Iron Uptake Genes Research Articles

Iron-responsive chromatin remodelling and MAPK signalling enhance adhesion in Candida albicans.

Recent cumulative data show that various transcription factors are recruited to the chromatin in an iron-responsive manner to affect diverse cellular functions in the pathogenic fungus Candida albicans. Here we identified groups of iron-responsive genes in C. albicans by chromatin remodelling analysis at gene promoters, using micrococcal nuclease (MNase) digestion followed by deep sequencing. Chromatin in the promoter regions of iron uptake and utilization genes showed repressed and active configuration, respectively, under iron-replete conditions. GO Term enrichment analysis of genes with differentially remodelled chromatin, in respective promoter locales, suggested that many genes involved in adhesion are also iron-responsive. C. albicans was observed to be more self-adherent (twofold increase) and formed higher biofilm mass (77% increase) in the presence of iron. Furthermore, we identified various known and novel adhesion-related genes with iron-dependent active chromatin profiles that are indicative of potential upregulation under iron-replete conditions. Transcription factor Cph1 that is activated upon Cek1 phosphorylation also showed an active chromatin profile under iron-replete conditions and cells showed iron-responsive Cek1 MAPK phosphorylation in the presence of iron. Thus, iron affects diverse biological functions by modulating chromatin profiles of large gene sets and by signalling through Cek1 MAPK in C. albicans.

Mediator subunit 16 functions in the regulation of iron uptake gene expression in Arabidopsis.

Iron is an essential nutrient for plant growth and development, and its absorption is tightly controlled. Under iron limitation, FIT dimerizes with the four Ib bHLH proteins and activates the expression of iron uptake genes. However, how the dimerized complex activates downstream genes remains unclear. Using forward genetics, a low-iron-sensitive mutant was screened. The corresponding gene (MED16) was isolated, and its biological functions in iron homeostasis were characterized using approaches such as gene expression, protein subcellular localization, protein-protein interaction and chromatin immunoprecipitation assay. Lesion of MED16 significantly reduced FRO2 and IRT1 expression in Arabidopsis roots. The MED16 mutants showed a low shoot iron concentration and severe leaf chlorosis under iron limitation, whereas it grew normally as wild-type under iron sufficiency. Furthermore, we showed that MED16 interacted with FIT and improved the binding of the FIT/Ib bHLH complex to FRO2 and IRT1 promoters under iron-deficient conditions. Additionally, we found that many iron-deficient response genes, which are regulated by FIT, were also controlled by MED16. In conclusion, MED16 is involved in the iron deficiency response, and modulates the iron uptake gene expression under iron limitation. Our results increase the understanding of the molecular regulation mechanisms underlying iron uptake and homeostasis in plants.

Increased Iron Sequestration in Alveolar Macrophages in Chronic Obtructive Pulmonary Disease

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer’s and Parkinson’s diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2–3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1–3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Futhermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.

AmrZ is a global transcriptional regulator implicated in iron uptake and environmental adaption in P. fluorescensF113

BackgroundAmrZ, a RHH transcriptional regulator, regulates motility and alginate production in pseudomonads. Expression of amrZ depends on the environmental stress sigma factor AlgU. amrZ and algU mutants have been shown to be impaired in environmental fitness in different pseudomonads with different lifestyles. Considering the importance of AmrZ for the ecological fitness of pseudomonads and taking advantage of the full sequencing and annotation of the Pseudomonas fluorescens F113 genome, we have carried out a ChIP-seq analysis from a pool of eight independent ChIP assays in order to determine the AmrZ binding sites and its implication in the regulation of genes involved in environmental adaption.Results154 enriched regions (AmrZ binding sites) were detected in this analysis, being 76% of them located in putative promoter regions. 18 of these peaks were validated in an independent ChIP assay by qPCR. The 154 peaks were assigned to genes involved in several functional classes such as motility and chemotaxis, iron homeostasis, and signal transduction and transcriptional regulators, including genes encoding proteins implicated in the turn-over of c-diGMP. A putative AmrZ binding site was also observed by aligning the 154 regions with the MEME software. This motif was present in 75% of the peaks and was similar to that described in the amrZ and algD promoters in P. aeruginosa. We have analyzed the role of AmrZ in the regulation of iron uptake genes, to find that AmrZ represses their expression under iron limiting conditions.ConclusionsThe results presented here show that AmrZ is an important global transcriptional regulator involved in environmental sensing and adaption. It is also a new partner in the complex iron homeostasis regulation.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity. Furthermore, we demonstrate that the [2Fe-2S]-bridged heterodimer formed between glutaredoxin-3 and the BolA-like protein Fe repressor of activation-2 transfers a [2Fe-2S] cluster to Aft2 that facilitates Aft2 dimerization. Previous in vivo findings strongly support the [2Fe-2S] cluster-induced dimerization model; however, given the available evidence, Fe(2+)-induced Aft2 dimerization cannot be completely ruled out as an alternative Aft2 inhibition mechanism. Taken together, these data provide insight into the molecular mechanism for iron-dependent transcriptional regulation of Aft2 and highlight the key role of Fe-S clusters as cellular iron signals.

