Dataset Information

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

ABSTRACT: We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism.

SUBMITTER: Stojiljkovic I

PROVIDER: S-EPMC178763 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

Stojiljkovic I I Srinivasan N N

Journal of bacteriology 19970201 3

We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementin ...[more]

PMID: 9006036

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Project description:Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [59Fe]apoferrichrome and 59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd ], 1 nM; Michaelis-Menten constant [KM ], 0.1 nM; Vmax, 19 pMol/109 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59Fe-citrate were similar to those of E. coli FecA (KM , 5.3 nM; Vmax, 29 pMol/109 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization).IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus, its heme/hemoglobin transporter (HutA).

Project description:Neisseria meningitidis requires iron, and in the absence of iron alters its gene expression to increase iron acquisition and to make the best use of the iron it has. During different stages of colonization and infection available iron sources differ, particularly the host iron-binding proteins haemoglobin, transferrin, and lactoferrin. This study compared the transcriptional responses of N. meningitidis, when grown in the presence of these iron donors and ferric iron, using microarrays.Specific transcriptional responses to the different iron sources were observed, including genes that are not part of the response to iron restriction. Comparisons between growth on haemoglobin and either transferrin or lactoferrin identified changes in 124 and 114 genes, respectively, and 33 genes differed between growth on transferrin or lactoferrin. Comparison of gene expression from growth on haemoglobin or ferric iron showed that transcription is also affected by the entry of either haem or ferric iron into the cytoplasm. This is consistent with a model in which N. meningitidis uses the relative availability of host iron donor proteins as niche indicators.Growth in the presence of haemoglobin is associated with a response likely to be adaptive to survival within the bloodstream, which is supported by serum killing assays that indicate growth on haemoglobin significantly increases survival, and the response to lactoferrin is associated with increased expression of epithelial cell adhesins and oxidative stress response molecules. The transferrin receptor is the most highly transcribed receptor and has the fewest genes specifically induced in its presence, suggesting this is the favoured iron source for the bacterium. Most strikingly, the responses to haemoglobin, which is associated with unrestricted growth, indicates a low iron transcriptional profile, associated with an aggressive phenotype that may be adaptive to access host iron sources but which may also underlie the lethal features of meningococcal septicaemia, when haemoglobin may become a major source of iron.

Dataset Information

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

Publications

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

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