Siderophore-mediated iron acquisition in the staphylococci

Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus.     

Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2.

Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide patterns were detected in response to iron stress. Finally, our data suggest that V. vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein.     

Yersiniabactin and other siderophores produced by clinical isolates of Enterobacter spp. and Citrobacter spp

We analyzed the ability of extraintestinal strains of Enterobacter spp. and Citrobacter spp. to employ different siderophore-mediated strategies of iron acquisition. All strains produced iron-chelating compounds. Cross-feeding assays indicated that most isolates of both Enterobacter spp. and Citrobacter spp. excreted catecholate siderophore enterobactin, less produced aerobactin, and single strains excreted hydroxamates different from aerobactin. Besides, we analyzed if the strains had the ability to produce the siderophore yersiniabactin coded by the Yersinia high-pathogenicity island (HPI). The presence of HPI genes was observed in single isolates of three species: E. cloaceae, E. aerogenes and C. koseri. A detailed polymerase chain reaction analysis revealed differences in the genetic organization of the HPIs; however, in a cross-feeding test we proved that yersiniabactin was produced and the island was functional.     

Siderophore-mediated uptake of iron in Azotobacter vinelandii

Azotobacter vinelandii produces two siderophores, N,N'-bis-(2,3-dihydroxybenzoyl)-L-lysine (azotochelin) and a yellow-green fluorescent peptide (azotobactin), under iron-limited growth conditions. 55Fe uptake was not observed until the substantial nonspecific binding of 55Fe to the cell surface was eliminated by the addition of 10 mM sodium citrate to the uptake medium. Citrate alone did not promote rapid 55Fe uptake in A. vinelandii, nor did it induce Fe-repressible outer membrane proteins. Siderophore-mediated 55Fe uptake appeared biphasic, with both the initial rapid and ensuing slower uptake being energy dependent. The purified siderophores demonstrated the same uptake pattern as the Fe-limited culture supernatant fluid, but either individually or in combination accounted for less than the total 55Fe uptake activity found in the latter. The purified siderophores appeared to be sensitive to acid, but the inhibition of 55Fe uptake was in fact caused by salt generated during neutralization. Similar 60% inhibition of 55Fe uptake activity was caused by the addition of 40 mM Na+, K+, Li+, or Mg2+ salts to the uptake medium. Ammonium was less inhibitory than the latter ions. 55Fe uptake mediated by azotobactin was more sensitive to added NaCl than was that mediated by azotochelin. Neither the chelation of iron nor the stability of the ferrisiderophore was affected by added NaCl.     

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent “carbon for iron mutualism”. While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in “algal-associated” VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal–bacterial “carbon for iron mutualism” hypothesis in the marine environment.     

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

The phenotypic and genotypic characterization of Korean isolates of Cronobacter spp. (Enterobacter sakazakii)

This study was conducted to investigate the phenotypic and genotypic characteristics of Korean isolates of Cronobacter spp. (Enterobacter sakazakii). A total of 43 Cronobacter spp., including 5 clinical isolates, 34 food isolates, 2 environmental isolates, and 2 reference strains (C. sakazakii ATCC 29004 and C. muytjensii ATCC51329) were used in this study. Korean isolates of Cronobacter spp. were divided into 11 biogroups according to their biochemical profiles and 3 genomic groups based on the analysis of their 16S rRNA gene sequences. Biogroups 1 and 2 contained the majority of isolates (n=26), most of which were contained in 16S rRNA cluster 1 (n=34). Korean isolates of Cronobacter spp. showed diverse biochemical profiles. Biogroup 1 contained C. sakazakii GIHE (Gyeonggido Research Institute of Health and Environment) 1 and 2, which were isolated from babies that exhibited symptoms of Cronobacter spp. infection such as gastroenteritis, sepsis, and meningitis. Our finding revealed that Biogroup 1, C. sakazakii, is more prevalent and may be a more pathogenic biogroup than other biogroups, but the pathogenic biogroup was not represented clearly among the 11 biogroups tested in this study. Thus, all biogroups of Cronobacter spp. were recognized as pathogenic bacteria, and the absence of Cronobacter spp. in infant foods should be constantly regulated to prevent food poisoning and infection caused by Cronobacter spp.     