Dynamics in the rhizosphere and iron-uptake gene expression in peanut induced by intercropping with maize: Role in improving iron nutrition in peanut

The intercropping of maize with peanuts is an effective cropping practice. Indeed, peanut/maize intercropping reportedly improves the iron nutrition of peanuts in calcareous soils. The limited evidence available suggests that the improved Fe nutrition in intercropping is largely attributable to a rhizosphere effect of maize. In this study, the effects of peanut/maize intercropping on the Fe nutritional status of peanut associated with the dynamics of the rhizosphere processes and Fe uptake gene expression induced by the interaction of the two species at various growth days were investigated. The results suggest that an interspecific rhizosphere effect improves Fe nutrition in peanut, as shown by changes in the rhizosphere available Fe concentration, pH, and Olsen-P concentration, based on time-course changes in peanut–maize interaction. The increase in available Fe in the rhizosphere of peanut ranged from 0.2 to 2.64 mg kg−1. The transition from the vegetative to reproductive stage was a key turning point in the time-course of changes in the rhizosphere processes in intercropping. There was more consistently positive effect of intercropping on peanut Fe nutrition after 53 days. Moreover, the expression of AhFRO1 and AhYSL1 was expressed at significantly higher level in intercropped peanuts compared to monocropped peanut at the vegetative stage, indicating a role for these genes in Fe improvement in intercropped peanuts. We conclude that the enhanced time-course changes in the rhizosphere processes and iron uptake gene expression with a consistent positive interspecific effect appear to be one of the mechanisms underlying the improved Fe nutrition in intercropped peanut plants.

The Basis for Evolution of DNA-Binding Specificity of the Aft1 Transcription Factor in Yeasts

The Saccharomyces cerevisiae Aft1 and Kluyveromyces lactis KlAft are orthologous yeast transcription activators that regulate the expression of the same group of iron-uptake genes but bind to the different DNA sites: TGCACCC for Aft1 and PuCACCC for KlAft. To establish whether the DNA-binding mechanisms of Aft1 and KlAft have diverged during the evolution of the Aft-type transcription factor, we examined the function of a nonconserved region in their DNA-binding domains. A large part of this region is composed of a sequence predicted to be disordered in structure and potentially phosphorylated. We show with deletion mutant analyses that this sequence is essential for the binding of Aft1 to its DNA site and for the iron uptake and growth of S. cerevisiae under iron-limited conditions. We constructed hybrid proteins by exchanging the nonconserved regions of Aft1 and KlAft. We show that the Aft1 region is necessary and sufficient for KlAft to bind efficiently to the Aft1 DNA site in S. cerevisiae and to complement the iron-dependent phenotype of the aft1Δaft2Δ mutant. This demonstrates that the changes in the nonconserved region of the Aft-type DNA-binding domain have led to changes in the DNA-binding specificity and have major consequences for the regulation of iron homeostasis. The combination of bioinformatic and experimental analyses indicates that the sequence TGCACCC is the most probable ancestral Aft-type element. Our findings suggest that the changes in the nonconserved region of the DNA-binding domain are responsible for the evolution of the TGCACCC sequence toward PuCACCC in the K. lactis species.

The LysR-type transcription factor HbrL is a global regulator of iron homeostasis and porphyrin synthesis in Rhodobacter capsulatus.

