in bacteria.
The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management
In iron-limited medium Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+ in a ligand:Fe ratio of 1:1 3:2 and 3:1 respectively. Protochelin had the highest affinity for Fe3+ with a calculated proton-independent solubility coefficient of 10439 comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition superoxide dismutase activity was greatly diminished in iron-limited cells whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates especially protochelin under oxidative stress conditions but had decreased activities of both superoxide dismutase and catalase and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O- 2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O- 2 and was a Fenton catalyst. These data suggest that under iron-limited conditions A. vinelandii suffers oxidative stress caused by.O- 2. The catecholate siderophores azotochelin and especially protochelin are hyper-produced to offer chemical protection from oxidative damage catalysed by.O- 2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.
Identification of a 29 kDa protein in the envelope of Mycobacterium smegmatis as a putative ferri-exochelin receptor
Evidence of a direct association between ferri-exochelin the major extracellular siderophore of Mycobacterium smegmatis and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of 55Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a complex with the non-denaturing detergent CHAPS and subsequent CHAPS-PAGE and autoradiography. (2) Affinity chromatography on a chemically synthesized ferri-exochelin-Sepharose 4B matrix led to the retention of the 29 kDa protein and a 25 kDa protein. The smaller protein was partially eluted with 1mM ferri-exochelin although it did not form a stable complex with ferri-exochelin. The 29 kDa protein could not be eluted from the affinity matrix with 1mM ferri-exochelin either alone or with 1 M NaCl. Only 2% (w/v) SDS could do this but resulted in protein denaturation. (3) Incubation of 55Fe-exochelin with CHAPS-solubilized envelope proteins in free solution followed by ion-exchange chromatography resolved three radioactive peaks; subsequent analysis by SDS-PAGE showed that the peak with the highest 55Fe-binding activity per unit protein contained both the 29 and 25 kDa proteins. A direct association was demonstrated between the 29 kDa protein and 55Fe-exochelin by gel filtration. The evidence suggests that the 29 kDa iron-regulated envelope protein of M. smegmatis is a ferri-exochelin-binding protein and that the 25 kDa protein which corresponds in size to a previously reported iron-regulated envelope protein in this bacterium may have a role in the formation or maintenance of this complex. Proteins extracted from the cell envelope of iron-deficient M. smegmatis with CHAPS were dialysed to remove the detergent incorporated into liposome suspensions and then incubated with 55Fe(III)-exochelin. This increased the retention of 55Fe by 133-fold compared to proteins not placed in liposomes. Retention of 55Fe was dependent upon the protein loading of the liposomes. Gel filtration confirmed that the iron was retained by these vesicles and even after dialysis the majority of 55Fe was still retained by the vesicles. Re-solubilization of the labelled proteo-liposomes in various detergents gave limited recovery of a ferri-exochelin-protein complex. Attempts to resolve this complex by Triton X-100 PAGE led to separation of the two entities. The complex was stable however in a CHAPS-PAGE system.