Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae

Harkness, Robin E.; Chong, Pele; Klein, Michel

doi:10.1128/jb.174.8.2425-2430.1992

Cited by 37 publications

(26 citation statements)

References 53 publications

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“…This is surprising given the fact that other proteins, such as a 31 kDa iron-regulated periplasmic protein found in both H . influenzae and P. haemolytica, appear to be strongly conserved with -95 % identity over available sequence (Harkness et al, 1992;Tabatabai & Frank, 1997). This would suggest that it is not lack of phylogenetic overlap which is responsible for the degree of sequence divergence in fbpA between species, but perhaps biological constraints dictating variation.…”

Section: Discussionmentioning

confidence: 99%

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue

et al. 1998

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In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasfeurella haemolytica grown under irondepleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATPbinding cassette (ABC)-type uptake systems, respectively. Based on amino acicUDNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Febinding protein ldiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Past eurellaceae or Neisseriaceae .~~

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Section: Discussionmentioning

confidence: 99%

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue

et al. 1998

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“…Low molecular weight protein standards for SDS-PAGE analysis were purchased from Pharmacia Biotech Inc. The radioisotopes [␣- Cloning of the hitA Gene Region and the Minimal hitABC Operon-A 700-bp hitA gene fragment was PCR amplified from H. influenzae DL63 chromosomal DNA by designing a primer based on the N-terminal amino acid sequence of a 40-kDa iron-regulated periplasmic protein suspected to be the H. influenzae Fbp analog (27) and by designing a second primer to a conserved region shared by the closely related sfuA (26) and gonococcal fbp genes (33). PCR reactions were performed in 50 l volumes by methods previously described (28).…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of a Haemophilus influenzae Periplasmic Iron Transport Operon

Adhikari

Kirby

Nowalk

et al. 1995

Journal of Biological Chemistry

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Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic ironbinding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.High affinity acquisition of iron from the host environment is a necessary determinant of virulence for pathogenic bacteria (1-9). This acquisition is vital for survival in the human host, where levels of extracellular iron are tightly controlled by the Transferrins (transferrin and lactoferrin), a family of ironbinding proteins that function in the extracellular chelation and transport of host iron (9). By binding iron with high affinity, Transferrins ensure that all extracellular iron is both efficiently sequestered from pathogenic invaders and mobilized for transport to host tissues. Microorganisms growing in the human host must therefore possess mechanisms for obtaining Transferrin-sequestered iron. For a number of pathogenic members of the Pasteurellaceae (H. influenzae) and Neisseriaceae (Neisseria meningitidis and Neisseria gonorrhoeae), iron acquisition is initiated by cell-surface receptors specific for the Transferrins (10 -16). Iron is removed from these proteins and transported across the outer membrane, presumably by an energy-dependent TonB-mediated process (17-19) involving gated-pore properties of the outer membrane receptor (18,20). The result is deposition of free iron within the periplasm, where it is separated from the cytosol, its eventual destination, by the cytoplasmic membrane (21).Transport of free iron from the periplasmic space into the cytoplasm is proposed to occur by a classic active transport process involving a periplasmic binding protein, a specific cytoplasmic permease, and an energy-supplying nucleotide-binding protein (22). Much of what is known about the biochemistry of a...

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“…However, the FeEnt-FepB system differs from other binding protein-dependent transport systems in several ways. First, FepB and another iron binding periplasmic protein of E. coli, FhuD (4,17) are synthesized at low levels relative to sugar (MalE [16]) and amino acid (HisJ [45]) binding proteins and the iron binding protein Fbp from Haemophilus influenzae (10). Even when derepressed by iron starvation, chromosomally encoded FepB was not detected in Coomassie blue-stained gels of periplasmic extracts.…”

Section: Discussionmentioning

confidence: 99%

Binding of Ferric Enterobactin by theEscherichia coliPeriplasmic Protein FepB

Sprencel

Cao

et al. 2000

J Bacteriol

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The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K d of the ferric enterobactinFepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K d ‫؍‬ 30 nM) and ferric enantioenterobactin (K d ‫؍‬ 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K d ‫؍‬ 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

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Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae

Cited by 37 publications

References 53 publications

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue

Biochemical Characterization of a Haemophilus influenzae Periplasmic Iron Transport Operon

Binding of Ferric Enterobactin by theEscherichia coliPeriplasmic Protein FepB

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