The purple bacterium Rhodobacter capsulatus is unique among Rhodobacteriacae as it contains a putative iron response regulator (Irr) but does not possess a copy of the ferric uptake regulator (Fur). Interestingly, an in-frame deletion mutant of Irr shows no major role in iron homeostasis. Instead, we showed that the previously identified activator of haem gene expression HbrL is a crucial regulator of iron homeostasis. We demonstrated that an HbrL deletion strain is unable to grow in iron-limited medium in aerobic, semi-aerobic and photosynthetic conditions and that suppressor strains can be isolated with mutations in iron uptake genes. Gene expression studies revealed that HbrL is a transcriptional activator of multiple ferrous and ferric iron uptake systems in addition to a haem uptake system. Finally, HbrL activates the expression of numerous haem biosynthesis genes. Thus, HbrL has a central role in controlling the amount of iron transport in conjunction with the synthesis of its cognate tetrapyrrole haem.

Replication proteinAsubunit 3 and the iron efficiency response in soybean

In soybean [Glycine max (L.) Merr.], iron deficiency results in interveinal chlorosis and decreased photosynthetic capacity, leading to stunting and yield loss. In this study, gene expression analyses investigated the role of soybean replication protein A (RPA) subunits during iron stress. Nine RPA homologs were significantly differentially expressed in response to iron stress in the near isogenic lines (NILs) Clark (iron efficient) and Isoclark (iron inefficient). RPA homologs exhibited opposing expression patterns in the two NILs, with RPA expression significantly repressed during iron deficiency in Clark but induced in Isoclark. We used virus induced gene silencing (VIGS) to repress GmRPA3 expression in the iron inefficient line Isoclark and mirror expression in Clark. GmRPA3-silenced plants had improved IDC symptoms and chlorophyll content under iron deficient conditions and also displayed stunted growth regardless of iron availability. RNA-Seq comparing gene expression between GmRPA3-silenced and empty vector plants revealed massive transcriptional reprogramming with differential expression of genes associated with defense, immunity, aging, death, protein modification, protein synthesis, photosynthesis and iron uptake and transport genes. Our findings suggest the iron efficient genotype Clark is able to induce energy controlling pathways, possibly regulated by SnRK1/TOR, to promote nutrient recycling and stress responses in iron deficient conditions.

The Gac/Rsm and cyclic‐di‐GMP signalling networks coordinately regulate iron uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile bacterial pathogen capable of occupying diverse ecological niches. To cope with iron limitation, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, whose ability to deliver iron to the cell is crucial for biofilm formation and pathogenicity. In this study, we describe a link between iron uptake and the Gac/Rsm system, a conserved signal transducing pathway of P. aeruginosa that controls the production of extracellular products and virulence factors, as well as the switch from planktonic to biofilm lifestyle. We have observed that pyoverdine and pyochelin production in P. aeruginosa is strongly dependent on the activation state of the Gac/Rsm pathway, which controls siderophore regulatory and biosynthetic genes at the transcriptional level, in a manner that does not involve regulation of ferric uptake regulator (Fur) expression. Gac/Rsm-mediated regulation of iron uptake genes appears to be conserved in different P. aeruginosa strains. Further experiments led to propose that the Gac/Rsm system regulates siderophore production through modulation of the intracellular levels of the second messenger c-di-GMP, indicating that the c-di-GMP and the Gac/Rsm regulatory networks essential for biofilm formation can also coordinately control iron uptake in P. aeruginosa.

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicansto iron availability

BackgroundIron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Δhog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module.ResultsWe observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 μM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Δhog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Δhog1 in comparison to wild type cells. This effect was independent of iron availability in growth media.ConclusionsIn C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to increased levels of components of the reductive iron uptake system in comparison to the wild-type, independent of iron concentrations in the media. However, the additional induction of this system by low iron concentrations was independent of HOG1.

Chapter Ten - Iron homeostasis in the Rhodobacter genus

Metals are utilized for a variety of critical cellular functions and are essential for survival. However cells are faced with the conundrum of needing metals coupled with e fact that some metals, iron in particular are toxic if present in excess. Maintaining metal homeostasis is therefore of critical importance to cells. In this review we have systematically analyzed sequenced genomes of three members of the Rhodobacter genus, R. capsulatus SB1003, R. sphaeroides 2.4.1 and R. ferroxidans SW2 to determine how these species undertake iron homeostasis. We focused our analysis on elemental ferrous and ferric iron uptake genes as well as genes involved in the utilization of iron from heme. We also discuss how Rhodobacter species manage iron toxicity through export and sequestration of iron. Finally we discuss the various putative strategies set up by these Rhodobacter species to regulate iron homeostasis and the potential novel means of regulation. Overall, this genomic analysis highlights surprisingly diverse features involved in iron homeostasis in the Rhodobacter genus.