Transferrin-mediated iron acquisition by pathogenic Neisseria

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.     

Cytotoxic activity of Enterobacter cloacae human isolates

We examined 55 Enterobacter cloacae isolates from clinical specimens for the production of cytotonic and cytotoxic toxins and the presence of the type III secretion system (TTSS). Twelve isolates (22%) revealed cytotoxic activity that caused destruction of Vero cells, whereas 28 (51%) strains induced lysis of the murine macrophage J774 cell line. TTSS genes were present in 27% of the isolates. The results indicated that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum β-lactamases, including bla_CTX-M-55 and bla_NDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of bla_NDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.     

Reduction of iron by extracellular iron reductases: implications for microbial iron acquisition

The extracellular enzymatic reduction of iron by microorganisms has not been appropriately considered. In this study the reduction and release of iron from ferrioxamine were examined using extracellular microbial iron reductases and compared to iron mobilization by chemical reductants, and to chelation by EDTA and desferrioxamine. A flavin semiquinone was formed during the enzymatic reduction of ferrioxamine, which was consistent with the 1 e(-) reduction of iron by an enzyme. The rates for the enzymatic reactions were substantially faster than both the 2 e(-) chemical reductions and the chelation reactions. The rapid rates of the enzymatic reduction reactions demonstrated that these enzymes are capable of accomplishing the extracellular mobilization of iron required by microorganisms. The data suggest that mechanistically there are two phases for the mobilization and transport of iron by those microorganisms that produce both extracellular iron reductases and siderophores, with reduction being the principle pathway.     

Genetic determinants of antibacterial resistance among nosocomial Escherichia coli, Klebsiella spp., and Enterobacter spp. isolates collected in Russia within 2003-2007

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.     

Serological cross-reactivity of environmental isolates of Enterobacter, Escherichia, Stenotrophomonas, and Aerococcus with Shigella spp.-specific antisera

Using protocols designed for the isolation of Shigella from environmental freshwater samples from different regions of Bangladesh, 11 bacterial strains giving rise to Shigella-like colonies on selective agar plates and showing serological cross-reaction with Shigella-specific antisera were isolated. Phylogenetic analyses revealed that three of the isolates were most closely related to Escherichia coli, four to Enterobacter sp., two to Stenotrophomonas, and two isolates belonged to the Gram-positive genus Aerococcus. The isolates cross-reacted with six different serotypes of Shigella and were, in each case, highly type-specific. Two of the isolates belonging to the Enterobacter and Escherichia genera gave extremely strong cross-reactivity with Shigella dysenteriae and Shigella boydii antisera, respectively. The Aerococcus isolates gave relatively weak but significant cross-reactions with S. dysenteriae. Western blot analysis revealed that a number of antigens from the isolates cross-react with Shigella spp. The results indicate that important Shigella spp. surface antigens are shared by a number of environmental bacteria, which have implications for the use of serological methods in attempts for the detection and recovery of Shigella from aquatic environments.     

Tracing of Enterobacter sakazakii isolates in infant milk formula processing by BOX-PCR genotyping

Aims:  Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe neonatal meningitis, bacteraemia and necrotizing enterocolitis. Contaminated powder infant formulae (PIF) have been identified as one of various infection routes. In this study, E. sakazakii was monitored in the processing environment of a PIF factory to identify possible dissemination routes.
Methods and Results:  The BOX-polymerase chain reaction (PCR), a fingerprinting technique which targets the repetitive BOX sequences, was used in routine to identify points of contamination and investigate clonal persistence. Two hundred E. sakazakii isolates were collected and typed. Most (70%) showed the same fingerprint that revealed the persistence of resident E. sakazakii strains in the processing environment. This method allowed to detect contamination of some PIF by dry-blending ingredients.
Conclusions:  Environment was the major cause for contamination of PIF and facilities. Some raw materials delivered as powder were also implicated.
Significance and Impact of the Study:  Routine BOX-PCR genotyping was very useful to trace and investigate in real-time dissemination of micro-organisms in the PIF plant and to implement a series of additional control measures to reduce the risk of final product contamination by E. sakazakii .     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.