Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12

Summary In Escherichia coli the iron uptake systems are regulated by the fur gene product. The synthesis of the outer membrane proteins fiu, fepA, fecA, fhuA, fhuE and cir is derepressed at low iron concentrations in the medium or constitutive in a fur mutant. The fur gene region cloned into pACYC184 was analysed by restriction analysis, Tn1000 mutagenesis and complementation studies. The presence of fur ⁺ plasmids repressed synthesis of the proteins fepA, fecA, fhuE and cir in a chromosomal fur mutant. More quantitatively, the repression to wild-type levels was shown with lac fusions to the genes fiu, fepA and cir. In minicells an 18,000 dalton protein was identified as the fur gene product. Correlated with the fur protein a slightly smaller protein, possibly a degradation product, was observed. The gene fur was mapped on the E. coli chromosome near nagA at about 15.5 min.     

Cloning and initial characterization of the Bordetella pertussis fur gene

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.     

Characterization oftrans-acting regulatory elements affecting the expression of Mn-superoxide dismutase (sodA) inEscherichia coli

Escherichia coli strains harboringtrans-acting mutations affecting the expression of Mn-superoxide dismutase (SOD) gene (sodA) were used to studysodA regulation. Complementation studies revealed that eitherarc (aerobic respiratory control) orfur (ferric uptake regulation) loci independently complemented anaerobic expression of asodA::lacZ protein fusion in one mutant strain (UV16). This mutant exhibited phenotypes (i.e., elevated outer membrane proteins, enzyme activity, and dye sensitivity) typical offur andarc mutants. When these mutations were introduced into an otherwise wild-type background, anaerobicsodA expression occurred only when botharc andfur mutations were present simultaneously, suggesting cooperative roles of Fur and Arc insodA repression. The reconstructedfur arcA andfur arcB double mutants were still inducible by iron chelators, suggesting the possible involvement of another iron-containing repressor protein. A second independent mutant strain harboring atrans-acting regulatory mutation (UV14) was only partially complemented by multicopy plasmids carryingfur ⁺ orarc ⁺ genes, implicating other genetic elements insodA regulation.     

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism

Summary A selection procedure using Mn²⁺ is described. A high percentage of the Mn²⁺ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of -galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur ⁺ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%–70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.     

Regulation of ferric iron transport in Escherichia coli K12: Isolation of a constitutive mutant

Summary The lac genes were inserted with phage Mu(Ap, lac) into the fhuA, fepA, cir and tonB genes which specify components of iron uptake systems. The expression of lac in all these operon fusions was controlled by the availability of iron to the cells, thereby facilitating a quick and simple measurement of the expression of the genes listed above. In an iron rich medium under anaerobic conditions all systems were strongly repressed. fhuA was depressed at higher iron concentration than was fepA or cir, and tonB was repressed only under anaerobic conditions and could be induced by iron limitation.Mutants constitutive for the expression of -galactosidase were selected in a fhuA-lac fusion strain. The outer membrane proteins Cir, FhuA, FecA, 76K and 83K were made constitutively in such mutant strains. Therefore, they were termed fur mutants. In these fur mutant strains, the synthesis of a 19K protein was reduced. Furthermore, it was found that transport of ferric enterochelin and ferrichrome was also constitutive in the fur mutant cells, and that ferric citrate uptake could be induced by only 10 M citrate in the growth medium in contrast to wild-type cells in which at least 100 M citrate was necessary. The fepA gene was concluded to be under an additional control, because it was not fully derepressed by the fur mutation.     

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence

The Neisseris meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.     

Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

Effect of fur mutation on acid-tolerance response and in vivo virulence of avian septicemic Escherichia coli

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.