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.

Post-Transcriptional Regulation of the Sef1 Transcription Factor Controls the Virulence of Candida albicans in Its Mammalian Host

The yeast Candida albicans transitions between distinct lifestyles as a normal component of the human gastrointestinal microbiome and the most common agent of disseminated fungal disease. We previously identified Sef1 as a novel Cys6Zn2 DNA binding protein that plays an essential role in C. albicans virulence by activating the transcription of iron uptake genes in iron-poor environments such as the host bloodstream and internal organs. Conversely, in the iron-replete gastrointestinal tract, persistence as a commensal requires the transcriptional repressor Sfu1, which represses SEF1 and genes for iron uptake. Here, we describe an unexpected, transcription-independent role for Sfu1 in the direct inhibition of Sef1 function through protein complex formation and localization in the cytoplasm, where Sef1 is destabilized. Under iron-limiting conditions, Sef1 forms an alternative complex with the putative kinase, Ssn3, resulting in its phosphorylation, nuclear localization, and transcriptional activity. Analysis of sfu1 and ssn3 mutants in a mammalian model of disseminated candidiasis indicates that these post-transcriptional regulatory mechanisms serve as a means for precise titration of C. albicans virulence.

Similarity and Divergence of Phylogenies, Antimicrobial Susceptibilities, and Virulence Factor Profiles of Escherichia coli Isolates Causing Recurrent Urinary Tract Infections That Persist or Result from Reinfection

In order to obtain a better molecular understanding of recurrent urinary tract infection (RUTI), we collected 75 cases with repeatedly occurring uncomplicated UTI. The genetic relationships among uropathogenic Escherichia coli (UPEC) isolates were analyzed by pulsed-field gel electrophoresis. While 39 (52%) of the RUTI cases were defined as "persistence" of the same strain as the primary infecting strain, 36 (48%) were characterized by "reinfection" with a new strain that is different from the primary strain. We then examined the antimicrobial susceptibilities and phylogenetic backgrounds of 39 persistence and 86 reinfection UPEC isolates, and screened 44 virulence factor (VF) genes. We found that isolates had significant differences in the following: placement in phylogenetic group B2 (41% versus 21%; P = 0.0193) and the presence of adhesin genes iha (49% versus 28%; P = 0.0233) and papG allele I' (51% versus 24%; P = 0.003), iron uptake genes fyuA (85% versus 58%; P = 0.0037), irp-2 (87% versus 65%; P = 0.0109), and iutA (87% versus 58%; P = 0.0014), and an aggregate VF score (median, 11 versus 9; P = 0.0030). In addition, 41% of persistence strains harbored three adhesin genes simultaneously, whereas 22% of reinfection isolates did (P = 0.0289). Moreover, 59% versus 29% (P = 0.0014) of persistence and reinfection isolates contained seven types of iron uptake genes. Taken together, the antimicrobial susceptibilities of UPEC isolates had little effect on the RUTI. Compared with reinfection strains, persistence UPEC isolates exhibited higher VF scores and carried more VF genes than may be involved in the development and progression of RUTI.

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload

Potato is one of the most important staple food in the world because it is a good source of vitamin C, vitamin B6 but also an interesting source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. The lack of iron constitutes the main form of micronutrient deficiency in the world, namely iron deficiency anemia, which strongly affects pregnant women and children from developing countries. Iron biofortification of major staple food such as potato is thus a crucial issue for populations from these countries. To better understand mechanisms leading to iron accumulation in potato, we followed in an invitro culture experiment, by qPCR, in the cultivar Désirée, the influence of media iron content on the expression of genes related to iron uptake, transport and homeostasis. As expected, plantlets grown in a low iron medium (1mgL(-1) FeNaEDTA) displayed a decreased iron content, a strong induction of iron deficiency-related genes and a decreased expression of ferritins. Inversely, plantlets grown in a high iron medium (120mgL(-1) FeNaEDTA) strongly accumulated iron in roots; however, no significant change in the expression of our set of genes was observed compared to control (40mgL(-1) FeNaEDTA).

