Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding ironcontaining proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.

Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation.Iron plays a critical role in the cellular metabolism of almost all living organisms. Iron is required for processes as diverse as the tricarboxylic acid (TCA) cycle, electron transport, DNA metabolism, and response to oxidative stress. Because iron has the potential for catalyzing production of reactive oxygen species, excess iron can also pose a significant problem. The influx and intracellular fate of iron must therefore be tightly regulated. This is achieved in part through the action of the irondependent negative regulator Fur, which functions to coordinate the iron status of the cell with the expression of genes involved in iron transport, storage, and metabolism. Under iron-replete conditions, Fur complexes with the ferrous ion and blocks transcription of its regulon by binding to conserved regions termed Fur boxes within the promoter region of these genes. There is another layer of complexity in the scheme of iron-and Fur-dependent regulation. In Escherichia coli, certain genes involved in iron storage, iron metabolism, and antioxidant defense appear to be positively regulated by Fur (6,36,42), and this was recently shown to be mediated through the action of a small RNA (sRNA), RyhB (26). RyhB negatively regulates the expression of sodB (encoding superoxide dismutase), ftn and bfr (encoding ferritin and bacterioferritin), and several iron-sulfur cluster-containing TCA cycle enzyme genes, including the sdh operon (encoding succinate dehydrogenase) and acnA (encoding aconitase). Because RyhB is itself negatively regulated by Fur, the net effect is positive regulation of these genes un...

SummaryThe two TonB systems in Vibrio cholerae were found to have unique as well as common functions. Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome. However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V. cholerae. Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization. This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source. Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size. An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2. A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low-osmolarity medium, but could not use haemin in high osmolarity. This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport. Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.

Interestingly, iron transport in bacteria that you really wait for now is coming. It's significant to wait for the representative and beneficial books to read. Every book that is provided in better way and utterance will be expected by many peoples. Even you are a good reader or not, feeling to read this book will always appear when you find it. But, when you feel hard to find it as yours, what to do? Borrow to your friends and don't know when to give back it to her or him.

Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

The ferrous iron transport system Feo is widely distributed among bacterial species, yet its physical structure and mechanism of iron transport are poorly understood. In Vibrio cholerae, the feo operon consists of three genes, feoABC. feoB encodes an 83-kDa protein with an amino-terminal GTPase domain and a carboxy-terminal domain predicted to be embedded in the inner membrane. While FeoB is believed to form the pore for iron transport, the roles of FeoA and FeoC are unknown. In this work, we show that FeoA and FeoC, as well as the more highly conserved FeoB, are all required for iron acquisition by V. cholerae Feo. An in-frame deletion of feoA, feoB, or feoC eliminated iron acquisition. The loss of transport activity in the feoA and feoC mutants was not due to reduced transcription of the feo operon, suggesting that these two small proteins are required for activity of the transporter. feoC was found to encode a protein that interacts with the cytoplasmic domain of FeoB, as determined using the BACTH bacterial two-hybrid system. Two conserved amino acids in FeoC were found to be necessary for the interaction with FeoB in the two-hybrid assay, and when either of these amino acids was mutated in the context of the entire feo operon, iron acquisition via Feo was reduced. No interaction of FeoA with FeoB or FeoC was detected in the BACTH two-hybrid assay.

The gram-negative enteric pathogen Vibrio cholerae requires iron for growth. V. cholerae has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make. One siderophore that V. cholerae transports, but does not make, is enterobactin. Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA. In this study, two candidate enterobactin receptor genes, irgA (VC0475) and vctA (VCA0232), were identified by analysis of the V. cholerae genomic sequence. A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin. When either irgA or vctA was supplied on a plasmid, the ability of the irgA vctA double mutant to use enterobactin was restored. This indicates that both VctA and IrgA transport enterobactin. We also identify the genes vctPDGC, which are linked to vctA and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230). An irgA::TnphoA mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of V. cholerae infection (M. B. Goldberg, V. J. DiRita, and S. B. Calderwood, Infect. Immun. 58:55-60, 1990). In this work, a new irgA mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose. These data indicate that the virulence defect in MBG40 is not due to the loss of irgA function and that irgA is unlikely to be an important virulence factor.Vibrio cholerae causes the severe diarrheal disease cholera (18, 32). Many of the genes required for this gram-negative pathogen to cause disease in humans or in animal models have been described. A key virulence factor is the cholera toxin, which is responsible for the severe voluminous diarrhea characteristic of cholera. The toxin-coregulated pilus is essential for colonization of the intestinal epithelium (65). The synthesis of this bundle-forming, type IV pilus is coordinately regulated with the synthesis of cholera toxin, and proper transcriptional control of this regulon is required for virulence. Another V. cholerae gene that is reported to be required for virulence is irgA. The irgA::TnphoA mutant strain MBG40 has a decreased competitive index and nearly a 100-fold-increased 50% lethal dose (LD 50 ) in an infant mouse model relative to its parental strain O395 (24). Other genes required for full virulence have been identified more recently, and these include genes for nutrient acquisition, stress response, and proper colonization of the lower small intestine (see references 18 and 32 for reviews).Genes for the acquisition of the nutrient iron play a critical role i...