Molecular cloning and expression of genetic determinants for the iron uptake system mediated by the Vibrio anguillarum plasmid pJM1

Plasmid pJM1 from an invasive strain of Vibrio anguillarum encodes an iron uptake system which mediates the biosynthesis of a siderophore and a membrane receptor for the iron-siderophore complex. This system has been associated with the ability of V. anguillarum to cause hemorrhagic septicemic disease in marine fish. Recombinant derivatives containing essential regions of the pJM1-mediated iron uptake system cloned into cosmid vector pVK102 were introduced into low-virulence iron uptake-deficient V. anguillarum strains by using a trifactor mating procedure with helper plasmid pRK2013. Three recombinant clones, pJHC-T7, pJHC-T11, and pJHC-T2612, possessed genetic determinants for receptor activity. Production of receptor activity was correlated in all three cases with the presence of OM2, an 86-kilodalton outer membrane protein which was induced under iron-limiting conditions. Two of the clones, pJHC-T7 and pJHC-T2612, also coded for the production of siderophore activity, although at a much lower level than the wild type. Strains harboring either of these two clones were still unable to grow under iron-limiting conditions. This inability was overcome only when other indigenous pJM1 derivatives were present in the cells in addition to the recombinant cosmids. This restoration of high siderophore production and ability to grow under iron-limiting conditions was achieved even when the indigenous plasmids possessed lesions in genes involved in siderophore activity or in both siderophore and receptor production. Thus, another function mediated by plasmid pJM1, possibly a transacting factor, may play a role in the regulation of siderophore production. Results of experimental infections demonstrated that restoration of the ability to grow under conditions of iron limitations by introduction of an recombinant clone into one of the low-virulence V. anguillarum strains was correlated with an increase in bacterial pathogenicity.     

Siderophore production by Fusarium venenatum A3/5

Fusarium venenatum A3/5 was grown in iron-restricted batch cultures and iron-limited chemostat cultures to determine how environmental conditions affected siderophore production. The specific growth rate in iron-restricted batch cultures was 0.22 h(-1), which was reduced to 0.12 h(-1) when no iron was added to the culture. D(crit) in iron-limited chemostat culture was 0.1 h(-1). Siderophore production was correlated with specific growth rate, with the highest siderophore production occurring at D=0.08 h(-1) and the lowest at D=0.03 h(-1). Siderophore production was greatest at pH 4.7 and was significantly reduced at pHs above 6.0. Siderophore production could be enhanced by providing insoluble iron instead of soluble iron in continuous flow cultures.     

Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production

We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA(+) gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.     

Siderophore cross-utilization amongst nodule isolates of the cowpea miscellany group and its effect on plant growth in the presence of antagonistic organisms

Nodule isolates from the cowpea miscellany group of legumes produced varying concentrations of catecholate and hydroxamate types of siderophores under iron-limiting conditions. The nodule isolates differed with respect to siderophore cross-utilizing abilities; some were proficient at using siderophores of other nodule isolates (homologous siderophores) while others could utilize siderophores produced by other rhizospheric bacteria (heterologous siderophores). Utilization of siderophore of rhizospheric bacterium PsB, a plant pathogen, benefited the nodule isolate G11 in terms of growth under iron-limiting laboratory conditions, while PsB was clearly inhibited in the presence of G11. Plate assays showed that siderophore of G11 could withhold iron from PsB and hence PsB was inhibited in the presence of G11. Isolates G11 and PsB when applied simultaneously to peanut seedlings under sterile soil conditions, provided a clear advantage to the plant in terms of reduction in the inhibitory effect of PsB. The count of the nodule isolate G11 increased in the soil when co-inoculated with PsB, as compared to when inoculated alone. Thus, the increased growth of the plant can be attributed to the iron sequestration and plant growth promoting properties of G11. The isolate G11 could utilize the siderophores produced by many other rhizospheric isolates while the siderophore of G11 was not being utilized by these rhizospheric isolates.     

Interactions between iron availability, aluminium toxicity and fungal siderophores

The influence of iron, aluminium and of the combined application of both metals on microbial biomass and production of siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and Hymenoscyphus ericae) were investigated. All three species showed a strong iron regulation and Al-sensitivity of siderophore biosynthesis although several differences remained species dependent. Inhibitory effects of Fe and Al on siderophore-production were additive and the higher binding capacity of siderophores towards iron could be compensated by a higher Al-availability. Although pH itself is also important for regulation of siderophore biosynthesis, an indirect effect of Al on siderophore production via an Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a reduced biomass production were compensated by high Fe-availability, whereas the addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Distinction of emigration by telotroch formation and death by predation in peritrich ciliates: SEM observations on the remaining stalk ends

Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according to the bioassays results. The same pattern of outer membrane proteins are synthesized under iron-limiting conditions in all species tested, which suggests a similarity of iron uptake systems in many species of Salmonella. This system could be also of great importance in the survival of these bacteria in natural waters, as well as in possible pathogenic mechanisms.     

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.     

The Fe Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe chelator proferrorosamine A, Fe -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.     

Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization

Vibrio fischeri is the sole species colonizing the light-emitting organ of the Hawaiian squid, Euprymna scolopes. Upon entering the nascent light organ of a newly hatched juvenile squid, the bacteria undergo morphological and physiological changes that include the loss of flagellation and the induction of bioluminescence. These and other events reveal a pattern of genetic regulation that is a response to the colonization of host tissue. In this study, we isolated and characterized a glnD:mTn5Cm mutant of V. fischeri. In addition to the predicted defects in the efficiency of nitrogen utilization, this glnD mutant had an unexpected reduction in the ability to produce siderophore and grow under iron-limiting conditions. Although the glnD mutant could colonize juvenile squid normally over the first 24 h, it was subsequently unable to persist in the light organ to the usual extent. This persistence phenotype was more severe if the mutant was pregrown under iron-limiting conditions before inoculation, but could be ameliorated by the presence of excess iron. These results indicate that the ability to respond to iron limitation may be an important requirement in the developing symbiosis. Supplying the glnD gene in trans restored normal efficiency of nitrogen use, iron sequestration and colonization phenotypes to the glnD:mTn5Cm mutant; thus, there appears to be a genetic and/or metabolic linkage between nitrogen sensing, siderophore synthesis and symbiosis competence in V. fischeri that involves the glnD gene.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.