Antibiofilm activity of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 against Pseudomonas aeruginosa

Members of the actinomycetes family are a rich source of bioactive compounds including diverse antibiotics. This study sought to identify novel and non-toxic biofilm inhibitors from the actinomycetes library for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. After the screening of 4104 actinomycetes strains, we found that the culture spent medium (1%, v/v) of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 inhibited P. aeruginosa biofilm formation by 90% without affecting the growth of planktonic P. aeruginosa cells, while the spent media enhanced the swarming motility of P. aeruginosa. Global transcriptome analyses revealed that the spent medium of Streptomyces sp. BFI 230 induced expression of phenazine, pyoverdine, pyochelin synthesis genes, and iron uptake genes in P. aeruginosa. The addition of exogenous iron restored the biofilm formation and swarming motility of P. aeruginosa in the presence of the spent medium of Streptomyces sp. BFI 230, which suggests that the Streptomyces sp. BFI 230 strain interfered iron acquisition in P. aeruginosa. Experiments on solvent extraction, heat treatment, and proteinase K treatment suggested that hydrophilic compound(s), possibly extracellular peptides or proteins from Streptomyces sp. BFI 230 cause the biofilm reduction of P. aeruginosa. Together, this study indicates that actinomycetes strains have an ability to control the biofilm of P. aeruginosa.

Capsular Types, Virulence Factors and DNA Types of <i>Klebsiella oxytoca </i> Strains Isolated from Blood and Bile

Klebsiella oxytoca is an opportunistic pathogen and is isolated at the second highest frequency among genus Klebsiella from hospitalized patients. According to previous reports, the major virulence factors of K. pneumoniae include capsules and several kinds of pill, whereas the virulence factors of K. oxytoca have not been well investigated. We noticed an increased frequency of K. oxytoca isolates from patients who had undergone a biliary tract operation in a general hospital from May through November, 2009. We then performed a PCR analysis of the virulence factors and an epidemiological analysis with capsular typing (serotyping) and pulsed field gel electrophoresis (PFGE) for K. oxytoca of 11 blood isolates and 10 bile isolates. As a result, serotypes of K9, K15, K26, K31, K43, K47, K55, K70, and K79 were identified in these strains, and K1 and K2 which are frequent serotypes in K. pneumoniae strains were not observed. Two blood isolates of the K55 serotype showed almost the same PFGE pattern, suggesting that these isolates were very closely related and caused cross-infection in a hospital ward. Strains of the K43 serotype were three blood isolates and 1 bile isolate, all of which showed different PFGE patterns. There were no common isolates among the blood and bile isolates. A PCR search revealed that fimH and mrkD genes which are relevant to type 1 and type 2 pili, respectively, were present in all strains, whereas kfuBC, an iron uptake gene, and cf29a were detected in only a few strains. Neither of the mucoid phenotype-related genes magA and rmpA was present in any strains. These results strongly suggest that type 1 and/or type 3 pili would have important roles in the pathogenesis of blood infection and bile infection caused by K. oxytoca.

Hunger for iron: the alternative siderophore iron scavenging systems in highly virulent Yersinia

Low molecular weight siderophores are used by many living organisms to scavenge scarcely available ferric iron. Presence of at least a single siderophore-based iron acquisition system is usually acknowledged as a virulence-associated trait and a pre-requisite to become an efficient and successful pathogen. Currently, it is assumed that yersiniabactin (Ybt) is the solely functional endogenous siderophore iron uptake system in highly virulent Yersinia (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica biotype 1B). Genes responsible for biosynthesis, transport, and regulation of the yersiniabactin (ybt) production are clustered on a mobile genetic element, the High-Pathogenicity Island (HPI) that is responsible for broad dissemination of the ybt genes in Enterobacteriaceae. However, the ybt gene cluster is absent from nearly half of Y. pseudotuberculosis O3 isolates and epidemic Y. pseudotuberculosis O1 isolates responsible for the Far East Scarlet-like Fever. Several potential siderophore-mediated iron uptake gene clusters are documented in Yersinia genomes, however, neither of them have been proven to be functional. It has been suggested that at least two siderophores alternative to Ybt may operate in the highly virulent Yersinia pestis/Y. pseudotuberculosis group, and are referred to as pseudochelin (Pch) and yersiniachelin (Ych). Furthermore, most sporadic Y. pseudotuberculosis O1 strains possess gene clusters encoding all three iron scavenging systems. Thus, the Ybt system appears not to be the sole endogenous siderophore iron uptake system in the highly virulent yersiniae and may be efficiently substituted and/or supplemented by alternative iron siderophore scavenging systems.